Polyfunctional Nanofibril Appendages Mediate Attachment, Filamentation, and Filament Adaptability in Leptothrix cholodnii
- Tatsuki Kunoh*Tatsuki Kunoh*Email: [email protected]Faculty, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8577, JapanMore by Tatsuki Kunoh,
- Kana MorinagaKana MorinagaGraduate School of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8577, JapanMore by Kana Morinaga,
- Shinya SugimotoShinya SugimotoDepartment of Bacteriology and Jikei Center for Biofilm Research and Technology, The Jikei University School of Medicine, 3-25-8, Nishi-Shimbashi, Minato-ku, Tokyo 105-8461, JapanMore by Shinya Sugimoto,
- Shun MiyazakiShun MiyazakiGraduate School of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8577, JapanMore by Shun Miyazaki,
- Masanori ToyofukuMasanori ToyofukuFaculty, Microbiology Research Center for Sustainability, , University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8577, JapanMore by Masanori Toyofuku,
- Kenji IwasakiKenji IwasakiLife Science Center for Survival Dynamics, Tsukuba Advanced Research Alliance, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8577, JapanMore by Kenji Iwasaki,
- Nobuhiko Nomura*Nobuhiko Nomura*Email: [email protected]Faculty, Microbiology Research Center for Sustainability, , University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8577, JapanMore by Nobuhiko Nomura, and
- Andrew S. Utada*Andrew S. Utada*Email: [email protected]Faculty, Microbiology Research Center for Sustainability, , University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8577, JapanMore by Andrew S. Utada
Copyright © 2019 American Chemical Society
Abstract
Leptothrix is a species of Fe/Mn-oxidizing bacteria known to form long filaments composed of chains of cells that eventually produce a rigid tube surrounding the filament. Prior to the formation of this brittle microtube, Leptothrix cells secrete hair-like structures from the cell surface, called nanofibrils, which develop into a soft sheath that surrounds the filament. To clarify the role of nanofibrils in filament formation in L. cholodnii SP-6, we analyze the behavior of individual cells and multicellular filaments in high-aspect ratio microfluidic chambers using time-lapse and intermittent in situ fluorescent staining of nanofibrils, complemented with atmospheric scanning electron microscopy. We show that in SP-6 nanofibrils are important for attachment and their distribution on young filaments post-attachment is correlated to the directionality of filament elongation. Elongating filaments demonstrate a surprising ability to adapt to their physical environment by changing direction when they encounter obstacles: they bend or reverse direction depending on the angle of the collision. We show that the forces involved in the collision can be used to predict the behavior of filament. Finally, we show that as filaments grow in length, the older region becomes confined by the sheath, while the newly secreted nanofibrils at the leading edge of the filament form a loose, divergent, structure from which cells periodically escape.
Microbial mats are macroscopic biofilms made of stratified communities of microorganisms that develop in mineral enriched aqueous environments, where they typically grow at the solid-liquid interface of stream beds. Like biofilms, these communities facilitate the sharing of resources, provide protection from predation(1,2) and changing environmental conditions, and enable synchronization of gene expression for their inhabitants.(3−5) Whereas most biofilms are densely packed, microbial mats are fundamentally different; these communities are porous structures composed of flocs woven together with extracellular polymeric substances (EPS) and rigid bacterial stalks and sheaths.(2,5,6) Microbial mats have received much less attention than traditional surface attached biofilm communities despite their significant role in the global cycling of minerals.(6)
The sheath-forming genus Leptothrix is a diverse group of bacteria that frequently dominate microbial mats in iron-rich aqueous environments.(7) These bacteria form linear cell-chains surrounded by an EPS sheath that can reach ∼100s of micrometers in length.(2,8) In the process of filament formation, the bacteria secrete proteins that oxidize dissolved Fe and Mn, which in turn generates metal oxide nanoparticles that accumulate in the sheath matrix;(7,9−11) this process transforms the soft sheath into a rigid mineral-encrusted hollow microtube, ∼1.5 μm in diameter. These rigid microtubes play important physical as well as ecological roles: they provide mechanical support for mat development, while simultaneously facilitating intramat migration of inhabitants toward more favorable environments.(2,8,10) In addition to structural support, the linear organization of these filamentous bacteria may indicate long-range electron or signal transport. For example, remarkable filamentous bacteria from the genus Desulfobulbaceae, known as “cable bacteria”, transmit electrons intercellularly over centimeters within an insulating sheath.(12−14) Whether Leptothrix possesses a similar transport mechanism within their sheath is unknown.
The sheaths themselves contain abundant COOH, NH2, and SH terminal functional groups that can act as a passive matrix that sorbs metals.(10,15−20) These reactive end-groups have been implicated in the biomineralization of a broad range of commercially important metals such as Ag, Au, Cu, Rh, Ru, Ti, Y, and Zr.(20,21) A fundamental understanding of sheath formation on filaments could enable the industrial harvesting of pigments and catalysts, lead to the development of novel amorphous iron oxides for lithium-ion battery anodes, and facilitate extraction of heavy and precious metals from surface waters through development of novel absorbents from the sheaths.(11,20−24)
The type species of the Leptothrix genus, L. ochracea, is a prolific sheath forming bacterium. However, it has proven resistant to laboratory cultivation due to intricate ecological interdependencies,(25) making real-time analysis of sheath development difficult.(8,26) Although there appear to be differences in the respective mechanisms of sheath formation, the related bacterium L. cholodnii has been used as a model organism to clarify much of what is known about sheath formation due to the ease of its cultivation.(1,27) Briefly, the L. cholodnii sheath gradually develops from physical entanglements, or possibly chemical cross-links,(1,28) between hair-like appendages that decorate the catenulate cells. These appendages, called nanofibrils, are glycoconjugates, which are mainly composed of complex amino-polysaccharides(19) that are ∼10 nm in diameter and ∼10 μm in length.(8,24,27) As cell division proceeds, new nanofibrils are continuously secreted and the entanglements are thought to irreversibly aggregate, eventually enclosing the cell filament in a sheath of woven nanofibrils.(1,24,27) Previous studies have clarified much of the qualitative description of sheath formation using a combination of traditional live-cell imaging methods in Petri dish cultures,(1,28,29) while atomic force microscopy (AFM),(8) scanning electron microscopy (SEM),(30) and transmission electron microscopy (TEM)(7,27) have been used to characterize the nanostructure of the sheath and microtubes. However, the role of nanofibrils in surface recognition, filament initiation, and sheath development remains unclear. Despite the importance of these different imaging methods, which have led to this qualitative picture, they are unable to provide access to the spatial and temporal resolutions required to analyze the behavior of the individual filament-founding bacteria.(31−33) This is crucial to elucidating the mechanisms of sheath and filament formation in these bacteria.
In this paper, we investigate the function of L. cholodnii nanofibrils in the context of surface attachment and filament formation using time-lapse microscopy in high-aspect ratio two-dimensional (2D) microfluidic culture chambers complemented with atmospheric scanning electron microscopy (ASEM).(34−36) Our microfluidic device allows us to circumvent the difficulties in tracking development of a bacterial colony that rapidly forms massive three-dimensional (3D) structures by directing growth in 2D, while simultaneously enabling multiple labeling and washing steps without disturbing the sample. We show that cell-associated nanofibrils are important for surface attachment and that their distribution on young filaments is correlated to the direction of filament elongation. We show that elongating filaments demonstrate the ability to adapt to obstructions by bending in a new direction or by reversing the direction of elongation; this behavior depends on the balance of bending and compressive forces experienced by the tip of the filament due to the angle of impact. Using ASEM we show that the nanofibril sheath envelops cell filaments as early as the second division, which limits cell escape to the elongating distal pole. Furthermore, we show that the nanofibril sheath displays a density gradient as a function of axial position, corresponding to the degree of sheath maturation. Near the initial attachment point, where the sheath is most mature, it has a dense tubular structure, whereas close to the distal tip, the relatively immature sheath has a diffuse, divergent structure. When cell growth outpaces sheath maturation, this divergent structure may be unable to retain the cells, resulting in cell-escape from the open end of the sheath.
Results and Discussion
ARTICLE SECTIONS
SP-6 Surface Attachment and Filament Formation in 2D Chambers
Leptothrix is known to form amorphous multicellular aggregates ranging in extent from centimeters to meters in the environment as well as in batch culture.(7,16,24,37)L. cholodnii SP-6 cells grown in 2 day batch-culture, similarly, develop into such 3D aggregates (see SI Figure S1). Confocal images of representative aggregates reveal that they are mainly composed of heavily entangled filaments. Due to the difficulty in analyzing dense 3D structures, which simultaneously obscure the dynamics of individual cells and the developmental progression of individual filaments, we utilize a microfluidic device to culture individual cells in high aspect-ratio “2D” chambers. Since the height of the chambers is only slightly larger than the diameter of SP-6 cells, the cells and filaments are always in focus and adjacent filaments are unable to grow over one another; this enables the tracking of filament evolution (see TOC graphic and schematic shown in SI Figure S2). A video of cells growing in a “2D” chamber is shown in SI Movie S1. In contrast, a video of several cells at the bottom of a glass-bottom dish demonstrates the difficulty in imaging unconfined cells; here some cells diffuse randomly, another demonstrates tethered swimming,(38,39) while another has attached to the surface (SI Movie S2).
We inoculate the device by infusing bacterial culture into the microfluidic channels, which typically carries a few cells into the chambers. Once there, cells swim about the chamber using a polar flagellum(7,40) before irreversibly(41−43) attaching to the surface, which we define as the starting time of the experiment (t = 0 min). Unattached cells are washed out of the device due to the flowing medium. We observe two distinct modes of elongation in the chambers: (1) unilateral, where a single pole of the filament elongates due to cell division, while the other pole remains fixed in the chamber; and (2) bilateral, where both poles simultaneously elongate, while the center of the founder cell remains fixed in the chamber. SP-6 cells exhibiting unilateral and bilateral elongation are shown in the time-lapse image sequences in Figure 1A,B, respectively. A kymograph showing the smooth elongation of a single filament is shown in SI Figure S3. In contrast, the “sheathless” variant SP-6 strain (SP-6 SL), which is unable to biosynthesize the sheath matrix,(7) lacks the ability to attach within the 2D chambers for the duration of the experiments. Although SL variants are reported to be able to form filaments in static conditions,(7) we find that filament formation is ablated; these cells simply roam about the chamber, periodically dividing, as shown in SI Movie S3 and Figure 1C. Aside from cellular elongation due to division, the average length of the SL cells remains constant at ∼4 μm over the course of the experiment. For the same time period, surface-attached SP-6 cells form cell filaments that increase in length monotonically. Assuming exponential cell growth within the filament, we fit the length with an exponential and find good agreement over this limited time frame (Figure 1D).
Figure 1
Figure 1. Time-lapse sequence showing cell growth in 2D chambers for cells exhibiting (A) unilateral elongation and (B) bilateral elongation. (C) Time-lapse sequence of a variant strain (SP-6 SL) unable to produce a sheath. Black arrowheads in (A,B) indicate the same spatial position in each image, the unfilled arrowheads indicate the position of the moving pole(s), and red arrows indicate the direction of elongation. Scale bars = 5 μm. The numbers refer to the time in minutes. (D) Filament length of SP-6 (●) and SP-6 SL (□) strains measured as a function of time within 2D chambers. The error bars represent the standard deviation (SD) taken from 10 filaments. The dashed exponential line is a best fit to the data (see Supporting Material and Methods S1.3 section).
These results indicate that cell-surface attachment is necessary for filament formation. They also suggest that, based on the differences between SP-6 and SL variants, the ability to synthesize a sheath appears to play a major role in surface attachment. This is not surprising since the sheaths of other species of Leptothrix are known to be adherent to surfaces, cells, and other filaments.(8,10,37) However, since surface attachment and time appear to be important for sheath development, it is likely that the surface attaching SP-6 cells are decorated with an incompletely formed sheath, composed of entangled nanofibrils; this suggests a direct role for nanofibrils in surface attachment. Importantly, we note that although SL variants do not synthesize a sheath, it is unknown if they can generate nanofibrils. In fact, the sheathless variant of a different L. cholodnii (OUMS1) strain can produce nanofibrils; however, in this case, the nanofibrils disassociate from the cells.(18,30) Thus, it is unknown if SP-6 SL variants do not produce a sufficient quantity of nanofibrils or if they are somehow lost. Taken together, these data suggest that in the case of SP-6, cell surface-associated nanofibrils appear to be important for initial surface attachment.
Direction of Elongation and the Distribution of Nanofibrils on Young Filaments
To clarify the role of the cell-associated nanofibrils in the unilateral and bilateral modes of elongation, we fluorescently label the nanofibril aggregates with an NH2-specific fluorescein reagent on live cells in batch culture(19) (see SI Figure S4A). We confirm the specificity of the fluorescein label by co-staining with a lectin, which recognizes sugar molecules contained in the nanofibrils. The overlap of fluorescence signals strongly suggests that the NH2-specific reagent is sufficiently specific to the nanofibrils (see Materials and Methods and SI Figure S5).
We find that SP-6 aggregates strongly fluoresce when labeled with the fluorescein reagent, indicating an abundance of nanofibrils, whereas SP-6 SL cells exposed to the same conditions remain unlabeled (see SI Figures S4B and S5). Filtering the batch culture to separate the large SP-6 aggregates from planktonic cells, we find that the free individuals in the filtrate have little to no fluorescence compared to the aggregates from the same culture. This difference in fluorescence suggests that prior to surface attachment, the free SP-6 cells in batch culture lack a sufficient amount of nanofibrils to be labeled; this result parallels the lack of fluorescence in the SP-6 SL cells (see SI Figure S4B, second and third columns).
To track the secretion of nanofibrils on these young SP-6 filaments from the filtrate, we culture cells within the “2D” microfluidic chambers, then label their nanofibrils. During this process, we incubate the cells long enough for them to lengthen by at least ∼50% prior to infusing the labeling reagent to allow sufficient accumulation of the nanofibrils to be labeled (see Materials and Methods). We find that unilaterally elongating filaments acquire fluorescence around the stationary pole, while the elongating pole remains unlabeled. In contrast, bilaterally elongating filaments acquire fluorescence in a central band, while the elongating poles remain unlabeled; this central band is centered around the location of the mother cell. An image sequence of uni- and bilaterally elongating filaments combined with fluorescence labeling is shown in Figure 2A. In contrast, cells that arrive in the “2D” chamber immediately prior to staining, conspicuously, remain free of fluorescence (see circled cells in Figure 2A, t = 200 min). Based on the width of the fluorescence on the labeled cells, it appears that >100 min are needed for the biosynthesis of a sufficient quantity of the nanofibrils to be labeled (see Figure 2A, t ≥ 215 min). Although we lack the spatial resolution to determine the 3D distribution of nanofibrils on individual cells, the fluorescence distribution on young filaments suggests that during unilateral elongation, the nanofibrils close one end of the sheath, whereas during bilateral elongation, nanofibrils are more centrally located, leaving both ends free to elongate.
Figure 2
Figure 2. Cellular distribution of nanofibrils. (A) Bright-field and fluorescence time-lapse sequence with stop-flow fluorescent labeling of the nanofibrils during that gap between 200 and 215 min. The upper cell demonstrates unilateral elongation, while the lower cell demonstrates bilateral elongation. Black arrowheads indicate the same spatial position in each image, red arrows indicate the direction of elongation, the yellow circle encloses cells that attach in the chamber midway through the imaging sequence, and the unfilled arrowheads indicate the position(s) of the moving pole(s). The numbers refer to the time in minutes. (B) ASEM images of SP-6 (left) and SP-6 SL (right) cells. The white arrow (left) indicates the nanofibrils. Scale bars = 5 μm.
Fluoresence labeling of the entangled nanofibrils enables the visualization of their approximate distribution on young filaments but is unable to access the submicron distribution on individual cells. To do so, we turn to ASEM, which can be used to image directly in aqueous solutions and thus is free from the artifacts associated with sample dehydration.(24) ASEM reveals that SP-6 cells are heterogeneously covered with nanofibrils, while in stark contrast, the sheathless variants lack visible nanofibrils altogether; representative ASEM images of SP-6 and SP-6 SL cells acquired under the same settings are shown in Figure 2B and SI Figure S6. The absence of fluorescently labeled nanofibrils on free SP-6 cells (SI Figure S4B) and the lack of nanofibrils on SL cells in the ASEM images (Figure 2B) collectively indicate that cell-associated nanofibrils are important for surface attachment.
Collisions between Filaments and Obstructions Lead to Bending and Reversal
In the absence of obstacles, elongating filaments grow approximately linearly. However, due to the finite dimensions of the chamber, growing filaments frequently collide with obstacles such as the chamber wall or other filaments. Obstacles can either deflect the direction of elongation, which causes filaments to bend, or they directly impede forward progress, which causes the direction of elongation to reverse, as shown in Figure 3A,B, respectively (see also SI Movies S4 and S5). Tracking filament evolution as a function of time reveals that the precollision shape in both bending and reversing filaments is unaffected by an impact (see colored traces overlaid in Figure 3A,B). In addition, we observe that elongating filament poles are unable to dislodge established sections of other filaments (see SI Movie S1). Axially compressed rods will generally undergo long-wavelength buckling unless the shaft is supported, when it can support higher curvature bends.(44) Since collisions appear to have no effect on the precollision shape, it appears that the filaments are supported against lateral deflection; this could be effected through attachment of the external face of the sheath to the chamber, while the cells move through the inner tube.
Figure 3
Figure 3. Filament bending and reversal. Time-lapse image sequence showing impact-induced filament (A) bending (at t = 380 min) and (B) reversal (at t = 255 min). The colored cells in (A,B) are overlays of the filament at different time points (labeled). (C) Model of the forces generated during a collision between a filament and obstacle as a function of the angle of impact, θ. The blue circles (●) represent fbend, the unfilled circles (○) represent the bending stiffness, and the blue shaded area indicates 30% variability in this value. The red triangles (▼) represent freverse, the unfilled triangles (▽) represent the nanofibril adhesion force, Fadh, and the red shaded area indicates 20% variability. (Inset) Schematic showing the collision between an elongating filament and an obstruction. (D) Binned probability densities of bending and reversing as a function of θ. The sum of the respective densities for bending and reversal equal the total, shown by the thick black line outlining the bars. The dashed (blue) and dotted (red) lines are simulated probability densities for bending and reversal, respectively, based on the model in panel C (see SI S1.4). (E,F) A time-lapse sequence combined with stop-flow fluorescence labeling of nanofibrils showing impact-induced bending and reversal, respectively. For (A,B) t = 0 defines the moment of cell-surface attachment, while in (E,F) it refers to start of the experiment upon completion of the nanofibril labeling. In all time-lapse sequences, the black arrowheads indicate the same spatial position in each image, the red arrows indicate the direction of elongation, unfilled arrowheads indicate the position of the moving pole(s), and the blue arrowheads indicate the location of a collision that causes reversal. The numbers refer to the time in minutes. Scale bars = 5 μm.
To elucidate the causes of filament bending versus reversal, we consider the component forces experienced by a filament during a collision. Filament elongation could arise from a number of sources such as an uncharacterized surface motility or through nanofibril extrusion that propels the enclosed cells forward similar to a model proposed for L. ochracea.(8) Although we do not rule out these other forces, it appears that SP-6 filament elongation is largely driven by cell division, directed away from the initial patch of attachment. The location of initial attachment likely harbors the oldest secreted nanofibrils and thus likely has the strongest adhesion energy.(45) We assume that during a collision, filament compression results from cellular division of the catenulate cells confined within the sheath, which pushes axially away from the initial attachment point. These assumptions limit the maximum force that a filament can exert using its adhesive patch of entangled nanofibrils at the initial attachment point, Fadh. During a collision, filaments experience varying degrees of bending and compressive loads depending on the angle of the impact. Forces directed perpendicularly to the axis of the filament act to bend the filament, while forces directed along the filament axis act to compress the filament, which can cause reversal. We define these forces as fbend = Fadh sin θ cos θ and freverse = Fadh(sin2θ – cos2θ), respectively, where θ is the angle between the filament axis and the obstacle, for 0° ≤ θ ≤ 90° (see inset in Figure 3C, SI S1.4, and SI Figure S7). We assume that filaments have an inherent stiffness that resists bending,(8,46) while their nanofibril at the initial attachment point resists reversal during a collision with an obstacle; however, when the bending or compressive force on the filament surpasses these resistive forces, they will either bend or reverse, respectively.
To describe the state of the system, we normalize all forces by Fadh and plot fbend and freverse as a function of θ, as shown in Figure 3C. According to this simple model, bending becomes possible as fbend becomes comparable to the bending stiffness for θ ≳ 10°, whereas filament reversal becomes possible as freverse becomes comparable to Fadh for θ ≳ 75°. In addition, in the range of θ ≈ 65–80°, there are θ values where both bending and reversal can occur. Finally, as θ → 90°, filament reversal is predicted to become dominant as freverse approaches unity. We note that the relative values of filament stiffness and nanofibril adhesion force are adjustable in the model and determine the specific crossover θ values. Moreover, to account for population level heterogeneity in bending stiffness and nanofibril adhesion force, we incorporate variability into these resistive model forces.
To test this simple model, we quantify the actual outcome of collisions by measuring the frequencies of bending and reversal as a function of θ. Supporting the general shape of the forces in the model, the probability densities show that bending occurs over a wide range of θ values, while filament reversals occur only when θ > 60° (Figure 3D). We simulate the outcomes of multiple collisions as a function of θ and generate probability densities for bending and reversal, shown by the dashed and dotted lines in Figure 3D (see SI S1.4). By adjusting the relative strength of the filament stiffness and nanofibril adhesive force as well as the variability of these resistive forces, we achieve good agreement between the data and the model. Surprisingly, when we remove all variability from the filament bending stiffness (blue filled area in Figure 3C), the model predicts the absence of filament bending for θ > 75°, which contradicts our observations (line-filled bars, Figure 3D). In fact, the significant number of filaments that bend for θ > 75° reflects heterogeneity in flexural rigidity of the filament. This heterogeneity could arise from the fact that the filament is composed of the sheath and enclosed cells, each of which have their own bending rigidities. According to recent reports, the flexural rigidity of the L. ochracea sheath(8) is comparable to that of Escherichia coli and Bacillus subtilis cells.(47,48) Assuming that the L. cholodnii sheath has a similar flexural rigidity, a large variability in bending rigidity of the filament could arise when bending occurs over the gap between two cells, where only the sheath supports the strain, compared to when both the sheath and cells are simultaneously bent.
As the data show, the probability of reversal increases when a larger fraction of the total collision force is converted into compressing the filament (θ > 60°). Since filaments do not buckle during collisions nor are the individual cells in the filament compressible, freverse is exerted directly on the nanofibril attachments that link the filaments to the surface at the initial attachment point. We suspect that during reversals, the entangled nanofibril sheath is unable to support the compressive load and is either pulled from the cell body or from the surface of the device, or both. To examine the effect of an impact on the nanofibril distribution at the point of initial attachment, we label the nanofibrils immediately after irreversible attachment (as in Figure 2A) then wait for collisions to occur. We find that the location of the labeled nanofibrils on bending filaments remains largely unaltered by collisions since there is no retrograde motion of the filament (see Figure 3E). Careful examination of the labeled nanofibrils reveals a small measure of fluorescence intensity loss, which could be due to the reagent washing away or a mixing of the labeled nanofibrils into the surrounding sheath as it matures. During reversal, we find a small measure of vertical displacement of the labeled nanofibril sheath; however, relative to the freed filament that slides past the initial attachment point, the nanofibril sheath remains adhered to the chamber in largely the same position (see Figure 3F). This indicates that the connections between the body of the founder cell and the nanofibrils rupture during reversal.
These data support our picture that filament bending and reversal depends on the component forces of impact at the filament tip. Moreover, SP-6 filaments demonstrate the ability to support high-curvature bending without catastrophic fragmentation as well as sustain compressive loads that reverse the direction of elongation without buckling. These characteristics suggest that at the time of collision, the filaments are already wrapped within an elastic sheath. In the case of filament bending, the combination of sheath elasticity and intercellular gaps enables bending without breaking, while in the case of reversal, the sheath prevents the cell chain from buckling under a compressive pressure.
Gradient in Sheath Maturation Starting from Initial Attachment Point
It is unclear when the entangled nanofibrils aggregate to form the sheath that encloses the filaments; however, during filament elongation, we frequently observe that the intercellular space between adjacent cells develops into wide gaps. Although these gaps can be as large as ∼2.5 μm, which is roughly twice the cell diameter, the cells remain aligned, even during cell division (see Figure 4A). The transience of these gaps suggests the presence of a sheath that maintains alignment of the cells in the filaments.
Figure 4
Figure 4. Intercellular gaps within filaments. (A) Time-lapse sequence showing the appearance and development of a large intercellular gap, followed by its disappearance during filament elongation. The black arrowheads indicate the same spatial position in each image, red arrows indicate direction of elongation, yellow lines and arrows indicate location and width of the gap between adjacent cells, the unfilled arrowheads indicate the position of the moving pole(s), and blue arrowheads indicate collision with an obstruction. The numbers refer to the time in minutes. (B) ASEM images showing the different stages of the immature sheath formation. White arrows indicate (left) diffuse nanofibrils on a young filament, (middle) a tighter distribution on a longer filament, and (right) the outline of the sheath on an even longer filament. Scale bars = 5 μm.
To directly image the state of the sheath, we again turn to ASEM. We culture SP-6 cells on an ASEM substrate long enough to ensure an abundance of filaments in various states of maturation. Although the young filaments, which are made of pairs of cells, tend to be surrounded by a diffuse web of nanofibrils, similar to the nanofibrils on individual cells, we find that the more mature filaments are surrounded by a tubular structure (compare Figure 2B to 4B). Notably, pronounced intercellular gaps are also present in the longer filaments. These ASEM snapshots indicate that the nanofibril entanglements form a sheath as early as the second cellular division. Furthermore, taken together with the images from Figure 2, these results suggest a gradient in the degree of sheath maturation along the filament that corresponds directly to its shape: the oldest sections are tubular, while the younger sections are a diffuse web of nanofibrils.
Although the ASEM snapshots provide no information on the dynamics of sheath maturation, since we know that the sheath eventually develops into an open-ended biomineralized microtube,(1,8) this suggests that the immature sheath itself is likely to be open-ended. Supporting this picture, cell escape occurs exclusively from the ends of the elongating filaments (Figure 5A). In light of the low nanofibril fluorescence intensity (Figure 2A, SI Figure S4) and the diffuse nanofibril distribution on both individual cells and young filaments (see Figures 2B and 4B), we hypothesize that cells at the elongating poles are able to escape the filament due to insufficient nanofibril coverage and sheath development.
Figure 5
Figure 5. Filament fragmentation and development of the sheath. (A) Escape of cells from the leading edge of the filament. The yellow arrowheads indicate the positions where filament fragmentation occurs, whereas the yellow arrows indicate the positions of the cells, after escaping from the filament. (B) Time-lapse sequence with stop-flow fluorescence labeling of the nanofibrils at two time-points during 265–280 (first) and 1495–1510 min (second), respectively. (Insets) Magnified, background-subtracted images showing the distribution of fluorescent nanofibrils on the cell filament. For (A,B) the black arrowheads indicate the same spatial position in each image, red arrows indicate the direction of elongation, blue arrowheads indicate the location of impact that causes reversal, unfilled arrowheads indicate the position of the moving pole(s), and blue arrowheads indicate collision with an obstruction. (C) (left) Fluorescence intensity profile measured along the axis of a representative filament. The width is defined as the peak-to-peak distance between intensity maxima. (right) Histogram of intensity-profile widths measured at different axial positions normalized by the width of the filament at its origin. For filaments shorter than 20 μm in length, the width is measured at the distal end and normalized by the width at the origin (unfilled). For filaments ≥20 μm in length, the widths are measured 20 μm from the origin of filament growth (filled, striped) and at the distal end of the filament (filled), then normalized by the width measured at its origin. The error bars represent one SD measured from 6 to 9 filaments. The stars represent statistical significance: (*) represents p < 0.01 and (**) represents p < 0.02. Scale bars = 5 μm.
To test this hypothesis, we exploit the flow control afforded by the 2D microfluidic environment to visualize sheath evolution by applying two iterations of stop-flow nanofibril fluorescent labeling. The first staining labels the initial distribution, while the second staining labels subsequently secreted nanofibrils (see Figure 5B, t = 280 and 1750 min). Surprisingly, the lateral extent of fluorescence surrounding the filament broadens in the direction of elongation (see Figure 5B, t = 1750 min). To quantify this broadening, we measure the intensity line-profile perpendicularly to the filament axis as a function of the distance from the filament origin, which is defined as the point of initial attachment (see Figure 5C, left). We find that filaments with lengths <20 μm have line-profile widths that are approximately constant, which indicates that the sheath is cylindrical. Filaments with lengths ≥20 μm are measured at two locations: (1) 20 μm from the origin and (2) at the distal end. The width at 20 μm has a cylindrical profile, similar to the shorter filaments, while the line-profile width measured at the distal end of the filament shows statistically significant broadening. A histogram of normalized intensity line-profiles along different filaments is shown in Figure 5C (right).
Although we are unable to image the degree of nanofibril entanglement, these data confirm the presence of a gradient in the organization of the nanofibrils as a function of filament length. This appears to correspond to a gradient of sheath maturity and is consistent with previous work that has shown that sheath synthesis occurs primarily at the elongating pole(s).(1) However, unlike prior work, because we confine the filaments to enable time-lapse imaging of filament elongation, we are able capture instances where the sheath has yet to evolve its characteristic cylindrical shape(1,2,7,8,11) as it matures. We speculate that as the cell train is driven forward, nanofibrils caught in entanglements would begin to experience tension along the axis of the filament; this pulling could lead to axial alignment of nanofibrils and simultaneously cause the older sections to “tighten” as they constrict around the cells. Atomic force microscopy (AFM) images of L. ochracea sheaths have shown that the nanofibrils can become aligned along the filament axis.(8) The nanofibrils secreted earlier in filament development become tubes that constrain and align the enclosed cells. These tubular sheaths are able to support bending and prevent filament buckling under compressive loading. However, the freshly secreted nanofibrils at the elongating pole of the filament spread outward, possibly with a conical shape, which may be less effective in preventing cell escape (Figure 5A). Our results suggest that the gradient in sheath development, leading to insufficient confinement at the elongating pole of the filament, may be a general phenomenon found in other filament producing bacteria that exhibit cell escape from the elongating pole.(1,2,20,49−52)
Conclusions
ARTICLE SECTIONS
Leptothrix plays an important role in the global iron cycle through iron oxidation in chalybeate freshwater. Their tendency to form entangled 3D aggregates has hindered analysis of the dynamics of filament formation in microbial mats. An understanding the bacterial filament lifestyle is important for understanding the behavior of this and other filamentous bacteria that consist of ecologically important groups. Our approach makes use of microfluidics due to its demonstrated utility in enabling precise control and structuring of microenvironments while facilitating imaging.(53) Leveraging these features, we designed a 2D chamber that facilitated trapping, culturing, and imaging multiple filaments over many hours and complemented these experiments with ASEM snapshots. We have shown that the L. cholodnii SP-6 nanofibrils are crucial for initial surface attachment and their distribution on young filaments appears to influence the direction of filament elongation. As the entangled nanofibrils develop into a sheath, it enables filaments to negotiate obstructions as it matures around the catenulate cells.
Although the role of the nanofibrils and sheath is becoming clear, it is still incomplete. Our data support the idea that at each stage of filamentation they impart adaptability to the environment. The nanofibrils appear to enable planktonic cells to remain in favorable environments with strong currents, while the sheathless variant lacks this ability. Upon surface attachment and filamentation, the developing nanofibril sheath protects the enclosed cells from predation and toxic biomineralization. Whole filaments, which are monoclonal colonies, due to their flexibility and reversibility have the ability to adapt to obstacles, and thereby elongate continuously; this contrasts with the relative immobility of surface-attached biofilm colonies encased in an EPS matrix. These features may enable Leptothrix to become one of the dominant microorganisms in microbial mats.
This developing picture has allowed us to ask more specific questions about Leptothrix filament formation, which will be interesting to test in future work. For example, are there conditions where filamentation is unfavorable, and if so, is it linked to environmental prosperity or adversity? In these instances, the catenulate cells require a means of escape, and as we have shown, SP-6 appears to possess it. We can imagine scenarios where nutrient-poor conditions could result in slowed nanofibril synthesis, leading to escape due to ineffective retention of cells at the distal pole, and nutrient-rich conditions could result in rapid division where cell growth may surpass sheath development, again, leading to escape. The features of nanofibril synthesis and sheath structure appear to define possible behaviors of the bacteria and require further investigation.
Our results provide a framework for understanding the role of appendages in L. cholodnii filament formation and, more generally, the behavior of other filamentous bacteria, which have shown promise in wastewater treatment,(54,55) bioremediation,(24,54,56) and biomineralization(20,57) applications. A deeper understanding of sheath formation combined with micropatterning to guide filament growth could enable the synthesis of individual, high-aspect ratio microtubes that may find application in the fabrication of microwires, biohybrid magnetic robots for targeted delivery,(58−60) or as a novel bioseparation material.(61)
Materials and Methods
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Strains and Culturing Conditions
In this study, we used L. cholodnii strain SP-6 (ATCC 51168) or its derivative strain SP-6SL (ATCC 51169), which is unable to synthesize the sheath skeleton.(7) We cultured these strains by transferring freeze stock to a MSVP agar plate,(7,16) followed by incubation at room temperature (RT). After culturing for a week, we transferred 1–3 single colonies to 25 mL of liquid MSVP medium and incubated in a reciprocating shaker at RT and 65 rpm for an additional 2 days. Following liquid culturing, cells were used immediately.
Microfluidic Device Fabrication and In Situ Culturing
We prepared microfluidic channels in polydimethylsiloxane (PDMS) replicated from a KMPR 1035 (Microchem, Westborough, MA, USA) master fabricated with two different heights. This was achieved by performing standard lithography two times(35,62,63) (see also SI S1.1). Briefly, we spin-coated a layer of photoresist and exposed through a photomask using a mask aligner (SÜSS MicroTec, Garching, Germany). Without developing the first layer, a second layer of photoresist was then deposited, and a second photomask was aligned to the first photoresist layer. We then exposed a second time and developed the two-level structure. The main channel dimensions are 500 μm wide × 70 μm deep, with a length of ∼5 mm, and contain a linear array of rectangular pillars, aligned along the central axis of this channel. The pillar dimensions are 100 μm wide × 70 μm tall. At the base of each pillar, there are 5 μm wide inlets and outlets; these entrances lead to a rectangular cavity with dimensions: 100 μm × 100 μm × ∼1.3 μm (see TOC graphic and schematic shown in SI Figure S2). After O2 plasma bonding the PDMS slab to glass, this cavity forms the quasi-2D chamber.
MSVP medium was infused into the PDMS devices using positive displacement syringe pumps (Harvard Apparatus, Holliston, MA, USA) at a rate of 100 μL/h. We inoculated a sufficient amount of 2 day culture into the main channel to achieve a seeding density of approximately 5–10 cells per 2D chamber. Images were taken on an Axio Observer.Z1 (Carl Zeiss, Oberkochen, Germany) at 5 min intervals. The microscope stage was equipped with a heating unit capable of maintaining a stable temperature of 25 ± 0.5 °C (TP-SQH26-C16N, TOKAI HIT, Fujinomiya, Japan).
Fluorescent Staining of Cytoplasmic Membrane and Nanofibril Amino Groups (NH2)
To confirm normal cell growth can occur within the 2D chambers, we labeled the plasma membrane of SP-6 cells with FM4-64 (Thermo Fisher, Waltham, MA, USA) and monitored the behavior of trapped cells for 3 h. We dissolved FM4-64 at a concentration of 0.5 μg/mL in MSVP medium and infused at a rate of 100 μL/h. A time-lapse sequence of the stained cells is shown in SI Figure S8A. As filaments elongate due to cellular division, distinct lines become visible along the filament. Staining the cytoplasmic membrane shows that these marks correspond to the septa between adjacent cells (see SI Figure S8B).
To visualize the presence of immature sheath skeletons, we labeled the terminal NH2 groups found within the nanofibrils(19) with fluorescein (Fluorescein Labeling Kit-NH2, DOJINDO, Kumamoto, Japan) (see SI Figure S4A). We stained SP-6 and SP-6 SL cells in 2 day liquid culture with the fluorescein reagent diluted 1:50 in medium, then incubated at 37 °C for 15 min.(18) The cells were collected, washed in MSVP medium, and then imaged using an Axio Observer.Z1 or LSM780 confocal laser scanning microscope (Carl Zeiss). Individual SP-6 cells were isolated from large cellular aggregates for fluorescence labeling by first filtering the 2 day culture with a 5 μm pore syringe filter then using the same staining protocol. However, this method suffers from the drawback in that it will label all NH2-rich regions.
To corroborate the specificity of the fluorescein reagent, we used the lectin Concanavalin A conjugated to Alexa594 (Con A-Alexa) (Thermo Fisher) to label glycosaminoglycans(64,65) found in the L. cholodnii nanofibril sheaths. The nanofibrils are made of structural glycoconjugates, which are a type of glycosaminoglycan.(19,66) By co-staining first with fluorescein and then with Con A-Alexa, we measured the amount of fluorescence overlap between NH2-rich regions and sugars (SI Figure S5). The working concentration of Con A-Alexa was 50 μg/mL. Con A appears to affect filament elongation in live-imaging experiments, so we used it simply to confirm label specificity.
To follow the development of the sheath skeletons that surround the cell filaments, we first inoculated cells into the 2D chambers, added 500 μL of MSVP containing the fluorescein reagent, at a 1:60 dilution, and then stopped the flow. We incubated the reagent mixture with the adherent cells in 2D chambers at RT for 15 min. We then restarted the flow of medium and resumed further time-lapse imaging. Bright-field images were taken at 5 min intervals and fluorescent images were taken at 25 min intervals.
Atmospheric Scanning Electron Microscopy (ASEM)
We used ASEM (JASM-6200, JEOL, Tokyo, Japan) to directly visualize cell-associated nanofibrils.(67) We were able to directly image the nanofibrils and their localization without problems associated with dehydration of the sample(24) since imaging can occur directly in an aqueous medium. We prepared the cells by transferring 3 mL of 2 day-culture to an ASEM membrane dish (35 mm in diameter, JEOL, Tokyo, Japan). After incubating the cells on the dish for 6–10 h, we removed the planktonic and weakly attached cells by vigorous pipetting in 3 mL of sterile phosphate-buffered saline (PBS) buffer, and we fixed the remaining adhered cells in 1 mL of 1 v/v glutaraldehyde in double distilled water (hereafter DDW) at RT for 10 min. We then quenched the fixing reaction by washing the specimens in 50 mM ammonium chloride followed by an additional washing in DDW. Secreted nanofibrils were visualized by staining with positively charged nanogold (PCG) and phosphotungstic acid (PTA). The DDW-washed specimens were incubated with a 6 μM PCG solution (Nanoprobes, Yaphank, NY, USA) for 20 min at RT; this was repeated once. The specimens were then washed in DDW, and the size of the gold nanoparticles was increased by gold enhancement (GoldEnhance, Nanoprobes) for 10 min at RT, followed by an additional washing in DDW. The resultant specimens were then incubated for 2 h at RT in 2 v/v PTA (TAAB Laboratories Equipment, Aldermaston, Berks, England) in DDW. After a final DDW-wash, specimens were soaked in 1 v/v ascorbic acid in DDW and observed using ASEM at an acceleration voltage of 30 kV.
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This material is available free of charge via the Internet at: . The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsnano.9b04663.
Figure S1: Microscopic images of DNA-stained cells in large aggregates cultured in a batch-culture. Figure S2: A schematic of the quasi-2D microfluidic chamber. Figure S3: Kymograph of an elongating filament. Figure S4: Fluorescent staining of NH2 groups in the nanofibrils. Figure S5: Fluorescent co-staining of sugars and NH2 groups in the nanofibrils. Figure S6: Additional ASEM images of SP-6 and SP-6SL cells. Figure S7: The radius of curvature measured as a function of measured for collisions between filaments and obstacles. Figure S8: Fluorescent staining of cytoplasmic membrane of SP-6 cells (PDF)
Movie S1: A representative time lapse video of SP-6 cells (AVI)
Movie S2: Tracking the centroid of SP-6 cells at the bottom of a glass bottom dish (AVI)
Movie S3: A representative time lapse video of SP-6 SL cells (AVI)
Movie S4: The time lapse video of a bending cell filaments (AVI)
Movie S5: The time lapse video of a reversing filaments (AVI)
Terms & Conditions
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Author Information
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- Tatsuki Kunoh - Faculty and , , and , University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8577, Japan;
http://orcid.org/0000-0002-8423-2903;
- Andrew S. Utada - Faculty, Microbiology Research Center for Sustainability, and , , and , University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8577, Japan;http://orcid.org/0000-0003-4542-6315;
- Kana Morinaga - Graduate School of Life and Environmental Sciences and , , and , University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8577, Japan;http://orcid.org/0000-0001-6739-7514
- Shinya Sugimoto - Department of Bacteriology and Jikei Center for Biofilm Research and Technology, The Jikei University School of Medicine, 3-25-8, Nishi-Shimbashi, Minato-ku, Tokyo 105-8461, Japan;http://orcid.org/0000-0002-3114-8513
These authors contributed equally.
The authors declare no competing financial interest.
Acknowledgments
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We thank T. Yamamoto for technical support and J. Fattaccioli, A. M. Spiesser, and D. P. Williford for a critical reading of the manuscript. We acknowledge financial support from Japan Science and Technology Agency (JST), ERATO (JPMJER1502) (N.N.), and the Cooperative Research Project Program of TARA Center, University of Tsukuba (T.K.). A.S.U., N.N., and K.M. are supported by grant-in-aid for Young Scientists (B) (17K15410), Scientific Research (S) (16H06382), and Scientific Research (16J00487) from the Japanese Society for the Promotion of Science (JSPS), respectively.
References
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This article references 67 other publications.
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- 11Hashimoto, H.; Kobayashi, G.; Sakuma, R.; Fujii, T.; Hayashi, N.; Suzuki, T.; Kanno, R.; Takano, M.; Takada, J. Bacterial Nanometric Amorphous Fe-Based Oxide: A Potential Lithium-Ion Battery Anode Material. ACS Appl. Mater. Interfaces 2014, 6, 5374– 5378, DOI: 10.1021/am500905y[ACS Full Text
], [CAS], Google Scholar11https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXltlymsbk%253D&md5=b4a29ef79208bd79a741475375aa7188Bacterial nanometric amorphous Fe-based oxide: a potential lithium-ion battery anode materialHashimoto, Hideki; Kobayashi, Genki; Sakuma, Ryo; Fujii, Tatsuo; Hayashi, Naoaki; Suzuki, Tomoko; Kanno, Ryoji; Takano, Mikio; Takada, JunACS Applied Materials & Interfaces (2014), 6 (8), 5374-5378CODEN: AAMICK; ISSN:1944-8244. (American Chemical Society)Amorphous Fe3+-based oxide nanoparticles produced by Leptothrix ochracea, aquatic bacteria living worldwide, show a potential as an Fe3+/Fe0 conversion anode material for lithium-ion batteries. The presence of minor components, Si and P, in the original nanoparticles leads to a specific electrode architecture with Fe-based electrochem. centers embedded in a Si, P-based amorphous matrix. - 12Lovley, D. R. Happy Together: Microbial Communities that Hook Up to Swap Electrons. ISME J. 2017, 11, 327– 336, DOI: 10.1038/ismej.2016.136[Crossref], [PubMed], [CAS], Google Scholar12https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhsFGlsLg%253D&md5=c86e142d350ba8648a5cfa9581909deeHappy together: microbial communities that hook up to swap electronsLovley, Derek R.ISME Journal (2017), 11 (2), 327-336CODEN: IJSOCF; ISSN:1751-7362. (Nature Publishing Group)A review. The discovery of direct interspecies electron transfer (DIET) and cable bacteria has demonstrated that microbial cells can exchange electrons over long distances (μm-cm) through elec. connections. For example, in the presence of cable bacteria electrons are rapidly transported over centimeter distances, coupling the oxidn. of reduced sulfur compds. in anoxic sediments to oxygen redn. in overlying surficial sediments. Bacteria and archaea wired for DIET are found in anaerobic methane-producing and methane-consuming communities. Elec. connections between gut microbes and host cells have also been proposed. Iterative environmental and defined culture studies on methanogenic communities revealed the importance of elec. conductive pili and c-type cytochromes in natural elec. grids, and demonstrated that conductive carbon materials and magnetite can substitute for these biol. connectors to facilitate DIET. This understanding has led to strategies to enhance and stabilize anaerobic digestion. Key unknowns warranting further investigation include elucidation of the archaeal elec. connections facilitating DIET-based methane prodn. and consumption; and the mechanisms for long-range electron transfer through cable bacteria. A better understanding of mechanisms for cell-to-cell electron transfer could facilitate the hunt for addnl. elec. connected microbial communities with omics approaches and could advance spin-off applications such as the development of sustainable bioelectronics materials and bioelectrochem. technologies.
- 13Nielsen, L. P.; Risgaard-Petersen, N.; Fossing, H.; Christensen, P. B.; Sayama, M. Electric Currents Couple Spatially Separated Biogeochemical Processes in Marine Sediment. Nature 2010, 463, 1071– 1074, DOI: 10.1038/nature08790[Crossref], [PubMed], [CAS], Google Scholar13https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXitl2gsr8%253D&md5=33967b4fb936e441ecc497ff82d9c811Electric currents couple spatially separated biogeochemical processes in marine sedimentNielsen, Lars Peter; Risgaard-Petersen, Nils; Fossing, Henrik; Christensen, Peter Bondo; Sayama, MikioNature (London, United Kingdom) (2010), 463 (7284), 1071-1074CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)Some bacteria are capable of extracellular electron transfer, thereby enabling them to use electron acceptors and donors without direct cell contact. Beyond the micrometer scale, however, no firm evidence has previously existed that spatially segregated biogeochem. processes can be coupled by elec. currents in nature. Based on studies on sediment samples from Aarhus harbor and bay, Denmark, the authors give evidence that elec. currents running through defaunated sediment couple oxygen consumption at the sediment surface to oxidn. of hydrogen sulfide and org. carbon deep within the sediment. Altering the oxygen concn. in the seawater overlying the sediment resulted in a rapid (<1-h) change in the hydrogen sulfide concn. within the sediment more than 12 mm below the oxic zone, a change explicable by transmission of electrons but not by diffusion of mols. Mass balances indicated that more than 40% of total oxygen consumption in the sediment was driven by electrons conducted from the anoxic zone. A distinct pH peak in the oxic zone could be explained by electrochem. oxygen redn., but not by any conventional sets of aerobic sediment processes. The authors suggest that the elec. current was conducted by bacterial nanowires combined with pyrite, sol. electron shuttles and outer-membrane cytochromes. Elec. communication between distant chem. and biol. processes in nature adds a new dimension to understanding of biogeochem. and microbial ecol.
- 14Pfeffer, C.; Larsen, S.; Song, J.; Dong, M.; Besenbacher, F.; Meyer, R. L.; Kjeldsen, K. U.; Schreiber, L.; Gorby, Y. A.; El-Naggar, M. Y.; Leung, K. M.; Schramm, A.; Risgaard-Petersen, N.; Nielsen, L. P. Filamentous Bacteria Transport Electrons Over Centimetre Distances. Nature 2012, 491, 218– 221, DOI: 10.1038/nature11586[Crossref], [PubMed], [CAS], Google Scholar14https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhsFOmt7fN&md5=53f87c4d3da6ef2fbcaa62459f2753e7Filamentous bacteria transport electrons over centimeter distancesPfeffer, Christian; Larsen, Steffen; Song, Jie; Dong, Mingdong; Besenbacher, Flemming; Meyer, Rikke Louise; Kjeldsen, Kasper Urup; Schreiber, Lars; Gorby, Yuri A.; El-Naggar, Mohamed Y.; Leung, Kar Man; Schramm, Andreas; Risgaard-Petersen, Nils; Nielsen, Lars PeterNature (London, United Kingdom) (2012), 491 (7423), 218-221CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)Oxygen consumption in marine sediments is often coupled to the oxidn. of sulfide generated by degrdn. of org. matter in deeper, oxygen-free layers. Geochem. observations have shown that this coupling can be mediated by elec. currents carried by unidentified electron transporters across centimeter-wide zones. Her, the authors present evidence that the native conductors are long, filamentous bacteria. They abounded in sediment zones with elec. currents and along their length they contained strings with distinct properties in accordance with a function as electron transporters. Living, elec. cables add a new dimension to the understanding of interactions in nature and may find use in technol. development.
- 15Chan, C. S.; Fakra, S. C.; Edwards, D. C.; Emerson, D.; Banfield, J. F. Iron Oxyhydroxide Mineralization on Microbial Extracellular Polysaccharides. Geochim. Cosmochim. Acta 2009, 73, 3807– 3818, DOI: 10.1016/j.gca.2009.02.036[Crossref], [CAS], Google Scholar15https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXmvVyktbo%253D&md5=0631525cf1a01cabfed340b2767843a5Iron oxyhydroxide mineralization on microbial extracellular polysaccharidesChan, Clara S.; Fakra, Sirine C.; Edwards, David C.; Emerson, David; Banfield, Jillian F.Geochimica et Cosmochimica Acta (2009), 73 (13), 3807-3818CODEN: GCACAK; ISSN:0016-7037. (Elsevier B.V.)Iron biominerals can form in neutral pH microaerophilic environments where microbes both catalyze iron oxidn. and create polymers that localize mineral pptn. To classify the microbial polymers that influence FeOOH mineralogy, the org. and mineral components of biominerals were studied using scanning transmission X-ray microscopy (STXM), micro X-ray fluorescence (μXRF) microscopy, and high-resoln. transmission electron microscopy (HRTEM). Focus was on iron microbial mat samples from a creek and abandoned mine; these samples are dominated by iron oxyhydroxide-coated structures with sheath, stalk, and filament morphologies. In addn., the mineralized products of an iron-oxidizing, stalk-forming bacterial culture isolated from the mine were characterized. In both natural and cultured samples, microbial polymers were found to be acidic polysaccharides with carboxyl functional groups, strongly spatially correlated with iron oxyhydroxide distribution patterns. Org. fibrils collect FeOOH and control its recrystn., in some cases resulting in oriented crystals with high aspect ratios. The impact of polymers is particularly pronounced as the materials age. Synthesis expts. designed to mimic the biomineralization processes show that the polysaccharide carboxyl groups bind dissolved iron strongly but release it as mineralization proceeds. Results suggest that carboxyl groups of acidic polysaccharides are produced by different microorganisms to create a wide range of iron oxyhydroxide biomineral structures. The intimate and potentially long-term assocn. controls the crystal growth, phase, and reactivity of iron oxyhydroxide nanoparticles in natural systems.
- 16Kunoh, T.; Hashimoto, H.; McFarlane, I. R.; Hayashi, N.; Suzuki, T.; Taketa, E.; Tamura, K.; Takano, M.; El-Naggar, M. Y.; Kunoh, H.; Takada, J. Abiotic Deposition of Fe Complexes onto Leptothrix Sheaths. Biology 2016, 5, 26, DOI: 10.3390/biology5020026[Crossref], [CAS], Google Scholar16https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXmsFSgsr0%253D&md5=e674a73d86b1d9a277bbc21a3068bcddAbiotic deposition of Fe complexes onto Leptothrix sheathsKunoh, Tatsuki; Hashimoto, Hideki; McFarlane, Ian R.; Hayashi, Naoaki; Suzuki, Tomoko; Taketa, Eisuke; Tamura, Katsunori; Takano, Mikio; El-Naggar, Mohamed Y.; Kunoh, Hitoshi; Takada, JunBiology (Basel, Switzerland) (2016), 5 (2), 26-42CODEN: BBSIBX; ISSN:2079-7737. (MDPI AG)Bacteria classified in species of the genus Leptothrix produce extracellular, microtubular, Fe-encrusted sheaths. The encrustation has been previously linked to bacterial Fe oxidases, which oxidize Fe(II) to Fe(III) and/or active groups of bacterial exopolymers within sheaths to attract and bind aq.-phase inorgs. When L. cholodnii SP-6 cells were cultured in media amended with high Fe(II) concns., Fe(III) ppts. visibly formed immediately after addn. of Fe(II) to the medium, suggesting prompt abiotic oxidn. of Fe(II) to Fe(III). Intriguingly, these ppts. were deposited onto the sheath surface of bacterial cells as the population was actively growing. When Fe(III) was added to the medium, similar ppts. formed in the medium first and were abiotically deposited onto the sheath surfaces. The ppts. in the Fe(II) medium were composed of assemblies of globular, amorphous particles (ca. 50 nm diam.), while those in the Fe(III) medium were composed of large, aggregated particles (≤3 μm diam.) with a similar amorphous structure. These ppts. also adhered to cell-free sheaths. We thus concluded that direct abiotic deposition of Fe complexes onto the sheath surface occurs independently of cellular activity in liq. media contg. Fe salts, although it remains unclear how this deposition is assocd. with the previously proposed mechanisms (oxidn. enzyme- and/or active group of org. components-involved) of Fe encrustation of the Leptothrix sheaths.
- 17Kunoh, T.; Hashimoto, H.; Suzuki, T.; Hayashi, N.; Tamura, K.; Takano, M.; Kunoh, H.; Takada, J. Direct Adherence of Fe (III) Particles Onto Sheaths of Leptothrix sp. Strain OUMS1 in Culture. Minerals 2016, 6, 4, DOI: 10.3390/min6010004[Crossref], [CAS], Google Scholar17https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXmt1yjurs%253D&md5=ce8920347e470172e944a7d66fa5fea4Direct adherence of Fe(III) particles onto sheaths of Leptothrix sp. strain OUMS1 in cultureKunoh, Tatsuki; Hashimoto, Hideki; Suzuki, Tomoko; Hayashi, Naoyuki; Tamura, Katsunori; Takano, Mikio; Kunoh, Hitoshi; Takada, JunMinerals (Basel, Switzerland) (2016), 6 (1), 4/1-4/15CODEN: MBSIBI; ISSN:2075-163X. (MDPI AG)Leptothrix species, one of the Fe/Mn-oxidizing bacteria, oxidize Fe(II) and produce extracellular, microtubuar, Fe-encrusted sheaths. Since protein(s) involved in Fe(II) oxidn. is excreted from Leptothrix cells, the oxidn. from Fe(II) to Fe(III) and subsequent Fe(III) deposition to sheaths have been thought to occur in the vicinity or within the sheaths. Previously, Fe(III) particles generated in MSVP medium amended with Fe(II) salts by abiotic oxidn. were directly recruited onto cell-encasing and/or -free sheaths of L. cholodnii SP-6. In this study, whether this direct Fe(III) adherence to sheaths also occurs in silicon-glucose-peptone (SGP) medium amended with Fe(0) (SGP + Fe) was investigated using another strain of Leptothrix sp., OUMS1. Prepn. of SGP + Fe with Fe powder caused turbidity within a few hours due to abiotic generation of Fe(III) particles via Fe(II), and the medium remained turbid until day 8. When OUMS1 was added to SGP + Fe, the turbidity of the medium cleared within 35 h as Fe(III) particles adhered to sheaths. When primitive sheaths, cell-killed, cell-free, or lysozyme/EDTA/SDS- and proteinase K-treated sheath remnants were mixed with Fe(III) particles, the particles immediately adhered to each. Thus, vital activity of cells was not required for the direct Fe(III) particle deposition onto sheaths regardless of Leptothrix strains.
- 18Kunoh, T.; Matsumoto, S.; Nagaoka, N.; Kanashima, S.; Hino, K.; Uchida, T.; Tamura, K.; Kunoh, H.; Takada, J. Amino Group in Leptothrix Sheath Skeleton Is Responsible for Direct Deposition of Fe(III) Minerals onto the Sheaths. Sci. Rep. 2017, 7, 6498, DOI: 10.1038/s41598-017-06644-8[Crossref], [PubMed], [CAS], Google Scholar18https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC1cfhsFelug%253D%253D&md5=3c69b73ea390d9adf54b549afb24d81dAmino group in Leptothrix sheath skeleton is responsible for direct deposition of Fe(III) minerals onto the sheathsKunoh Tatsuki; Matsumoto Syuji; Tamura Katsunori; Kunoh Hitoshi; Takada Jun; Kunoh Tatsuki; Matsumoto Syuji; Uchida Tetsuya; Tamura Katsunori; Kunoh Hitoshi; Takada Jun; Nagaoka Noriyuki; Kanashima Shoko; Hino KatsuhikoScientific reports (2017), 7 (1), 6498 ISSN:.Leptothrix species produce microtubular organic-inorganic materials that encase the bacterial cells. The skeleton of an immature sheath, consisting of organic exopolymer fibrils of bacterial origin, is formed first, then the sheath becomes encrusted with inorganic material. Functional carboxyl groups of polysaccharides in these fibrils are considered to attract and bind metal cations, including Fe(III) and Fe(III)-mineral phases onto the fibrils, but the detailed mechanism remains elusive. Here we show that NH2 of the amino-sugar-enriched exopolymer fibrils is involved in interactions with abiotically generated Fe(III) minerals. NH2-specific staining of L. cholodnii OUMS1 detected a terminal NH2 on its sheath skeleton. Masking NH2 with specific reagents abrogated deposition of Fe(III) minerals onto fibrils. Fe(III) minerals were adsorbed on chitosan and NH2-coated polystyrene beads but not on cellulose and beads coated with an acetamide group. X-ray photoelectron spectroscopy at the N1s edge revealed that the terminal NH2 of OUMS1 sheaths, chitosan and NH2-coated beads binds to Fe(III)-mineral phases, indicating interaction between the Fe(III) minerals and terminal NH2. Thus, the terminal NH2 in the exopolymer fibrils seems critical for Fe encrustation of Leptothrix sheaths. These insights should inform artificial synthesis of highly reactive NH2-rich polymers for use as absorbents, catalysts and so on.
- 19Takeda, M.; Kondo, K.; Yamada, M.; Koizumi, J.; Mashima, T.; Matsugami, A.; Katahira, M. Solubilization and Structural Determination of a Glycoconjugate Which Is Assembled into the Sheath of Leptothrix cholodnii. Int. J. Biol. Macromol. 2010, 46, 206– 211, DOI: 10.1016/j.ijbiomac.2009.12.006[Crossref], [PubMed], [CAS], Google Scholar19https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhs1ygurw%253D&md5=64fecc7ed10706358ed22f2c160f8192Solubilization and structural determination of a glycoconjugate which is assembled into the sheath of Leptothrix cholodniiTakeda, Minoru; Kondo, Keiko; Yamada, Mina; Koizumi, Jun-ichi; Mashima, Tsukasa; Matsugami, Akimasa; Katahira, MasatoInternational Journal of Biological Macromolecules (2010), 46 (2), 206-211CODEN: IJBMDR; ISSN:0141-8130. (Elsevier B.V.)The sheath of Leptothrix cholodnii is constructed from a structural glycoconjugate, a straight-chained amphoteric heteropolysaccharide modified with glycine and cysteine. Though the structure of the glycan core is already detd., its modifications with amino acids and other mols. are not fully resolved. In this study, we aimed to det. the chem. structure of the glycoconjugate as a whole. Enantiomeric detn. of cysteine in the sheath was performed and as a result, -cysteine was detected. NMR spectroscopy was endeavored to det. overall structure of the glycoconjugate. Prior to NMR anal., solubilization of the glycoconjugate was attempted by adding denaturing reagents or by derivatization. As far as tested, sulfonation by performic acid oxidn. was suitable for solubilization, but further improvement was achieved by N-acetylation. The approx. mol. wt. of the deriv. was estd. to be 4.5 × 104 by size-exclusion chromatog. The NMR studies for the sulfonated glycoconjugate and its N-acetylated deriv. revealed that the sheath glycoconjugate is a glycosaminoglycan consisting of a pentasaccharide repeating unit which is substoichiometrically esterified with 3-hydroxypropionic acid and stoichiometrically amidated with acetic acid and glycyl-L-cysteine.
- 20Kunoh, T.; Nakanishi, M.; Kusano, Y.; Itadani, A.; Ando, K.; Matsumoto, S.; Tamura, K.; Kunoh, H.; Takada, J. Biosorption of Metal Elements by Exopolymer Nanofibrils Excreted From Leptothrix Cells. Water Res. 2017, 122, 139– 147, DOI: 10.1016/j.watres.2017.05.003[Crossref], [PubMed], [CAS], Google Scholar20https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXps1KntLs%253D&md5=a7f57920037f6ca10e103f90e3bcf8a0Biosorption of metal elements by exopolymer nanofibrils excreted from Leptothrix cellsKunoh, Tatsuki; Nakanishi, Makoto; Kusano, Yoshihiro; Itadani, Atsushi; Ando, Kota; Matsumoto, Syuji; Tamura, Katsunori; Kunoh, Hitoshi; Takada, JunWater Research (2017), 122 (), 139-147CODEN: WATRAG; ISSN:0043-1354. (Elsevier Ltd.)Leptothrix species, aquatic Fe-oxidizing bacteria, excrete nano-scaled exopolymer fibrils. Once excreted, the fibrils weave together and coalesce to form extracellular, microtubular, immature sheaths encasing catenulate cells of Leptothrix. The immature sheaths, composed of aggregated nanofibrils with a homogeneous-looking matrix, attract and bind aq.-phase inorgs., esp. Fe, P, and Si, to form seemingly solid, mature sheaths of a hybrid org.-inorg. nature. To verify our assumption that the org. skeleton of the sheaths might sorb a broad range of other metallic and nonmetallic elements, we examd. the sorption potential of chem. and enzymically prepd. protein-free org. sheath remnants for 47 available elements. The sheath remnants were found by XRF to sorb each of the 47 elements, although their sorption degree varied among the elements: >35% at. percentages for Ti, Y, Zr, Ru, Rh, Ag, and Au. Electron microscopy, energy dispersive x-ray spectroscopy, electron and x-ray diffractions, and Fourier transform IR spectroscopy analyses of sheath remnants that had sorbed Ag, Cu, and Pt revealed that (i) the sheath remnants comprised a 5-10 nm thick aggregation of fibrils, (ii) the test elements were distributed almost homogeneously throughout the fibrillar aggregate, (iii) the nanofibril matrix sorbing the elements was nearly amorphous, and (iv) these elements plausibly were bound to the matrix by ionic binding, esp. via OH. The present results show that the constitutive protein-free exopolymer nanofibrils of the sheaths can contribute to creating novel filtering materials for recovering and recycling useful and/or hazardous elements from the environment.
- 21Kunoh, T.; Kusano, Y.; Takeda, M.; Nakanishi, M.; Matsumoto, S.; Suzuki, I.; Takano, M.; Kunoh, H.; Takada, J. Formation of Gold Particles via Thiol Groups on Glycoconjugates Comprising the Sheath Skeleton of Leptothrix. Geomicrobiol. J. 2019, 36, 251– 260, DOI: 10.1080/01490451.2018.1550127[Crossref], [CAS], Google Scholar21https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhvVChtbo%253D&md5=caad0348dc25b013f66b9aaaf56c8a45Formation of Gold Particles via Thiol Groups on Glycoconjugates Comprising the Sheath Skeleton of LeptothrixKunoh, Tatsuki; Kusano, Yoshihiro; Takeda, Minoru; Nakanishi, Makoto; Matsumoto, Syuji; Suzuki, Ichiro; Takano, Mikio; Kunoh, Hitoshi; Takada, JunGeomicrobiology Journal (2019), 36 (3), 251-260CODEN: GEJODG; ISSN:0149-0451. (Taylor & Francis, Inc.)Leptothrix, iron-oxidizing bacterium, produces microtubular sheaths that surround the catenulate cells. Org. nanofibrils excreted from the cell surfaces interweave and coalesce to form immature sheaths, which attract aq.-phase inorgs. to eventually form mature org.-inorg. sheaths. Such inorg. encrustation of the sheaths results from interactions between functional groups in the sheath skeleton and inorgs. Based on our previous findings that Leptothrix sheath skeleton sorbed 47 inorgs. (Au was one of the most abundant adsorbates), we examd. the sorption status of Au cations on cell-enclosing sheaths and their protein-free remnants and found that nano to sub-micron Au particles (AuNPs and AuSMPs, resp.) formed on the sheath-forming polymer consisting of a glycoconjugate (an amphoteric glycan modified with cysteine, glycine, and 3-hydroxypropionic acid). When the purified polymer was incubated in HAuCl4 soln., AuNPs and AuSMPs formed on the polymer surfaces. Both particles formed also on cell-enclosing sheaths and protein-free sheath remnants incubated in HAuCl4 soln. When SH groups in the cell-enclosing sheaths were masked with a fluorescent protein, Au particles did not form after incubation in HAuCl4 soln. Results implicate that SH groups are at least partially involved in the redn. of Au cations to metallic Au and eventual formation of Au particles.
- 22Ema, T.; Miyazaki, Y.; Taniguchi, T.; Takada, J. Robust Porphyrin Catalysts Immobilized on Biogenous Iron Oxide for the Repetitive Conversions of Epoxides and CO2 Into Cyclic Carbonates. Green Chem. 2013, 15, 2485– 2492, DOI: 10.1039/c3gc41055b[Crossref], [CAS], Google Scholar22https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXht1Oqtr%252FJ&md5=c8f9dd55c74bc1db2de970b5a6708f61Robust porphyrin catalysts immobilized on biogenous iron oxide for the repetitive conversions of epoxides and CO2 into cyclic carbonatesEma, Tadashi; Miyazaki, Yuki; Taniguchi, Tomoya; Takada, JunGreen Chemistry (2013), 15 (9), 2485-2492CODEN: GRCHFJ; ISSN:1463-9262. (Royal Society of Chemistry)Porphyrin magnesium and zinc complexes I [R = (HO)3Si(CH2)3NMe2+(CH2)6 Br-; R1 = H, R; M = Mg, Zn] bound to biogenic iron oxide derived from the iron-oxidizing bacterium Leptothrix ochracea were prepd. and used as supported catalysts for the ring-expansion of primary epoxides II [R2 = Bu, Me, Me(CH2)7, Me(CH2)11, Ph, MeOCH2, PhOCH2, ClCH2] to yield dioxolanones III [R2 = Bu, Me, Me(CH2)7, Me(CH2)11, Ph, MeOCH2, PhOCH2, ClCH2]. In particular, 0.1 mol% I [R = R1 = (HO)3Si(CH2)3NMe2+(CH2)6 Br-; M = Zn] was an effective catalyst for the ring-expansion of II [R2 = Bu, Me, Me(CH2)7, Me(CH2)11, Ph, MeOCH2, PhOCH2, ClCH2] with carbon dioxide at 120° and 17 atm to give III [R2 = Bu, Me, Me(CH2)7, Me(CH2)11, Ph, MeOCH2, PhOCH2, ClCH2] in 41-93% yields under solvent-free conditions. Ring expansion of a deuterated epoxide indicated that initial ring opening occurred at the least hindered epoxide carbon.
- 23Hashimoto, H.; Asaoka, H.; Nakano, T.; Kusano, Y.; Ishihara, H.; Ikeda, Y.; Nakanishi, M.; Fujii, T.; Yokoyama, T.; Horiishi, N.; Nanba, T.; Takada, J. Preparation, Microstructure, and Color Tone of Microtubule Material Composed of Hematite/Amorphous-Silicate Nanocomposite from Iron Oxide of Bacterial Origin. Dyes Pigm. 2012, 95, 639– 643, DOI: 10.1016/j.dyepig.2012.06.024[Crossref], [CAS], Google Scholar23https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhtlWlurvI&md5=629e52be853e2928fb1b407275376c2fPreparation, microstructure, and color tone of microtubule material composed of hematite/amorphous-silicate nanocomposite from iron oxide of bacterial originHashimoto, Hideki; Asaoka, Hiroshi; Nakano, Takuya; Kusano, Yoshihiro; Ishihara, Hiromichi; Ikeda, Yasunori; Nakanishi, Makoto; Fujii, Tatsuo; Yokoyama, Tadanori; Horiishi, Nanao; Nanba, Tokuro; Takada, JunDyes and Pigments (2012), 95 (3), 639-643CODEN: DYPIDX; ISSN:0143-7208. (Elsevier Ltd.)By heating an amorphous iron oxide produced by Leptothrix ochracea, an iron-oxidizing bacterium species, at 600-1100 °C in air for 2 h, vivid red-colored powd. materials including α-Fe2O3 (hematite) and amorphous silicate with high thermostability were prepd. which offer potential for use as overglaze enamels on porcelain. The precise color tone of the materials greatly depends on the heat-treatment temp. The most strikingly beautiful sample, heat-treated at 800 °C, is light yellowish-red in color (L* = 47.3, a* = 34.1, and b* = 34.6), has a unique microstructure, and does not fade in color even with reheating at 800 °C, which is the firing temp. for overglaze enamel on porcelain. The sample primarily consists of cryst. hematite particles ∼40 nm in diam. with slightly longer axis unit-cell parameters than those of pure hematite. The particles are covered with amorphous silicate phase ∼5 nm in thickness and are intricately interconnected into microtubules with an av. diam. of 1.26 μm. The attractive color of this material is due to the following structural features: small particle size (∼40 nm), nanocomposite of hematite and amorphous silicate, and a microtubule structure that inhibits aggregation of individual hematite particles and microtubules.
- 24Kunoh, T.; Kunoh, H.; Takada, J. Perspectives on the Biogenesis of Iron Oxide Complexes Produced by Leptothrix, an Iron-Oxidizing Bacterium and Promising Industrial Applications for Their Functions. J. Microb. Biochem. Technol. 2015, 7, 419– 426, DOI: 10.4172/1948-5948.1000249[Crossref], [CAS], Google Scholar24https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXnslers7c%253D&md5=aebc5493dac0b1ad6a7fd7eba9af446aPerspectives on the biogenesis of iron oxide complexes produced by leptothrix, an iron-oxidizing bacterium and promising industrial applications for their functionsKunoh, Tatsuki; Kunoh, Hitoshi; Takada, JunJournal of Microbial & Biochemical Technology (2015), 7 (6), 419-426CODEN: JMBTA9; ISSN:1948-5948. (OMICS Publishing Group)Leptothrix species, one of the Fe-/Mn-oxidizing bacteria, are ubiquitous in aq. environments, esp. at sites characterized by a circumneutral pH, an oxygen gradient and a source of reduced Fe and Mn minerals. Characteristic traits that distinguish the genus Leptothrix from other phylogenetically related species are its filamentous growth and ability to form uniquely shaped microtubular sheaths through the pptn. of copious amts. of oxidized Fe or Mn. The sheath is an ingenious hybrid of org. and inorg. materials produced through the interaction of bacterial exopolymers with aq.-phase inorgs. Intriguingly, we discovered that Leptothrix sheaths have a variety of unexpected functions that are suitable for industrial applications such as material for lithium battery electrode, a catalyst enhancer, pottery pigment among others. This review focuses on the structural and chem. properties of the Leptothrix sheaths and their noteworthy functions that show promise for development of cost-effective, eco-friendly industrial applications.
- 25Fleming, E. J.; Langdon, A. E.; Martinez-Garcia, M.; Stepanauskas, R.; Poulton, N. J.; Masland, E. D. P.; Emerson, D. What’s New Is Old: Resolving the Identity of Leptothrix ochracea Using Single Cell Genomics, Pyrosequencing and FISH. PLoS One 2011, 6, e17769 DOI: 10.1371/journal.pone.0017769[Crossref], [PubMed], [CAS], Google Scholar25https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXktVWhsbo%253D&md5=48ad56f01304dcc6438c19cc040c4a8dWhat's new is old: Resolving the identity of Leptothrix ochracea using single cell genomics, pyrosequencing and FISHFleming, Emily J.; Langdon, Amy E.; Martinez-Garcia, Manuel; Stepanauskas, Ramunas; Poulton, Nicole J.; Masland, E. Dashiell P.; Emerson, DavidPLoS One (2011), 6 (3), e17769CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)Leptothrix ochracea is a common inhabitant of freshwater iron seeps and iron-rich wetlands. Its defining characteristic is copious prodn. of extracellular sheaths encrusted with iron oxyhydroxides. Surprisingly, over 90% of these sheaths are empty; hence, what appears to be an abundant population of iron-oxidizing bacteria consists of relatively few cells. Because L. ochracea has proven difficult to cultivate, its identification is based solely on habitat preference and morphol. The authors utilized cultivation-independent techniques to resolve this long-standing enigma. By selecting the actively growing edge of a Leptothrix-contg. iron mat, a conventional SSU rRNA gene clone library was obtained that had 29 clones (42% of the total library) related to the Leptothrix/Sphaerotilus group (≤96% identical to cultured representatives). A pyrotagged library of the V4 hypervariable region constructed from the bulk mat showed that 7.2% of the total sequences also belonged to the Leptothrix/Sphaerotilus group. Sorting of individual L. ochracea sheaths, followed by whole genome amplification (WGA) and PCR identified a SSU rRNA sequence that clustered closely with the putative Leptothrix clones and pyrotags. Using these data, a fluorescence in-situ hybridization (FISH) probe, Lepto175, was designed that bound to ensheathed cells. Quant. use of this probe demonstrated that up to 35% of microbial cells in an actively accreting iron mat were L. ochracea. The SSU rRNA gene of L. ochracea shares 96% homol. with its closet cultivated relative, L. cholodnii, This establishes that L. ochracea is indeed related to this group of morphol. similar, filamentous, sheathed microorganisms.
- 26Fleming, E.; Woyke, T.; Donatello, R.; Kuypers, M.; Sczyrba, A.; Littmann, S.; Emerson, D. Insights Into the Fundamental Physiology of the Uncultured Fe-Oxidizing Bacterium Leptothrix ochracea. Appl. Environ. Microbiol. 2018, 84, e02239 DOI: 10.1128/AEM.02239-17[Crossref], [PubMed], [CAS], Google Scholar26https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhslGks7bF&md5=83b3ec04c76700fbc73b236017cb1b3cInsights into the fundamental physiology of the uncultured Fe-oxidizing bacterium Leptothrix ochraceaFleming, E. J.; Woyke, T.; Donatello, R. A.; Kuypers, M. M. M.; Sczyrba, A.; Littmann, S.; Emerson, D.Applied and Environmental Microbiology (2018), 84 (9), e02239-17/1-e02239-17/15CODEN: AEMIDF; ISSN:1098-5336. (American Society for Microbiology)Leptothrix ochracea is known for producing large vols. of iron oxyhydroxide sheaths that alter wetland biogeochem. For over a century, these delicate structures have fascinated microbiologists and geoscientists. Because L. ochracea still resists long-term in vitro culture, the debate regarding its metabolic classification dates back to 1885. We developed a novel culturing technique for L. ochracea using in situ natural waters and coupled this with single-cell genomics and nanoscale secondary-ion mass spectrophotometry (nanoSIMS) to probe L. ochracea's physiol. In microslide cultures L. ochracea doubled every 5.7 h and had an abs. growth requirement for ferrous iron, the genomic capacity for iron oxidn., and a branched electron transport chain with cytochromes putatively involved in lithotrophic iron oxidn. Addnl., its genome encoded several electron transport chain proteins, including a molybdopterin alternative complex III (ACIII), a cytochrome bd oxidase reductase, and several terminal oxidase genes. L. ochracea contained two key autotrophic proteins in the Calvin-Benson-Bassham cycle, a form II ribulose bisphosphate carboxylase, and a phosphoribulose kinase. L. ochracea also assimilated bicarbonate, although calcns. suggest that bicarbonate assimilation is a small fraction of its total carbon assimilation. Finally, L. ochracea's fundamental physiol. is a hybrid of those of the chemolithotrophic Gallionella-type ironoxidizing bacteria and the sheathed, heterotrophic filamentous metal-oxidizing bacteria of the Leptothrix-Sphaerotilus genera. This allows L. ochracea to inhabit a unique niche within the neutrophilic iron seeps. IMPORTANCE Leptothrix ochracea was one of three groups of organisms that Sergei Winogradsky used in the 1880s to develop his hypothesis on chemolithotrophy. L. ochracea continues to resist cultivation and appears to have an abs. requirement for org.-rich waters, suggesting that its true physiol. remains unknown. Further, L. ochracea is an ecol. engineer; a few L. ochracea cells can generate prodigious vols. of iron oxyhydroxides, changing the ecosystem's geochem. and ecol. Therefore, to det. L. ochracea's basic physiol., we employed new single-cell techniques to demonstrate that L. ochracea oxidizes iron to generate energy and, despite having predicted genes for autotrophic growth, assimilates a fraction of the total CO2 that autotrophs do. Although not a true chemolithoautotroph, L. ochracea's physiol. strategy allows it to be flexible and to extensively colonize iron-rich wetlands.
- 27Furutani, M.; Suzuki, T.; Ishihara, H.; Hashimoto, H.; Kunoh, H.; Takada, J. Initial Assemblage of Bacterial Saccharic Fibrils and Element Deposition to Form an Immature Sheath in Cultured Leptothrix sp Strain OUMS1. Minerals 2011, 1, 157– 166, DOI: 10.3390/min1010157[Crossref], [CAS], Google Scholar27https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XjtlantLw%253D&md5=a845c5b878a15816aa6042a2a080c8d5Initial assemblage of bacterial saccharic fibrils and element deposition to form an immature sheath in cultured Leptothrix sp. strain OUMS1Furutani, Mitsuaki; Suzuki, Tomoko; Ishihara, Hiromichi; Hashimoto, Hideki; Kunoh, Hitoshi; Takada, JunMinerals (Basel, Switzerland) (2011), 1 (1), 157-166CODEN: MBSIBI; ISSN:2075-163X. (MDPI AG)In an aquatic environment, the genus Leptothrix produces an extracellular Fe- or Mn-encrusted tubular sheath composed of a complex hybrid of bacterial exopolymers and aq.-phase inorg. elements. This ultrastructural study investigated initial assemblage of bacterial saccharic fibrils and subsequent deposition of aq.-phase inorg. elements to form the immature sheath skeleton of cultured Leptothrix sp. strain OUMS1. After one day of culture, a globular and/or thread-like secretion was obsd. on the surface of the bacterial cell envelope, and secreted bodies were transported across the intervening space away from the cell to form an immature sheath skeleton comprising assembled and intermingled fibrils. Energy dispersive X-ray microanal. and specific Bi-staining detected a distinguishable level of P, trace Si, and a notable amt. of carbohydrates in the skeleton, but not Fe. By the second day, the skeleton was prominently thickened with an inner layer of almost parallel aligned fibrils, along with low level of Fe deposition, whereas an outer intermingled fibrous layer exhibited heavy deposition of Fe along with significant deposition of P and Si. These results indicate that basic sheath-construction proceeds in two steps under culture conditions: an initial assemblage of bacterial saccharic fibrils originated from the cell envelope and the subsequent deposition of aq.-phase Fe, P, and Si.
- 28Emerson, D.; Ghiorse, W. C. Role of Disulfide Bonds in Maintaining the Structural Integrity of the Sheath of Leptothrix discophora SP-6. J. Bacteriol. 1993, 175, 7819– 7827, DOI: 10.1128/jb.175.24.7819-7827.1993[Crossref], [PubMed], [CAS], Google Scholar28https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2cXkvFOltw%253D%253D&md5=9d7c9282b4acddbd53762aed510f71a4Role of disulfide bonds in maintaining the structural integrity of the sheath of Leptothrix discophora SP-6Emerson, David; Ghiorse, William C.Journal of Bacteriology (1993), 175 (24), 7819-27CODEN: JOBAAY; ISSN:0021-9193.Isolated sheaths of Leptothrix discophora SP-6 (ATCC 51168) were tested for susceptibility to degrdn. by a variety of chem. denaturants and lytic enzymes and found to be resistant to many reagents and enzyme treatments. However, disulfide bond-reducing agents such as dithiothreitol (DTT), β-mercaptoethanol, sodium cyanide, and sodium sulfite degraded the sheath, esp. at elevated pH (pH 9) and temp. (50°). DTT and β-mercaptoethanol caused more rapid degrdn. of the sheath than cyanide or sulfite. Treatment of the sheath with 1N NaOH resulted in rapid breakdown, while treatment with 1N HCl resulted in slow but significant hydrolysis. Transmission electron microscopy showed that the 6.5-nm fibrils previously shown to be an integral structural element of the sheath fabric (D. Emerson and W. C. Ghiorse, 1993) were progressively dissocd. into random masses during DTT-induced degrdn. Quantitation of disulfide bonds with DTT showed that the sheaths contained approx. 2.2 μmol of disulfides per mg of sheath protein. Reaction with 5,5'-dithio-bis-(2-nitrobenzoic acid) showed that sheaths also contained approx. 0.8 μmol of free sulfhydryls per mg of protein. A sulfhydryl-specific fluorescent probe (fluorescein 5-maleimide) showed that the free sulfhydryls in sheathed cell filaments were evenly distributed throughout the sheath. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis autoradiog. of [14C]iodoacetamide-labeled sheaths and DTT-dissocd. sheath fibril suspensions showed that the majority of 14C-labeled sulfhydryls in the sheaths did not enter the gel. However, low-mol.-mass silver-staining bands (14 to 45 kDa) did appear in the gels after iodoacetic acid or iodoacetamide alkylation of the dissocd. fibrils. These bands did not stain with Coomassie blue. Their migration in gels was slightly affected by digestion with pronase. The fibrils contained 20-25% protein. These results confirm that the sheath fibrils consist of high-mol.-wt. heteropolysaccharide-protein complexes. Evidently, proteins in the fibril complexes provide interfibril crosslinking to maintain the structural integrity of the sheath.
- 29Emerson, D.; Ghiorse, W. C. Ultrastructure and Chemical-Composition of the Sheath of Leptothrix discophora SP-6. J. Bacteriol. 1993, 175, 7808– 7818, DOI: 10.1128/jb.175.24.7808-7818.1993[Crossref], [PubMed], [CAS], Google Scholar29https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2cXkvFOltg%253D%253D&md5=1cb9d3e52ef4f2322b6ce15c6acee738Ultrastructure and chemical composition of sheath of Leptothrix discophora SP-6Emerson, David; Ghiorse, William C.Journal of Bacteriology (1993), 175 (24), 7808-18CODEN: JOBAAY; ISSN:0021-9193.Light microscopy and transmission electron microscopy of thin sections and metal-shadowed specimens showed that the sheath of Leptothrix discophora SP-6 (ATCC 51168) is a tube-like extracellular polymeric structure consisting of a condensed fabric of 6.5-nm-diam. fibrils underlying a more diffuse outer capsular layer. In thin sections, outer membrane bridges seen to contact the inner sheath layer suggested that the sheath fabric was attached to the outer layer of the gram-neg. cell wall. The capsular polymers showed an affinity for cationic colloidal iron and polycationic ferritin, indicating that they carry a neg. charge. Cell-free sheaths were isolated by treatment with a mixt. of lysozyme, EDTA, and N-lauroylsarcosine (Sarkosyl) or sodium dodecyl sulfate (SDS). Both Sarkosyl- and SDS-isolated sheaths were indistinguishable in microscopic appearance. However, the Mn-oxidizing activity of Sarkosyl-isolated sheaths was more stable than that of SDS-isolated sheaths. The Sarkosyl-isolated sheaths also contained more 2-keto-3-deoxyoctanoic acid and more outer membrane protein than SDS-isolated sheaths. The oven-dried mass of detergent-isolated sheaths represented approx. 9% of the total oven-dried biomass of SP-6 cultures; the oven-dried sheaths contained 38% C, 6.9% N, 6% H, and 2.1% S and approx. 34 to 35% carbohydrte (polysaccharide), 23 to 25% protein, 8% lipid, and 4% inorg. ash. Gas-liq. chromatog. showed that the polysaccharide was an approx. 1:1 mixt. of uronic acids (glucuronic, galacturonic, and mannuronic acids and at least one other unidentified uronic acid) and an amino sugar (galactosamine). Neutral sugars were not detected. Amino acid anal. showed that sheath proteins were enriched in cysteine (6 mol%). The cysteine residues in the sheath proteins probably provide sulfhydryls for disulfide bonds that play an important role in maintaining the structural integrity of the sheath.
- 30Ishihara, H.; Suzuki, T.; Hashimoto, H.; Kunoh, H.; Takada, J. Initial Parallel Arrangement of Extracellular Fibrils Holds a Key for Sheath Frame Construction by Leptothrix sp. Strain OUMS1. Minerals 2013, 3, 73– 81, DOI: 10.3390/min3010073[Crossref], [CAS], Google Scholar30https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXlsFCrs7w%253D&md5=5e74a7afc074b0c47df1f5d9b887caf5Initial parallel arrangement of extracellular fibrils holds a key for sheath frame construction by Leptothrix sp. strain OUMS1Ishihara, Hiromichi; Suzuki, Tomoko; Hashimoto, Hideki; Kunoh, Hitoshi; Takada, JunMinerals (Basel, Switzerland) (2013), 3 (1), 73-81CODEN: MBSIBI; ISSN:2075-163X. (MDPI AG)Early stages of sheath formation by Leptothrix sp. strain OUMS1 and its deriv. sheathless mutant grown in media with or without Fe were examd. by light and electron microscopy. Results showed that the initial parallel arrangement of fibrils excreted from the cells holds a key for subsequent construction of the sheath frame and that aq.-phase Fe interacts with excreted fibrils whether fibrils are parallel-arranged or simply-intermingled.
- 31Hol, F. J. H.; Dekker, C. Zooming in to See the Bigger Picture: Microfluidic and Nanofabrication Tools to Study Bacteria. Science 2014, 346, 1251821, DOI: 10.1126/science.1251821[Crossref], [PubMed], [CAS], Google Scholar31https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC2M3jtV2ntQ%253D%253D&md5=3ee5fcb84fe48324170050e3f82bb53cZooming in to see the bigger picture: microfluidic and nanofabrication tools to study bacteriaHol Felix J H; Dekker CeesScience (New York, N.Y.) (2014), 346 (6208), 1251821 ISSN:.The spatial structure of natural habitats strongly affects bacterial life, ranging from nanoscale structural features that individual cells exploit for surface attachment, to micro- and millimeter-scale chemical gradients that drive population-level processes. Nanofabrication and microfluidics are ideally suited to manipulate the environment at those scales and have emerged as powerful tools with which to study bacteria. Here, we review the new scientific insights gained by using a diverse set of nanofabrication and microfluidic techniques to study individual bacteria and multispecies communities. This toolbox is beginning to elucidate disparate bacterial phenomena-including aging, electron transport, and quorum sensing-and enables the dissection of environmental communities through single-cell genomics. A more intimate integration of microfluidics, nanofabrication, and microbiology will enable further exploration of bacterial life at the smallest scales.
- 32Nagy, K.; Abraham, A.; Keymer, J. E.; Galajda, P. Application of Microfluidics in Experimental Ecology: The Importance of Being Spatial. Front. Microbiol. 2018, 9, 496, DOI: 10.3389/fmicb.2018.00496[Crossref], [PubMed], [CAS], Google Scholar32https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC1MnoslOmuw%253D%253D&md5=a1e25e8fcde515bb0338578105effcdaApplication of Microfluidics in Experimental Ecology: The Importance of Being SpatialNagy Krisztina; Abraham Agnes; Galajda Peter; Abraham Agnes; Keymer Juan EFrontiers in microbiology (2018), 9 (), 496 ISSN:1664-302X.Microfluidics is an emerging technology that is used more and more in biology experiments. Its capabilities of creating precisely controlled conditions in cellular dimensions make it ideal to explore cell-cell and cell-environment interactions. Thus, a wide spectrum of problems in microbial ecology can be studied using engineered microbial habitats. Moreover, artificial microfluidic ecosystems can serve as model systems to test ecology theories and principles that apply on a higher level in the hierarchy of biological organization. In this mini review we aim to demonstrate the versatility of microfluidics and the diversity of its applications that help the advance of microbiology, and in more general, experimental ecology.
- 33Aleklett, K.; Kiers, E. T.; Ohlsson, P.; Shimizu, T. S.; Caldas, V. E.; Hammer, E. C. Build Your Own Soil: Exploring Microfluidics to Create Microbial Habitat Structures. ISME J. 2018, 12, 312– 319, DOI: 10.1038/ismej.2017.184[Crossref], [PubMed], [CAS], Google Scholar33https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC1M3htVCjsA%253D%253D&md5=83f9848fafa3fe58dda05716c8f2ffc5Build your own soil: exploring microfluidics to create microbial habitat structuresAleklett Kristin; Hammer Edith C; Kiers E Toby; Caldas Victor Ea; Ohlsson Pelle; Shimizu Thomas S; Caldas Victor EaThe ISME journal (2018), 12 (2), 312-319 ISSN:.Soil is likely the most complex ecosystem on earth. Despite the global importance and extraordinary diversity of soils, they have been notoriously challenging to study. We show how pioneering microfluidic techniques provide new ways of studying soil microbial ecology by allowing simulation and manipulation of chemical conditions and physical structures at the microscale in soil model habitats.
- 34Volfson, D.; Cookson, S.; Hasty, J.; Tsimring, L. S. Biomechanical Ordering of Dense Cell Populations. Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 15346– 15351, DOI: 10.1073/pnas.0706805105[Crossref], [PubMed], [CAS], Google Scholar34https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BD1cnkslSitQ%253D%253D&md5=3aa430ec2ae45f77b262314c1bc3cafeBiomechanical ordering of dense cell populationsVolfson Dmitri; Cookson Scott; Hasty Jeff; Tsimring Lev SProceedings of the National Academy of Sciences of the United States of America (2008), 105 (40), 15346-51 ISSN:.The structure of bacterial populations is governed by the interplay of many physical and biological factors, ranging from properties of surrounding aqueous media and substrates to cell-cell communication and gene expression in individual cells. The biomechanical interactions arising from the growth and division of individual cells in confined environments are ubiquitous, yet little work has focused on this fundamental aspect of colony formation. We analyze the spatial organization of Escherichia coli growing in a microfluidic chemostat. We find that growth and expansion of a dense colony of cells leads to a dynamical transition from an isotropic disordered phase to a nematic phase characterized by orientational alignment of rod-like cells. We develop a continuum model of collective cell dynamics based on equations for local cell density, velocity, and the tensor order parameter. We use this model and discrete element simulations to elucidate the mechanism of cell ordering and quantify the relationship between the dynamics of cell proliferation and the spatial structure of the population.
- 35Grunberger, A.; Paczia, N.; Probst, C.; Schendzielorz, G.; Eggeling, L.; Noack, S.; Wiechert, W.; Kohlheyer, D. A Disposable Picolitre Bioreactor for Cultivation and Investigation of Industrially Relevant Bacteria on the Single Cell Level. Lab Chip 2012, 12, 2060– 2068, DOI: 10.1039/c2lc40156h[Crossref], [PubMed], [CAS], Google Scholar35https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC38rlvVCntw%253D%253D&md5=b04291ec1337d92cf5eaf13a44b9cf92A disposable picolitre bioreactor for cultivation and investigation of industrially relevant bacteria on the single cell levelGrunberger Alexander; Paczia Nicole; Probst Christopher; Schendzielorz Georg; Eggeling Lothar; Noack Stephan; Wiechert Wolfgang; Kohlheyer DietrichLab on a chip (2012), 12 (11), 2060-8 ISSN:.In the continuously growing field of industrial biotechnology the scale-up from lab to industrial scale is still a major hurdle to develop competitive bioprocesses. During scale-up the productivity of single cells might be affected by bioreactor inhomogeneity and population heterogeneity. Currently, these complex interactions are difficult to investigate. In this report, design, fabrication and operation of a disposable picolitre cultivation system is described, in which environmental conditions can be well controlled on a short time scale and bacterial microcolony growth experiments can be observed by time-lapse microscopy. Three exemplary investigations will be discussed emphasizing the applicability and versatility of the device. Growth and analysis of industrially relevant bacteria with single cell resolution (in particular Escherichia coli and Corynebacterium glutamicum) starting from one single mother cell to densely packed cultures is demonstrated. Applying the picolitre bioreactor, 1.5-fold increased growth rates of C. glutamicum wild type cells were observed compared to typical 1 litre lab-scale batch cultivation. Moreover, the device was used to analyse and quantify the morphological changes of an industrially relevant l-lysine producer C. glutamicum after artificially inducing starvation conditions. Instead of a one week lab-scale experiment, only 1 h was sufficient to reveal the same information. Furthermore, time lapse microscopy during 24 h picolitre cultivation of an arginine producing strain containing a genetically encoded fluorescence sensor disclosed time dependent single cell productivity and growth, which was not possible with conventional methods.
- 36Cho, H.; Jönsson, H.; Campbell, K.; Melke, P.; Williams, J. W.; Jedynak, B.; Stevens, A. M.; Groisman, A.; Levchenko, A. Self-Organization in High-Density Bacterial Colonies: Efficient Crowd Control. PLoS Biol. 2007, 5, e302 DOI: 10.1371/journal.pbio.0050302[Crossref], [PubMed], [CAS], Google Scholar36https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BD2sjgslymsQ%253D%253D&md5=be7321f0d9f65b8992dcc1e4ae988379Self-organization in high-density bacterial colonies: efficient crowd controlCho HoJung; Jonsson Henrik; Campbell Kyle; Melke Pontus; Williams Joshua W; Jedynak Bruno; Stevens Ann M; Groisman Alex; Levchenko AndrePLoS biology (2007), 5 (11), e302 ISSN:.Colonies of bacterial cells can display complex collective dynamics, frequently culminating in the formation of biofilms and other ordered super-structures. Recent studies suggest that to cope with local environmental challenges, bacterial cells can actively seek out small chambers or cavities and assemble there, engaging in quorum sensing behavior. By using a novel microfluidic device, we showed that within chambers of distinct shapes and sizes allowing continuous cell escape, bacterial colonies can gradually self-organize. The directions of orientation of cells, their growth, and collective motion are mutually correlated and dictated by the chamber walls and locations of chamber exits. The ultimate highly organized steady state is conducive to a more-organized escape of cells from the chambers and increased access of nutrients into and evacuation of waste out of the colonies. Using a computational model, we suggest that the lengths of the cells might be optimized to maximize self-organization while minimizing the potential for stampede-like exit blockage. The self-organization described here may be crucial for the early stage of the organization of high-density bacterial colonies populating small, physically confined growth niches. It suggests that this phenomenon can play a critical role in bacterial biofilm initiation and development of other complex multicellular bacterial super-structures, including those implicated in infectious diseases.
- 37Kunoh, T.; Nagaoka, N.; McFarlane, I. R.; Tamura, K.; El-Naggar, M. Y.; Kunoh, H.; Takada, J. Dissociation and Re-Aggregation of Multicell-Ensheathed Fragments Responsible for Rapid Production of Massive Clumps of Leptothrix Sheaths. Biology 2016, 5, 32, DOI: 10.3390/biology5030032[Crossref], [CAS], Google Scholar37https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXms1OitLo%253D&md5=898bf1601d3d9fd1640c63083627058fDissociation and re-aggregation of multicell-ensheathed fragments responsible for rapid production of massive clumps of leptothrix sheathsKunoh, Tatsuki; Nagaoka, Noriyuki; McFarlane, Ian R.; Tamura, Katsunori; El-Naggar, Mohamed Y.; Kunoh, Hitoshi; Takada, JunBiology (Basel, Switzerland) (2016), 5 (3), 32/1-32/9CODEN: BBSIBX; ISSN:2079-7737. (MDPI AG)Species of the Fe/Mn-oxidizing bacteria Leptothrix produce tremendous amts. of microtubular, Fe/Mn-encrusted sheaths within a few days in outwells of groundwater that can rapidly clog water systems. To understand this mode of rapid sheath prodn. and define the timescales involved, behaviors of sheath-forming Leptothrix sp. strain OUMS1 were examd. using time-lapse video at the initial stage of sheath formation. OUMS1 formed clumps of tangled sheaths. Electron microscopy confirmed the presence of a thin layer of bacterial exopolymer fibrils around catenulate cells (corresponding to the immature sheath). In time-lapse videos, numerous sheath filaments that extended from the periphery of sheath clumps repeatedly fragmented at the apex of the same fragment, the fragments then aggregated and again elongated, eventually forming a large sheath clump comprising tangled sheaths within two days. In this study, we found that fast microscopic fragmentation, dissocn., re-aggregation and re-elongation events are the basis of the rapid, massive prodn. of Leptothrix sheaths typically obsd. at macroscopic scales.
- 38Bennett, R. R.; Lee, C. K.; De Anda, J.; Nealson, K. H.; Yildiz, F. H.; O’Toole, G. A.; Wong, G. C.; Golestanian, R. Species-Dependent Hydrodynamics of Flagellum-Tethered Bacteria in Early Biofilm Development. J. R. Soc., Interface 2016, 13, 20150966, DOI: 10.1098/rsif.2015.0966[Crossref], [PubMed], [CAS], Google Scholar38https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC28nps1ShtA%253D%253D&md5=4b84034d8dd578d99d29e79bb1f7d3ceSpecies-dependent hydrodynamics of flagellum-tethered bacteria in early biofilm developmentBennett Rachel R; Lee Calvin K; De Anda Jaime; Wong Gerard C L; Nealson Kenneth H; Yildiz Fitnat H; O'Toole George A; Golestanian RaminJournal of the Royal Society, Interface (2016), 13 (115), 20150966 ISSN:.Monotrichous bacteria on surfaces exhibit complex spinning movements. Such spinning motility is often a part of the surface detachment launch sequence of these cells. To understand the impact of spinning motility on bacterial surface interactions, we develop a hydrodynamic model of a surface-bound bacterium, which reproduces behaviours that we observe in Pseudomonas aeruginosa, Shewanella oneidensis and Vibrio cholerae, and provides a detailed dictionary for connecting observed spinning behaviour to bacteria-surface interactions. Our findings indicate that the fraction of the flagellar filament adhered to the surface, the rotation torque of this appendage, the flexibility of the flagellar hook and the shape of the bacterial cell dictate the likelihood that a microbe will detach and the optimum orientation that it should have during detachment. These findings are important for understanding species-specific reversible attachment, the key transition event between the planktonic and biofilm lifestyle for motile, rod-shaped organisms.
- 39De Anda, J.; Lee, E. Y.; Lee, C. K.; Bennett, R. R.; Ji, X.; Soltani, S.; Harrison, M. C.; Baker, A. E.; Luo, Y.; Chou, T. High-Speed “4D” Computational Microscopy of Bacterial Surface Motility. ACS Nano 2017, 11, 9340– 9351, DOI: 10.1021/acsnano.7b04738[ACS Full Text], [CAS], Google Scholar39https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhtlyhsL%252FM&md5=c0b8c2763726329389fb18c8fb4ff8e9High-Speed "4D" Computational Microscopy of Bacterial Surface Motilityde Anda, Jaime; Lee, Ernest Y.; Lee, Calvin K.; Bennett, Rachel R.; Ji, Xiang; Soltani, Soheil; Harrison, Mark C.; Baker, Amy E.; Luo, Yun; Chou, Tom; O'Toole, George A.; Armani, Andrea M.; Golestanian, Ramin; Wong, Gerard C. L.ACS Nano (2017), 11 (9), 9340-9351CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)Bacteria exhibit surface motility modes that play pivotal roles in early-stage biofilm community development, such as type IV pili-driven "twitching" motility and flagellum-driven "spinning" and "swarming" motility. Appendage-driven motility is controlled by mol. motors, and anal. of surface motility behavior is complicated by its inherently 3D nature, the speed of which is too fast for confocal microscopy to capture. Here, the authors combine electromagnetic field computation and statistical image anal. to generate 3D movies close to a surface at 5 ms time resoln. using conventional inverted microscopes. The authors treat each bacterial cell as a spherocylindrical lens and use finite element modeling to solve Maxwell's equations and compute the diffracted light intensities assocd. with different angular orientations of the bacterium relative to the surface. By performing cross-correlation calcns. between measured 2D microscopy images and a library of computed light intensities, near-surface 3D movies of Pseudomonas aeruginosa translational and rotational motion are possible at high temporal resoln. Comparison between computational reconstructions and detailed hydrodynamic calcns. reveals that P. aeruginosa act like low Reynolds no. spinning tops with unstable orbits, driven by a flagellum motor with a torque output of ∼2 pN μm. The authors' anal. reveals that P. aeruginosa can undergo complex flagellum-driven dynamical behavior, including precession, nutation, and an unexpected taxonomy of surface motility mechanisms, including upright-spinning bacteria that diffuse laterally across the surface, and horizontal bacteria that follow helicoidal trajectories and exhibit superdiffusive movements parallel to the surface.
- 40Adams, L. F.; Ghiorse, W. C. Influence of Manganese on Growth of a Sheathless Strain of Leptothrix discophora. Appl. Environ. Microbiol. 1985, 49, 556– 562[Crossref], [PubMed], [CAS], Google Scholar40https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL2MXhsVGqtrc%253D&md5=44e448f8e54750d18053f7facfb6dc19Influence of manganese on growth of a sheathless strain of Leptothrix discophoraAdams, Lee F.; Ghiorse, William C.Applied and Environmental Microbiology (1985), 49 (3), 556-62CODEN: AEMIDF; ISSN:0099-2240.Mn2+ exerted various effects on the growth of L. discophora strain SS-1 in batch cultures, depending on the concn. added to the medium. Concns. of 0.55-5.5 μM Mn2+, comparable to those in the environment from which SS-1 was isolated, decreased cell yield and prolonged stationary-phase survival, but did not affect growth rate. Elevated concns. of 55-910 μM Mn2+ also decreased cell yield and prolonged survival, but growth rate was decreased as well. The addn. of 1820 μM Mn2+ caused a decline in cell nos., followed by an exponential rise after 80 h of incubation, indicating the development of a population of cells resistant to Mn2+ toxicity. When ≤360 μM Mn2+ was added to growth flasks, Mn2+ was oxidized to Mn oxide (MnOx, where x is ∼2) which appeared as brown particles in the medium. Quantification of Mn oxidn. during growth of cultures to which 55 μM Mn2+ was added showed that nearly all of the Mn2+ was oxidized at the beginning of the stationary phase of growth (15-25 h). Thus, the decrease in cell yield obsd. at low and moderate concns. of Mn2+ was related to the formation of MnOx, which may have bound cationic nutrients essential to the growth of SS-1. The addn. of excess Fe3+ to cultures contg. 55 μM Mn2+ increased cell yield to levels near those found in cultures with no added Mn2+, indicating that Fe deprivation by MnOx was at least partly responsible for the decreased cell yield.
- 41Sauer, K.; Camper, A. K.; Ehrlich, G. D.; Costerton, J. W.; Davies, D. G. Pseudomonas aeruginosa Displays Multiple Phenotypes During Development As a Biofilm. J. Bacteriol. 2002, 184, 1140– 1154, DOI: 10.1128/jb.184.4.1140-1154.2002[Crossref], [PubMed], [CAS], Google Scholar41https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XhtVeisrg%253D&md5=dc6e3a7aeed3d4bce4f507db4690d1e5Pseudomonas aeruginosa displays multiple phenotypes during development as a biofilmSauer, Karin; Camper, Anne K.; Ehrlich, Garth D.; Costerton, J. William; Davies, David G.Journal of Bacteriology (2002), 184 (4), 1140-1154CODEN: JOBAAY; ISSN:0021-9193. (American Society for Microbiology)Complementary approaches were employed to characterize transitional episodes in Pseudomonas aeruginosa biofilm development using direct observation and whole-cell protein anal. Microscopy and in situ reporter gene anal. were used to directly observe changes in biofilm physiol. and to act as signposts to standardize protein collection for two-dimensional electrophoretic anal. and protein identification in chemostat and continuous-culture biofilm-grown populations. Using these approaches, we characterized five stages of biofilm development: (i) reversible attachment, (ii) irreversible attachment, (iii) maturation-1, (iv) maturation-2, and (v) dispersion. Biofilm cells were shown to change regulation of motility, alginate prodn., and quorum sensing during the process of development. The av. difference in detectable protein regulation between each of the five stages of development was 35% (approx. 525 proteins). When planktonic cells were compared with maturation-2 stage biofilm cells, more than 800 proteins were shown to have a sixfold or greater change in expression level (over 50% of the proteome). This difference was higher than when planktonic P. aeruginosa were compared with planktonic cultures of Pseudomonas putida. Las quorum sensing was shown to play no role in early biofilm development but was important in later stages. Biofilm cells in the dispersion stage were more similar to planktonic bacteria than to maturation-2 stage bacteria. These results demonstrate that P. aeruginosa displays multiple phenotypes during biofilm development and that knowledge of stage-specific physiol. may be important in detecting and controlling biofilm growth.
- 42Hinsa, S. M.; Espinosa-Urgel, M.; Ramos, J. L.; O’Toole, G. A. Transition from Reversible to Irreversible Attachment During Biofilm Formation by Pseudomonas fluorescens WCS365 Requires an ABC Transporter and a Large Secreted Protein. Mol. Microbiol. 2003, 49, 905– 918, DOI: 10.1046/j.1365-2958.2003.03615.x[Crossref], [PubMed], [CAS], Google Scholar42https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXmslKltL8%253D&md5=eabbe27d3809bdb88357fb219e6bcff7Transition from reversible to irreversible attachment during biofilm formation by Pseudomonas fluorescens WCS365 requires an ABC transporter and a large secreted proteinHinsa, Shannon M.; Espinosa-Urgel, Manuel; Ramos, Juan L.; O'Toole, George A.Molecular Microbiology (2003), 49 (4), 905-918CODEN: MOMIEE; ISSN:0950-382X. (Blackwell Publishing Ltd.)We report the identification of an ATP-binding cassette (ABC) transporter and an assocd. large cell-surface protein that are required for biofilm formation by Pseudomonas fluorescens WCS365. The genes coding for these proteins are designated lap for large adhesion protein. The LapA protein, with a predicted mol. wt. of ∼900 kDa, is found to be loosely assocd. with the cell surface and present in the culture supernatant. The LapB, LapC and LapE proteins are predicted to be the cytoplasmic membrane-localized ATPase, membrane fusion protein and outer membrane protein component, resp., of an ABC transporter. Consistent with this prediction, LapE, like other members of this family, is localized to the outer membrane. We propose that the lapEBC-encoded ABC transporter participates in the secretion of LapA, as strains with mutations in the lapEBC genes do not have detectable LapA assocd. with the cell surface or in the supernatant. The lap genes are conserved among environmental pseudomonads such as P. putida KT2440, P. fluorescens PfO1 and P. fluorescens WCS365, but are absent from pathogenic pseudomonads such as P. aeruginosa and P. syringae. The wild-type strain of P. fluorescens WCS365 and its lap mutant derivs. were assessed for their biofilm forming ability in static and flow systems. The lap mutant strains are impaired in an early step in biofilm formation and are unable to develop the mature biofilm structure seen for the wild-type bacterium. Time-lapse microscopy studies detd. that the lap mutants are unable to progress from reversible (or transient) attachment to the irreversible attachment stage of biofilm development. The lap mutants were also defective in attachment to quartz sand, an abiotic surface these organisms likely encounter in the environment.
- 43Tuson, H. H.; Weibel, D. B. Bacteria–Surface Interactions. Soft Matter 2013, 9, 4368– 4380, DOI: 10.1039/c3sm27705d[Crossref], [PubMed], [CAS], Google Scholar43https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXltVOrurg%253D&md5=d0757729a24782a2faa7d4b022be150dBacteria-surface interactionsTuson, Hannah H.; Weibel, Douglas B.Soft Matter (2013), 9 (17), 4368-4380CODEN: SMOABF; ISSN:1744-683X. (Royal Society of Chemistry)A review. The interaction of bacteria with surfaces has important implications in a range of areas, including bioenergy, biofouling, biofilm formation, and the infection of plants and animals. Many of the interactions of bacteria with surfaces produce changes in the expression of genes that influence cell morphol. and behavior, including genes essential for motility and surface attachment. Despite the attention that these phenotypes have garnered, the bacterial systems used for sensing and responding to surfaces are still not well understood. An understanding of these mechanisms will guide the development of new classes of materials that inhibit and promote cell growth, and complement studies of the physiol. of bacteria in contact with surfaces. Recent studies from a range of fields in science and engineering are poised to guide future investigations in this area. This review summarizes recent studies on bacteria-surface interactions, discusses mechanisms of surface sensing and consequences of cell attachment, provides an overview of surfaces that have been used in bacterial studies, and highlights unanswered questions in this field.
- 44Brangwynne, C. P.; MacKintosh, F. C.; Kumar, S.; Geisse, N. A.; Talbot, J.; Mahadevan, L.; Parker, K. K.; Ingber, D. E.; Weitz, D. A. Microtubules Can Bear Enhanced Compressive Loads in Living Cells Because of Lateral Reinforcement. J. Cell Biol. 2006, 173, 733– 741, DOI: 10.1083/jcb.200601060[Crossref], [PubMed], [CAS], Google Scholar44https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28Xls1Shsrc%253D&md5=0119caf6de99e66dce86a304946d4053Microtubules can bear enhanced compressive loads in living cells because of lateral reinforcementBrangwynne, Clifford P.; MacKintosh, Frederick C.; Kumar, Sanjay; Geisse, Nicholas A.; Talbot, Jennifer; Mahadevan, L.; Parker, Kevin K.; Ingber, Donald E.; Weitz, David A.Journal of Cell Biology (2006), 173 (5), 733-741CODEN: JCLBA3; ISSN:0021-9525. (Rockefeller University Press)Cytoskeletal microtubules have been proposed to influence cell shape and mechanics based on their ability to resist large-scale compressive forces exerted by the surrounding contractile cytoskeleton. Consistent with this, cytoplasmic microtubules are often highly curved and appear buckled because of compressive loads. However, the results of in vitro studies suggest that microtubules should buckle at much larger length scales, withstanding only exceedingly small compressive forces. This discrepancy calls into question the structural role of microtubules, and highlights our lack of quant. knowledge of the magnitude of the forces they experience and can withstand in living cells. We show that intracellular microtubules do bear large-scale compressive loads from a variety of physiol. forces, but their buckling wavelength is reduced significantly because of mech. coupling to the surrounding elastic cytoskeleton. We quant. explain this behavior, and show that this coupling dramatically increases the compressive forces that microtubules can sustain, suggesting they can make a more significant structural contribution to the mech. behavior of the cell than previously thought possible.
- 45Duvernoy, M.-C.; Mora, T.; Ardré, M.; Croquette, V.; Bensimon, D.; Quilliet, C.; Ghigo, J.-M.; Balland, M.; Beloin, C.; Lecuyer, S.; Desprat, N. Asymmetric Adhesion of Rod-Shaped Bacteria Controls Microcolony Morphogenesis. Nat. Commun. 2018, 9, 1120, DOI: 10.1038/s41467-018-03446-y[Crossref], [PubMed], [CAS], Google Scholar45https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC1MnitFersw%253D%253D&md5=1d0011ddb7e7089b6091609ccd6a26c7Asymmetric adhesion of rod-shaped bacteria controls microcolony morphogenesisDuvernoy Marie-Cecilia; Mora Thierry; Ardre Maxime; Croquette Vincent; Bensimon David; Desprat Nicolas; Duvernoy Marie-Cecilia; Quilliet Catherine; Balland Martial; Lecuyer Sigolene; Duvernoy Marie-Cecilia; Ardre Maxime; Croquette Vincent; Bensimon David; Desprat Nicolas; Bensimon David; Ghigo Jean-Marc; Beloin Christophe; Desprat NicolasNature communications (2018), 9 (1), 1120 ISSN:.Surface colonization underpins microbial ecology on terrestrial environments. Although factors that mediate bacteria-substrate adhesion have been extensively studied, their spatiotemporal dynamics during the establishment of microcolonies remains largely unexplored. Here, we use laser ablation and force microscopy to monitor single-cell adhesion during the course of microcolony formation. We find that adhesion forces of the rod-shaped bacteria Escherichia coli and Pseudomonas aeruginosa are polar. This asymmetry induces mechanical tension, and drives daughter cell rearrangements, which eventually determine the shape of the microcolonies. Informed by experimental data, we develop a quantitative model of microcolony morphogenesis that enables the prediction of bacterial adhesion strength from simple time-lapse measurements. Our results demonstrate how patterns of surface colonization derive from the spatial distribution of adhesive factors on the cell envelope.
- 46Landau, L. D.; Lifshitz, E. M. Theory of Elasticity, 3rd ed.; Elsevier: New York, 1986; Vol. 7.Google ScholarThere is no corresponding record for this reference.
- 47Amir, A.; Babaeipour, F.; McIntosh, D. B.; Nelson, D. R.; Jun, S. Bending Forces Plastically Deform Growing Bacterial Cell Walls. Proc. Natl. Acad. Sci. U. S. A. 2014, 111, 5778– 5783, DOI: 10.1073/pnas.1317497111[Crossref], [PubMed], [CAS], Google Scholar47https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXmtlWntLo%253D&md5=04c501958cff07e96b03df45712bde40Bending forces plastically deform growing bacterial cell wallsAmir, Ariel; Babaeipour, Farinaz; McIntosh, Dustin B.; Nelson, David R.; Jun, SuckjoonProceedings of the National Academy of Sciences of the United States of America (2014), 111 (16), 5778-5783CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Cell walls define a cell's shape in bacteria. The walls are rigid to resist large internal pressures, but remarkably plastic to adapt to a wide range of external forces and geometric constraints. Currently, it is unknown how bacteria maintain their shape. In this paper, we develop exptl. and theor. approaches and show that mech. stresses regulate bacterial cell wall growth. By applying a precisely controllable hydrodynamic force to growing rod-shaped Escherichia coli and Bacillus subtilis cells, we demonstrate that the cells can exhibit two fundamentally different modes of deformation. The cells behave like elastic rods when subjected to transient forces, but deform plastically when significant cell wall synthesis occurs while the force is applied. The deformed cells always recover their shape. The exptl. results are in quant. agreement with the predictions of the theory of dislocation-mediated growth. In particular, we find that a single dimensionless parameter, which depends on a combination of independently measured phys. properties of the cell, can describe the cell's responses under various exptl. conditions. These findings provide insight into how living cells robustly maintain their shape under varying phys. environments.
- 48Wang, S.; Arellano-Santoyo, H.; Combs, P. A.; Shaevitz, J. W. Actin-Like Cytoskeleton Filaments Contribute to Cell Mechanics in Bacteria. Proc. Natl. Acad. Sci. U. S. A. 2010, 107, 9182– 9185, DOI: 10.1073/pnas.0911517107[Crossref], [PubMed], [CAS], Google Scholar48https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXmslGrt70%253D&md5=d65bddef3d6e8c44fc09719c3b90861aActin-like cytoskeleton filaments contribute to cell mechanics in bacteriaWang, Siyuan; Arellano-Santoyo, Hugo; Combs, Peter A.; Shaevitz, Joshua W.Proceedings of the National Academy of Sciences of the United States of America (2010), 107 (20), 9182-9185, S9182/1-S9182/6CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)A filamentous cytoskeleton largely governs the phys. shape and mech. properties of eukaryotic cells. In bacteria, proteins homologous to all three classes of eukaryotic cytoskeletal filaments have recently been discovered. These proteins are essential for the maintenance of bacterial cell shape and have been shown to guide the localization of key cell-wall-modifying enzymes. However, whether the bacterial cytoskeleton is stiff enough to affect the overall mech. rigidity of a cell has not been probed. Here, we used an optical trap to measure the bending rigidity of live Escherichia coli cells. We find that the actin-homolog MreB contributes nearly as much to the stiffness of a cell as the peptidoglycan cell wall. By quant. modeling these measurements, our data indicate that the MreB is rigidly linked to the cell wall, increasing the mech. stiffness of the overall system. These data are the first evidence that the bacterial cytoskeleton contributes to the mech. integrity of a cell in much the same way as it does in eukaryotes.
- 49Kawasaki, Y.; Kurosaki, K.; Kan, D.; Borges, I. K.; Atagui, A. S.; Sato, M.; Kondo, K.; Katahira, M.; Suzuki, I.; Takeda, M. Identification and Characterization of the S-Layer Formed on the Sheath of Thiothrix nivea. Arch. Microbiol. 2018, 200, 1257– 1265, DOI: 10.1007/s00203-018-1543-x[Crossref], [PubMed], [CAS], Google Scholar49https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXht1Smsb3O&md5=fe1da3fb23ad7254ce10edfb970738ecIdentification and characterization of the S-layer formed on the sheath of Thiothrix niveaKawasaki, Yuta; Kurosaki, Kaishi; Kan, Daisuke; Borges, Isabele Kazahaya; Atagui, Ayumi Satake; Sato, Michio; Kondo, Keiko; Katahira, Masato; Suzuki, Ichiro; Takeda, MinoruArchives of Microbiology (2018), 200 (8), 1257-1265CODEN: AMICCW; ISSN:0302-8933. (Springer)Thiothrix nivea is a filamentous sulfur-oxidizing bacterium common in activated sludge and its filament is covered with a polysaccharide layer called sheath. In this study, we found that T. nivea aggregates under acidic conditions. A hexagonal lattice pattern, a typical morphol. feature of proteinaceous S-layers, was newly obsd. on the surface of the sheath by transmission electron microscopy. The pattern and the acid-dependent aggregation were not obsd. in T. fructosivorans, a relative sheath-forming bacterium of T. nivea. The putative S-layer of T. nivea was detached by washing with unbuffered tris(hydroxymethyl)aminomethane base (Tris) soln. and a protein of 160 kDa was detected by electrophoresis. Based on partial amino acid sequences of the protein, its structural gene was identified. The gene encodes an acidic protein which has a putative secretion signal and a Ca2+-binding domain. The protein was solubilized with urea followed by dialysis in the presence of calcium. A hexagonal lattice pattern was obsd. in the aggregates formed during dialysis, revealing that the protein is responsible for S-layer formation. Biosorption ability of copper, zinc, and cadmium onto the T. nivea filament decreased upon pretreatment with Tris, demonstrating that the S-layer was involved in metal adsorption. Moreover, aggregation of Escherichia coli was promoted by acidification in the presence of the S-layer protein, suggesting that the protein is potentially applicable as an acid-driven flocculant for other bacteria.
- 50Suzuki, T.; Kanagawa, T.; Kamagata, Y. Identification of a Gene Essential for Sheathed Structure Formation in Sphaerotilus natans, a Filamentous Sheathed Bacterium. Appl. Environ. Microbiol. 2002, 68, 365– 371, DOI: 10.1128/AEM.68.1.365-371.2002[Crossref], [PubMed], [CAS], Google Scholar50https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38Xjt1Wlsw%253D%253D&md5=03f0274274fafb3c2a7108e3de9f8bfeIdentification of a gene essential for sheathed structure formation in Sphaerotilus natans, a filamentous sheathed bacteriumSuzuki, Toshihiko; Kanagawa, Takahiro; Kamagata, YoichiApplied and Environmental Microbiology (2002), 68 (1), 365-371CODEN: AEMIDF; ISSN:0099-2240. (American Society for Microbiology)Sphaerotilus natans, a filamentous bacterium that causes bulking in activated sludge processes, can assume two distinct morphologies, depending on the substrate concn. for growth; in substrate-rich media it grows as single rod-shaped cells, whereas in substrate-limited media it grows as filaments. To identify genes responsible for sheath formation, we carried out transposon Tn5 mutagenesis. Of the approx. 20,000 mutants obtained, 7 did not form sheathed structures. Sequencing of the Tn5-flanking regions showed that five of the seven Tn5 insertions converged at the same open reading frame, designated sthA. The deduced amino acids encoded by sthA were found to be homologous to glycosyltransferase, which is known to be involved in linking sugars to lipid carriers during bacterial exopolysaccharide biosynthesis. Disruption of the gene of the wild-type strain by inserting a kanamycin resistance gene cassette also resulted in sheathless growth under either type of nutrient condition. These findings indicate that sthA is a crucial component responsible for sheath formation.
- 51Kawasaki, Y.; Endo, T.; Fujiwara, A.; Kondo, K.; Katahira, M.; Nittami, T.; Sato, M.; Takeda, M. Elongation Pattern and Fine Structure of the Sheaths Formed by Thiothrix nivea and Thiothrix fructosivorans. Int. J. Biol. Macromol. 2017, 95, 1280– 1288, DOI: 10.1016/j.ijbiomac.2016.11.025[Crossref], [PubMed], [CAS], Google Scholar51https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhvVGgt77I&md5=c40e9a5d8d4a6d7ac0ef74716310b4b8Elongation pattern and fine structure of the sheaths formed by Thiothrix nivea and Thiothrix fructosivoransKawasaki, Yuta; Endo, Tomoyuki; Fujiwara, Atsuo; Kondo, Keiko; Katahira, Masato; Nittami, Tadashi; Sato, Michio; Takeda, MinoruInternational Journal of Biological Macromolecules (2017), 95 (), 1280-1288CODEN: IJBMDR; ISSN:0141-8130. (Elsevier B.V.)Thiothrix strains are filamentous sulfur-oxidizing bacteria common in activated sludge. Some of the members, including Thiothrix nivea and T. fructosivorans, are known to form a microtubular sheath that covers a line of cells. The sheaths are assemblages of [→4)-β-D-GlcN-(1 → 4)-β-D-Glc-(1→]n modified with unusual deoxy sugars. To elucidate the sheath-forming mechanism, the patterns of sheath formation and cell proliferation were detd. Prior to anal., both sheaths were confirmed to be highly de-N-acetylated. Sheaths in viable filaments were N-biotinylated followed by cultivation and then fluorescently immunostained. Epifluorescence microscopy of the filaments revealed ubiquitous elongation of the sheaths. For visualization of the cell proliferation pattern, the cell membrane was fluorescently stained. The epifluorescence images demonstrated that cell proliferation also proceeds ubiquitously, suggesting that sheath elongation proceeds surrounding an elongating cell. In addn., the fine structure of the Thiothrix filaments was analyzed by transmission electron microscopy employing a freeze-substitution technique. The micrographs of freeze-substituted filaments showed that the sheaths were thin and single layered. In contrast, the sheaths in chem. fixed filaments appeared thick and multilayered. Treatment with glutaraldehyde probably caused deformation of the sheaths. Supporting this possibility, the sheaths were found to be deformed or solubilized by N-acetylation.
- 52Kondo, K.; Umezu, T.; Shimura, S.; Narizuka, R.; Koizumi, J.-i.; Mashima, T.; Katahira, M.; Takeda, M. Structure of Perosamine-Containing Polysaccharide, a Component of the Sheath of Thiothrix fructosivorans. Int. J. Biol. Macromol. 2013, 59, 59– 66, DOI: 10.1016/j.ijbiomac.2013.04.013[Crossref], [PubMed], [CAS], Google Scholar52https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXptFOms7w%253D&md5=9cbbc84059ef6889e1dee235484b9990Structure of perosamine-containing polysaccharide, a component of the sheath of Thiothrix fructosivoransKondo, Keiko; Umezu, Takuto; Shimura, Shoichi; Narizuka, Rie; Koizumi, Jun-ichi; Mashima, Tsukasa; Katahira, Masato; Takeda, MinoruInternational Journal of Biological Macromolecules (2013), 59 (), 59-66CODEN: IJBMDR; ISSN:0141-8130. (Elsevier B.V.)A sheath-forming and sulfur-oxidizing bacterium, Thiothrix fructosivorans, was heterotrophically cultured. The sheath, which is an extracellular microtube, was prepd. by selectively removing the cells using lysozyme, sodium dodecyl sulfate, and sodium hydroxide. Solid-state 13C-NMR spectrum revealed that the sheath is assembled from a glycan possessing acetyl and Me groups. When the sheath was deacetylated, the original microtube structure was lost and the sheath became sol. under acidic conditions, revealing the importance of acetyl groups in maintaining the sheath structure. Equimolar D-glucose, D-glucosamine, and L-fucose were detected in the acid hydrolyzate of the sheath by gas liq. chromatog. In addn. to these sugars, β-GlcN-(1 → 4)-Glc and unidentified sugar were detected by analyzing the hydrolyzate using HPLC anal. 1H and 13C NMR spectroscopy was used to identify a disaccharide composed of 4-deoxy-4-aminorhamnose (perosamine, Rha4N) and fucose. N-Acetyl-perosamine prepd. from the disaccharide was polarimetric and exhibited a D-configuration. The previously unidentified disaccharide is α-D-Rhap4N-(1→3)-D-Fuc. According to 1H and 13C NMR analyses, the deacetylated sheath-forming polysaccharide was found to h have a main chain of [(→4)-β-D-GlcpN-(1 → 4)-β-D-Glcp-(1→)]n, to which disaccharide side chains of α-D-Rhap4N-(1→3)-α-L-Fucp-(1 →) were attached at position 3 of Glc.
- 53Stanley, C. E.; Grossmann, G.; Casadevall i Solvas, X.; deMello, A. J. Soil-On-A-Chip: Microfluidic Platforms for Environmental Organismal Studies. Lab Chip 2016, 16, 228– 241, DOI: 10.1039/C5LC01285F[Crossref], [PubMed], [CAS], Google Scholar53https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhvFWlu7vO&md5=064f9995395a44eab18893c8a99f5e01Soil-on-a-Chip: microfluidic platforms for environmental organismal studiesStanley, Claire E.; Grossmann, Guido; Casadevall i Solvas, Xavier; deMello, Andrew J.Lab on a Chip (2016), 16 (2), 228-241CODEN: LCAHAM; ISSN:1473-0189. (Royal Society of Chemistry)Soil is the habitat of countless organisms and encompasses an enormous variety of dynamic environmental conditions. While it is evident that a thorough understanding of how organisms interact with the soil environment may have substantial ecol. and economical impact, current lab.-based methods depend on reductionist approaches that are incapable of simulating natural diversity. The application of Lab-on-a-Chip or microfluidic technologies to organismal studies is an emerging field, where the unique benefits afforded by system miniaturization offer new opportunities for the experimentalist. Indeed, precise spatiotemporal control over the microenvironments of soil organisms in combination with high-resoln. imaging has the potential to provide an unprecedented view of biol. events at the single-organism or single-cell level, which in turn opens up new avenues for environmental and organismal studies. Herein we review some of the most recent and interesting developments in microfluidic technologies for the study of soil organisms and their interactions with the environment. We discuss how so-called "Soil-on-a-Chip" technol. has already contributed significantly to the study of bacteria, nematodes, fungi and plants, as well as inter-organismal interactions, by advancing exptl. access and environmental control. Most crucially, we highlight where distinct advantages over traditional approaches exist and where novel biol. insights will ensue.
- 54Nelson, Y. M.; Lion, L. W.; Shuler, M. L.; Ghiorse, W. C. Effect of Oxide Formation Mechanisms on Lead Adsorption by Biogenic Manganese (Hydr)Oxides, Iron (Hydr)Oxides, and Their Mixtures. Environ. Sci. Technol. 2002, 36, 421– 425, DOI: 10.1021/es010907c[ACS Full Text], [CAS], Google Scholar54https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XhsVWisQ%253D%253D&md5=2afadf2f0552d2d976909c749e3ee3dcEffect of Oxide Formation Mechanisms on Lead Adsorption by Biogenic Manganese (Hydr)oxides, Iron (Hydr)oxides, and Their MixturesNelson, Yarrow M.; Lion, Leonard W.; Shuler, Michael L.; Ghiorse, William C.Environmental Science and Technology (2002), 36 (3), 421-425CODEN: ESTHAG; ISSN:0013-936X. (American Chemical Society)The effects of iron and manganese (hydr)oxide formation processes on the trace metal adsorption properties of these metal (hydr)oxides and their mixts. was investigated by measuring Pb adsorption by iron and manganese (hydr)oxides prepd. by a variety of methods. Amorphous iron (hydr)oxide formed by fast pptn. at pH 7.5 exhibited greater Pb adsorption (Γmax = 50 mmol of Pb/mol of Fe at pH 6.0) than iron (hydr)oxide formed by slow, diffusion-controlled oxidn. of Fe(II) at pH 4.5-7.0 or goethite. Biogenic manganese(III/IV) (hydr)oxide prepd. by enzymic oxidn. of Mn(II) by the bacterium Leptothrix discophora SS-1 adsorbed five times more Pb (per mole of Mn) than an abiotic manganese (hydr)oxide prepd. by oxidn. of Mn(II) with permanganate, and 500-5000 times more Pb than pyrolusite oxides (β-MnO2). X-ray crystallog. indicated that biogenic manganese (hydr)oxide and iron (hydr)oxide were predominantly amorphous or poorly cryst. and their x-ray diffraction patterns were not significantly affected by the presence of the other (hydr)oxide during formation. When iron and manganese (hydr)oxides were mixed after formation, or for Mn biol. oxidized with iron(III) (hydr)oxide present, the obsd. Pb adsorption was similar to that expected for the mixt. based on Langmuir parameters for the individual (hydr)oxides. These results indicate that interactions in iron/manganese (hydr)oxide mixts. related to the formation process and sequence of formation such as site masking, alterations in sp. surface area, or changes in cryst. structure either did not occur or had a negligible effect on Pb adsorption by the mixts.
- 55Katsoyiannis, I. A.; Zouboulis, A. I. Application of Biological Processes for the Removal of Arsenic From Groundwaters. Water Res. 2004, 38, 17– 26, DOI: 10.1016/j.watres.2003.09.011[Crossref], [PubMed], [CAS], Google Scholar55https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BD3srltF2ltg%253D%253D&md5=08fece0d01f1658ff4223d2bf0979bccApplication of biological processes for the removal of arsenic from groundwatersKatsoyiannis Ioannis A; Zouboulis Anastasios IWater research (2004), 38 (1), 17-26 ISSN:0043-1354.Bacteria are widespread, abundant, geochemically reactive components of aquatic environments. In particular, iron-oxidizing bacteria, are involved in the oxidation and subsequent precipitation of ferrous ions. Due to this property, they have been applied in drinking water treatment processes, in order to accelerate the removal of ferrous iron from groundwaters. Iron also exerts a strong influence on arsenic concentrations in groundwater sources, while iron oxides are efficient adsorbents in arsenic removal processes. In the present study, the removal of arsenic (III and V), during biological iron oxidation has been investigated. The results showed that both inorganic forms of arsenic could be efficiently treated, for the concentration range of interest in drinking water (50-200microg/L). In addition, the oxidation of trivalent arsenic was found to be catalyzed by bacteria, leading to enhanced overall arsenic removal, because arsenic in the form of arsenites cannot be efficiently sorbed onto iron oxides. This method comprises a cost competitive technology, which can find application in treatment of groundwaters with elevated concentrations of iron and arsenic.
- 56Ghiorse, W. Biology of Iron- and Manganese-Depositing Bacteria. Annu. Rev. Microbiol. 1984, 38, 515– 550, DOI: 10.1146/annurev.mi.38.100184.002503[Crossref], [PubMed], [CAS], Google Scholar56https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL2cXmtVOht74%253D&md5=358a2be031bfbf201a76ada3ef999287Biology of iron- and manganese-depositing bacteriaGhiorse, W. C.Annual Review of Microbiology (1984), 38 (), 515-50CODEN: ARMIAZ; ISSN:0066-4227.A review with 200 refs.
- 57Rhoads, A.; Beyenal, H.; Lewandowski, Z. Microbial Fuel Cell Using Anaerobic Respiration as an Anodic Reaction and Biomineralized Manganese as a Cathodic Reactant. Environ. Sci. Technol. 2005, 39, 4666– 4671, DOI: 10.1021/es048386r[ACS Full Text], [CAS], Google Scholar57https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXktFWksLY%253D&md5=332aab447fb9215e6bf4947063a01f45Microbial Fuel Cell using Anaerobic Respiration as an Anodic Reaction and Biomineralized Manganese as a Cathodic ReactantRhoads, Allison; Beyenal, Haluk; Lewandowski, ZbigniewEnvironmental Science and Technology (2005), 39 (12), 4666-4671CODEN: ESTHAG; ISSN:0013-936X. (American Chemical Society)The authors have operated a microbial fuel cell in which glucose was oxidized by Klebsiella pneumoniae in the anodic compartment, and biomineralized manganese oxides, deposited by Leptothrix discophora, were electrochem. reduced in the cathodic compartment. In the anodic compartment, to facilitate the electron transfer from glucose to the graphite electrode, the authors added a redox mediator, 2-hydroxy-1,4-naphthoquinone. The authors did not add any redox mediator to the cathodic compartment because the biomineralized manganese oxides were deposited on the surface of a graphite electrode and were reduced directly by electrons from the electrode. Biomineralized manganese oxides are superior to oxygen when used as cathodic reactants in microbial fuel cells. The c.d. delivered by using biomineralized manganese oxides as the cathodic reactant was almost 2 orders of magnitude higher than that delivered using oxygen. Several fuel cells were operated for 500 h, reaching anodic potentials of -441.5 ± 31 mVSCE and cathodic potentials of +384.5 ± 64 mVSCE. When the electrodes were connected by a 50 Ω resistor, the fuel cell delivered the peak power d. of 126.7 ± 31.5 mW/m2.
- 58Yan, X.; Zhou, Q.; Vincent, M.; Deng, Y.; Yu, J.; Xu, J.; Xu, T.; Tang, T.; Bian, L.; Wang, Y.-X. J. Multifunctional Biohybrid Magnetite Microrobots for Imaging-Guided Therapy. Science Robotics 2017, 2, eaaq1155, DOI: 10.1126/scirobotics.aaq1155
- 59Bente, K.; Codutti, A.; Bachmann, F.; Faivre, D. Biohybrid and Bioinspired Magnetic Microswimmers. Small 2018, 14, 1704374, DOI: 10.1002/smll.201704374
- 60Sanchez, S.; Solovev, A. A.; Schulze, S.; Schmidt, O. G. Controlled Manipulation of Multiple Cells Using Catalytic Microbots. Chem. Commun. 2011, 47, 698– 700, DOI: 10.1039/C0CC04126B[Crossref], [PubMed], [CAS], Google Scholar60https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhsFGrtb3J&md5=eb971f1b9123eb8c433fe70e6328fcedControlled manipulation of multiple cells using catalytic microbotsSanchez, Samuel; Solovev, Alexander A.; Schulze, Sabine; Schmidt, Oliver G.Chemical Communications (Cambridge, United Kingdom) (2011), 47 (2), 698-700CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)Self-propelled microjet engines (microbots) can transport multiple cells into specific locations in a fluid. The motion is externally controlled by a magnetic field which allows to selectively load, transport and deliver the cells.
- 61Son, S. J.; Reichel, J.; He, B.; Schuchman, M.; Lee, S. B. Magnetic Nanotubes for Magnetic-Field-Assisted Bioseparation, Biointeraction, and Drug Delivery. J. Am. Chem. Soc. 2005, 127, 7316– 7317, DOI: 10.1021/ja0517365[ACS Full Text], [CAS], Google Scholar61https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXjslKqt78%253D&md5=3443abb4a5a2c85c3c1d299663bc3589Magnetic nanotubes for magnetic-field-assisted bioseparation, biointeraction, and drug deliverySon, Sang Jun; Reichel, Jonathan; He, Bo; Schuchman, Mattan; Lee, Sang BokJournal of the American Chemical Society (2005), 127 (20), 7316-7317CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Tubular structure of nanoparticles is highly attractive due to their structural attributes, such as the distinctive inner and outer surfaces, over conventional spherical nanoparticles. Inner voids can be used for capturing, concg., and releasing species ranging in size from large proteins to small mols. Distinctive outer surfaces can be differentially functionalized with environment-friendly and/or probe mols. to a specific target. Magnetic particles have been extensively studied in the field of biomedical and biotechnol. applications, including drug delivery, biosensors, chem. and biochem. sepn. and concn. of trace amts. of specific targets, and contrast enhancement in magnetic resonance imaging (MRI). Therefore, by combining the attractive tubular structure with magnetic property, the magnetic nanotube (MNT) can be an ideal candidate for the multifunctional nanomaterial toward biomedical applications, such as targeting drug delivery with MRI capability. Here, we successfully synthesized magnetic silica-iron oxide composite nanotubes and demonstrated the magnetic-field-assisted chem. and biochem. sepns., immunobinding, and drug delivery.
- 62Qin, D.; Xia, Y.; Whitesides, G. M. Soft Lithography for Micro- and Nanoscale Patterning. Nat. Protoc. 2010, 5, 491– 502, DOI: 10.1038/nprot.2009.234[Crossref], [PubMed], [CAS], Google Scholar62https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXislOmsbs%253D&md5=ddc10ed2bb9b84f4799c8ec3b59a473eSoft lithography for micro- and nanoscale patterningQin, Dong; Xia, Younan; Whitesides, George M.Nature Protocols (2010), 5 (3), 491-502CODEN: NPARDW; ISSN:1750-2799. (Nature Publishing Group)This protocol provides an introduction to soft lithog.-a collection of techniques based on printing, molding and embossing with an elastomeric stamp. Soft lithog. provides access to three-dimensional and curved structures, tolerates a wide variety of materials, generates well-defined and controllable surface chemistries, and is generally compatible with biol. applications. It is also low in cost, exptl. convenient and has emerged as a technol. useful for a no. of applications that include cell biol., microfluidics, lab-on-a-chip, microelectromech. systems and flexible electronics/photonics. As examples, here we focus on three of the commonly used soft lithog. techniques: (i) microcontact printing of alkanethiols and proteins on gold-coated and glass substrates; (ii) replica molding for fabrication of microfluidic devices in poly(di-Me siloxane), and of nanostructures in polyurethane or epoxy; and (iii) solvent-assisted micromolding of nanostructures in poly(Me methacrylate).
- 63Rotem, A.; Abate, A. R.; Utada, A. S.; Van Steijn, V.; Weitz, D. A. Drop Formation in Non-Planar Microfluidic Devices. Lab Chip 2012, 12, 4263– 4268, DOI: 10.1039/c2lc40546f[Crossref], [PubMed], [CAS], Google Scholar63https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhsVCqu7vM&md5=a8ad19fd65e0af7f14fd6eb13566204dDrop formation in non-planar microfluidic devicesRotem, Assaf; Abate, Adam R.; Utada, Andrew S.; Van Steijn, Volkert; Weitz, David A.Lab on a Chip (2012), 12 (21), 4263-4268CODEN: LCAHAM; ISSN:1473-0189. (Royal Society of Chemistry)Microfluidic devices can be used to produce single or multiple emulsions with remarkably precise control of both the contents and size of the drops. Since each level of a multiple emulsion is formed by a distinct fluid stream, very efficient encapsulation of materials can be achieved. To obtain high throughput, these devices can be fabricated lithog., allowing many devices to operate in parallel. However, to form multiple emulsions using a planar microfluidic device, the wettability of its surface must switch from hydrophobic to hydrophilic on the scale of micrometers where the drops are formed; this makes the fabrication of the devices very difficult. To overcome this constraint, non-planar microfluidic devices are introduced with graduated thicknesses; these can make drops even when their wetting properties do not favor drop formation. Nevertheless, the dependence of drop formation on the device geometry, the flow rates, and the properties of the fluids, particularly in the case of unfavorable wetting, is very complex, making the successful design of these devices more difficult. Here it is shown that there exists a crit. value of flow of the continuous phase above which drop formation occurs; this value decreases by two orders of magnitude as the wetting to the device wall of the continuous phase improves. How this new understanding can be used to optimize device design is demonstrated for efficient prodn. of double or multiple emulsions.
- 64Strathmann, M.; Wingender, J.; Flemming, H.-C. Application of Fluorescently Labelled Lectins for the Visualization and Biochemical Characterization of Polysaccharides in Biofilms of Pseudomonas aeruginosa. J. Microbiol. Methods 2002, 50, 237– 248, DOI: 10.1016/S0167-7012(02)00032-5[Crossref], [PubMed], [CAS], Google Scholar64https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XjvVejtrs%253D&md5=e3b42845df9f2eb4805b8bcc44eedd6dApplication of fluorescently labelled lectins for the visualization and biochemical characterization of polysaccharides in biofilms of Pseudomonas aeruginosaStrathmann, Martin; Wingender, Jost; Flemming, Hans-CurtJournal of Microbiological Methods (2002), 50 (3), 237-248CODEN: JMIMDQ; ISSN:0167-7012. (Elsevier Science B.V.)Fluorescently labeled lectins were used in combination with epifluorescence microscopy and confocal laser scanning microscopy to allow the visualization and characterization of carbohydrate-contg. extracellular polymeric substances (EPS) in biofilms of Pseudomonas aeruginosa. A mucoid strain characterized by an overprodn. of the exopolysaccharide alginate, and an isogenic, non-mucoid strain were used. Model biofilms grown on polycarbonate filters were treated with lectins Con A (ConA) and wheat germ agglutinin (WGA) that were fluorescently labeled with fluorescein isothiocyanate or tetra-Me rhodamine isothiocyanate. Fluorescently labeled ConA yielded cloud-like regions that were heterogeneously distributed within mucoid biofilms, whereas these structures were only rarely present in biofilms of the non-mucoid strain. The bacteria visualized with the fluorochrome SYTO 9 were localized both within and between the ConA-stained regions. In WGA-treated biofilms, the lectin was predominantly assocd. with bacterial cells. Alginate seemed to be involved in the interaction of ConA with the EPS matrix, since (i) pre-treatment of biofilms with an alginate lyase resulted in a loss of ConA biofilm staining, and (ii) using an enzyme-linked lectinsorbent assay (ELLA), ConA was shown to bind to purified alginate, but not to alginate that was degraded by alginate lyase. The application of fluorescently labeled lectins in combination with ELLA was found to be useful for the visualization and characterization of extracellular polysaccharide structures in P. aeruginosa biofilms.
- 65Toda, N.; Doi, A.; Jimbo, A.; Matsumoto, I.; Seno, N. Interaction of Sulfated Glycosaminoglycans with Lectins. J. Biol. Chem. 1981, 256, 5345– 5349[PubMed], [CAS], Google Scholar65https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL3MXkt1aksbw%253D&md5=25de5ab7d967117035a0f6edba455bbeInteraction of sulfated glycosaminoglycans with lectinsToda, Noriko; Doi, Akiko; Jimbo, Akiko; Matsumoto, Isamu; Seno, NobukoJournal of Biological Chemistry (1981), 256 (11), 5345-9CODEN: JBCHA3; ISSN:0021-9258.The sulfated glycosaminoglycans, such as keratan sulfate and chitin sulfate having 3-hydroxyl free N-acetyl-β-D-glucosaminyl residues as constituents, reacted with wheat germ agglutinin and Solanum tuberosum agglutinin by sugar-specific interaction. The glycosaminoglycans showed different inhibitory activities to the hemagglutination reaction of these lectins and keratan sulfate and its modified products formed insol. complexes with both of the lectins at pH 7.0 in physiol. saline soln. (0.15M NaCl). S. tuberosum Agglutinin was pptd. within a particularly narrow concn. range of keratan sulfate, and the formation of a sol. complex was obsd. by gel chromatog. These interactions were specifically inhibited by N,N'-diacetylchitobiose but not by 2M NaCl. The specific interactions of the glycosaminoglycans with S. tuberosum agglutinin were confirmed by their UV difference spectra with 2 peaks at 285 and 293 nm attributable to the tryptophan residues in the binding site of the agglutinin. S. tuberosum Agglutinin and wheat germ agglutinin have different binding specificities. The presence of sulfate groups in either keratan sulfate or chitin sulfate did not interfere with their specific interactions with S. tuberosum agglutinin as strongly as with wheat germ agglutinin. The N-acetylneuraminic acid residues in keratan sulfate were receptor sites for wheat germ agglutinin but not for S. tuberosum agglutinin.
- 66Takeda, M.; Makita, H.; Ohno, K.; Nakahara, Y.; Koizumi, J.-i. Structural Analysis of the Sheath of a Sheathed Bacterium, Leptothrix cholodnii. Int. J. Biol. Macromol. 2005, 37, 92– 98, DOI: 10.1016/j.ijbiomac.2005.09.002[Crossref], [PubMed], [CAS], Google Scholar66https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXhtFKnsbvP&md5=8e6b7d175ea42bf3bb5022cca65551e3Structural analysis of the sheath of a sheathed bacterium, Leptothrix cholodniiTakeda, Minoru; Makita, Hiroko; Ohno, Katsutoshi; Nakahara, Yuichi; Koizumi, Jun-ichiInternational Journal of Biological Macromolecules (2005), 37 (1-2), 92-98CODEN: IJBMDR; ISSN:0141-8130. (Elsevier B.V.)L. cholodnii is an aerobic sheath-forming bacterium often found in oligotrophic and metal-rich aquatic environments. The sheath of this bacterium was isolated by selectively lysing the cells. Glycine and cysteine were the major amino acids of the sheath. The sheath was readily dissolved in hydrazine, and a polysaccharide substituted with cysteine was recovered from the soln. Galactosamine, glucosamine and galacturonic acid were detected in the hydrazinolyzate by gas liq. chromatog. anal. FAB-MS anal. of the hydrazinolyzate suggested a sugar sequence of HexN-GalA-HexN-HexN. Methylation linkage anal. revealed the presence of 4-linked GalA, 3-linked HexN and 4-linked HexN. The sulfhydryl groups of the sheath were used for labeling with the fluorogenic reagent 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (ABD-F). The labeled sheath (ABD-sheath) was partially hydrolyzed and 3 fluorescent fragments were purified by HPLC. One of them was identified as ABD-cysteine. The 2nd was the ABD-cysteine tetramer. Another fragment was indicated to be a pentasaccharide substituted with ABD-cysteine by NMR anal. It can be assumed that the polysaccharide and peptide moieties of the sheath are connected by a cysteine residue. NMR anal. of the hydrazinolyzate revealed that the polysaccharide moiety of the sheath was constructed from a pentasaccharide repeating unit contg. 2-amino-2-deoxygalacturonic acid (GalNA): →4)-α-GalNA-(1→4)-α-D-GalN(p)-(1→4)-α-D-GalA(p)-(1→4)-β-D-GlcN(p)-(1→3)-β-D-GalN(p)-(1→.
- 67Sugimoto, S.; Okuda, K.; Miyakawa, R.; Sato, M.; Arita-Morioka, K.; Chiba, A.; Yamanaka, K.; Ogura, T.; Mizunoe, Y.; Sato, C. Imaging of Bacterial Multicellular Behaviour in Biofilms in Liquid by Atmospheric Scanning Electron Microscopy. Sci. Rep. 2016, 6, 25889, DOI: 10.1038/srep25889[Crossref], [PubMed], [CAS], Google Scholar67https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XotFOiu7s%253D&md5=a8394ff0a1cdec458a2e84413c140fceImaging of bacterial multicellular behaviour in biofilms in liquid by atmospheric scanning electron microscopySugimoto, Shinya; Okuda, Ken-ichi; Miyakawa, Reina; Sato, Mari; Arita-Morioka, Ken-ichi; Chiba, Akio; Yamanaka, Kunitoshi; Ogura, Teru; Mizunoe, Yoshimitsu; Sato, ChikaraScientific Reports (2016), 6 (), 25889CODEN: SRCEC3; ISSN:2045-2322. (Nature Publishing Group)Biofilms are complex communities of microbes that attach to biotic or abiotic surfaces causing chronic infectious diseases. Within a biofilm, microbes are embedded in a self-produced soft extracellular matrix (ECM), which protects them from the host immune system and antibiotics. The nanoscale visualisation of delicate biofilms in liq. is challenging. Here, we develop atm. SEM (ASEM) to visualise Gram-pos. and -neg. bacterial biofilms immersed in aq. soln. Biofilms cultured on electron-transparent film were directly imaged from below using the inverted SEM, allowing the formation of the region near the substrate to be studied at high resoln. We visualised intercellular nanostructures and the exocytosis of membrane vesicles, and linked the latter to the trafficking of cargos, including cytoplasmic proteins and the toxins hemolysin and coagulase. A thick dendritic nanotube network was obsd. between microbes, suggesting multicellular communication in biofilms. A universal immuno-labeling system was developed for biofilms and tested on various examples, including S. aureus biofilms. In the ECM, fine DNA and protein networks were visualised and the precise distribution of protein complexes was detd. (e.g., straight curli, flagella, and excreted cytoplasmic mol. chaperones). Our observations provide structural insights into bacteria-substratum interactions, biofilm development and the internal microbe community.
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Abstract
Figure 1
Figure 1. Time-lapse sequence showing cell growth in 2D chambers for cells exhibiting (A) unilateral elongation and (B) bilateral elongation. (C) Time-lapse sequence of a variant strain (SP-6 SL) unable to produce a sheath. Black arrowheads in (A,B) indicate the same spatial position in each image, the unfilled arrowheads indicate the position of the moving pole(s), and red arrows indicate the direction of elongation. Scale bars = 5 μm. The numbers refer to the time in minutes. (D) Filament length of SP-6 (●) and SP-6 SL (□) strains measured as a function of time within 2D chambers. The error bars represent the standard deviation (SD) taken from 10 filaments. The dashed exponential line is a best fit to the data (see Supporting Material and Methods S1.3 section).
Figure 2
Figure 2. Cellular distribution of nanofibrils. (A) Bright-field and fluorescence time-lapse sequence with stop-flow fluorescent labeling of the nanofibrils during that gap between 200 and 215 min. The upper cell demonstrates unilateral elongation, while the lower cell demonstrates bilateral elongation. Black arrowheads indicate the same spatial position in each image, red arrows indicate the direction of elongation, the yellow circle encloses cells that attach in the chamber midway through the imaging sequence, and the unfilled arrowheads indicate the position(s) of the moving pole(s). The numbers refer to the time in minutes. (B) ASEM images of SP-6 (left) and SP-6 SL (right) cells. The white arrow (left) indicates the nanofibrils. Scale bars = 5 μm.
Figure 3
Figure 3. Filament bending and reversal. Time-lapse image sequence showing impact-induced filament (A) bending (at t = 380 min) and (B) reversal (at t = 255 min). The colored cells in (A,B) are overlays of the filament at different time points (labeled). (C) Model of the forces generated during a collision between a filament and obstacle as a function of the angle of impact, θ. The blue circles (●) represent fbend, the unfilled circles (○) represent the bending stiffness, and the blue shaded area indicates 30% variability in this value. The red triangles (▼) represent freverse, the unfilled triangles (▽) represent the nanofibril adhesion force, Fadh, and the red shaded area indicates 20% variability. (Inset) Schematic showing the collision between an elongating filament and an obstruction. (D) Binned probability densities of bending and reversing as a function of θ. The sum of the respective densities for bending and reversal equal the total, shown by the thick black line outlining the bars. The dashed (blue) and dotted (red) lines are simulated probability densities for bending and reversal, respectively, based on the model in panel C (see SI S1.4). (E,F) A time-lapse sequence combined with stop-flow fluorescence labeling of nanofibrils showing impact-induced bending and reversal, respectively. For (A,B) t = 0 defines the moment of cell-surface attachment, while in (E,F) it refers to start of the experiment upon completion of the nanofibril labeling. In all time-lapse sequences, the black arrowheads indicate the same spatial position in each image, the red arrows indicate the direction of elongation, unfilled arrowheads indicate the position of the moving pole(s), and the blue arrowheads indicate the location of a collision that causes reversal. The numbers refer to the time in minutes. Scale bars = 5 μm.
Figure 4
Figure 4. Intercellular gaps within filaments. (A) Time-lapse sequence showing the appearance and development of a large intercellular gap, followed by its disappearance during filament elongation. The black arrowheads indicate the same spatial position in each image, red arrows indicate direction of elongation, yellow lines and arrows indicate location and width of the gap between adjacent cells, the unfilled arrowheads indicate the position of the moving pole(s), and blue arrowheads indicate collision with an obstruction. The numbers refer to the time in minutes. (B) ASEM images showing the different stages of the immature sheath formation. White arrows indicate (left) diffuse nanofibrils on a young filament, (middle) a tighter distribution on a longer filament, and (right) the outline of the sheath on an even longer filament. Scale bars = 5 μm.
Figure 5
Figure 5. Filament fragmentation and development of the sheath. (A) Escape of cells from the leading edge of the filament. The yellow arrowheads indicate the positions where filament fragmentation occurs, whereas the yellow arrows indicate the positions of the cells, after escaping from the filament. (B) Time-lapse sequence with stop-flow fluorescence labeling of the nanofibrils at two time-points during 265–280 (first) and 1495–1510 min (second), respectively. (Insets) Magnified, background-subtracted images showing the distribution of fluorescent nanofibrils on the cell filament. For (A,B) the black arrowheads indicate the same spatial position in each image, red arrows indicate the direction of elongation, blue arrowheads indicate the location of impact that causes reversal, unfilled arrowheads indicate the position of the moving pole(s), and blue arrowheads indicate collision with an obstruction. (C) (left) Fluorescence intensity profile measured along the axis of a representative filament. The width is defined as the peak-to-peak distance between intensity maxima. (right) Histogram of intensity-profile widths measured at different axial positions normalized by the width of the filament at its origin. For filaments shorter than 20 μm in length, the width is measured at the distal end and normalized by the width at the origin (unfilled). For filaments ≥20 μm in length, the widths are measured 20 μm from the origin of filament growth (filled, striped) and at the distal end of the filament (filled), then normalized by the width measured at its origin. The error bars represent one SD measured from 6 to 9 filaments. The stars represent statistical significance: (*) represents p < 0.01 and (**) represents p < 0.02. Scale bars = 5 μm.
References
ARTICLE SECTIONSThis article references 67 other publications.
- 1Takeda, M.; Kawasaki, Y.; Umezu, T.; Shimura, S.; Hasegawa, M.; Koizumi, J. Patterns of Sheath Elongation, Cell Proliferation, and Manganese(II) Oxidation in Leptothrix cholodnii. Arch. Microbiol. 2012, 194, 667– 673, DOI: 10.1007/s00203-012-0801-6[Crossref], [PubMed], [CAS], Google Scholar1https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhtVCmurjP&md5=ebf71cd8b937dd1b5b58a8c1cb6194d8Patterns of sheath elongation, cell proliferation, and manganese(II) oxidation in Leptothrix cholodniiTakeda, Minoru; Kawasaki, Yuta; Umezu, Takuto; Shimura, Shoichi; Hasegawa, Makoto; Koizumi, Jun-ichiArchives of Microbiology (2012), 194 (8), 667-673CODEN: AMICCW; ISSN:0302-8933. (Springer)Leptothrix cholodnii is a Mn(II)-oxidizing and sheath-forming member of the class β-Proteobacteria. Its sheath is a microtube-like filament that contains a chain of cells. From a chem. perspective, the sheath can be described as a supermol. composed of a cysteine-rich polymeric glycoconjugate, called thiopeptidoglycan. However, the mechanism that controls the increase in sheath length is unknown. In this study, the authors attempted to detect sheath elongation through microscopic examn. by using conventional reagents. Selective fluorescent labeling of preexisting or newly formed regions of the sheath was accomplished using combinations of biotin-conjugated maleimide, propionate-conjugated maleimide, and a fluorescent antibiotin antibody. Epifluorescence microscopy indicated that the sheath elongates at the terminal regions. On the bases of this observation, the authors assumed that the newly secreted thiopeptidoglycan mols. are integrated into the preexisting sheath at its terminal ends. Successive phase-contrast microscopy revealed that all cells proliferate at nearly the same rate regardless of their positions within the sheath. Mn(II) oxidn. in microcultures was also examd. with respect to cultivation time. The results suggested that the deposition of Mn oxides is notable in the aged regions. The combined data reveal the spatiotemporal relationships among sheath elongation, cell proliferation, and Mn oxide deposition in L. cholodnii.
- 2Chan, C. S.; McAllister, S. M.; Leavitt, A. H.; Glazer, B. T.; Krepski, S. T.; Emerson, D. The Architecture of Iron Microbial Mats Reflects the Adaptation of Chemolithotrophic Iron Oxidation in Freshwater and Marine Environments. Front. Microbiol. 2016, 7, 796, DOI: 10.3389/fmicb.2016.00796[Crossref], [PubMed], [CAS], Google Scholar2https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC2s7ht1aquw%253D%253D&md5=73069fbaa4cbfb64322610eb908745daThe Architecture of Iron Microbial Mats Reflects the Adaptation of Chemolithotrophic Iron Oxidation in Freshwater and Marine EnvironmentsChan Clara S; McAllister Sean M; Leavitt Anna H; Emerson David; Glazer Brian T; Krepski Sean TFrontiers in microbiology (2016), 7 (), 796 ISSN:1664-302X.Microbes form mats with architectures that promote efficient metabolism within a particular physicochemical environment, thus studying mat structure helps us understand ecophysiology. Despite much research on chemolithotrophic Fe-oxidizing bacteria, Fe mat architecture has not been visualized because these delicate structures are easily disrupted. There are striking similarities between the biominerals that comprise freshwater and marine Fe mats, made by Beta- and Zetaproteobacteria, respectively. If these biominerals are assembled into mat structures with similar functional morphology, this would suggest that mat architecture is adapted to serve roles specific to Fe oxidation. To evaluate this, we combined light, confocal, and scanning electron microscopy of intact Fe microbial mats with experiments on sheath formation in culture, in order to understand mat developmental history and subsequently evaluate the connection between Fe oxidation and mat morphology. We sampled a freshwater sheath mat from Maine and marine stalk and sheath mats from Loihi Seamount hydrothermal vents, Hawaii. Mat morphology correlated to niche: stalks formed in steeper O2 gradients while sheaths were associated with low to undetectable O2 gradients. Fe-biomineralized filaments, twisted stalks or hollow sheaths, formed the highly porous framework of each mat. The mat-formers are keystone species, with nascent marine stalk-rich mats comprised of novel and uncommon Zetaproteobacteria. For all mats, filaments were locally highly parallel with similar morphologies, indicating that cells were synchronously tracking a chemical or physical cue. In the freshwater mat, cells inhabited sheath ends at the growing edge of the mat. Correspondingly, time lapse culture imaging showed that sheaths are made like stalks, with cells rapidly leaving behind an Fe oxide filament. The distinctive architecture common to all observed Fe mats appears to serve specific functions related to chemolithotrophic Fe oxidation, including (1) removing Fe oxyhydroxide waste without entombing cells or clogging flow paths through the mat and (2) colonizing niches where Fe(II) and O2 overlap. This work improves our understanding of Fe mat developmental history and how mat morphology links to metabolism. We can use these results to interpret biogenicity, metabolism, and paleoenvironmental conditions of Fe microfossil mats, which would give us insight into Earth's Fe and O2 history.
- 3Hornlein, C.; Confurius-Guns, V.; Stal, L. J.; Bolhuis, H. Daily Rhythmicity in Coastal Microbial Mats. npj Biofilms Microbiomes 2018, 4, 11, DOI: 10.1038/s41522-018-0054-5[Crossref], [PubMed], [CAS], Google Scholar3https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC1MfmslamsA%253D%253D&md5=ff9e1e38d559b604e15279af86ff97ccDaily rhythmicity in coastal microbial matsHornlein Christine; Confurius-Guns Veronique; Stal Lucas J; Bolhuis Henk; Stal Lucas JNPJ biofilms and microbiomes (2018), 4 (), 11 ISSN:2055-5008.Cyanobacteria are major primary producers in coastal microbial mats and provide biochemical energy, organic carbon, and bound nitrogen to the mat community through oxygenic photosynthesis and dinitrogen fixation. In order to anticipate the specific requirements to optimize their metabolism and growth during a day-and-night cycle, Cyanobacteria possess a unique molecular timing mechanism known as the circadian clock that is well-studied under laboratory conditions but little is known about its function in a natural complex community. Here, we investigated daily rhythmicity of gene expression in a coastal microbial mat community sampled at 6 time points during a 24-h period. In order to identify diel expressed genes, meta-transcriptome data was fitted to periodic functions. Out of 24,035 conserved gene transcript clusters, approximately 7% revealed a significant rhythmic expression pattern. These rhythmic genes were assigned to phototrophic micro-eukaryotes, Cyanobacteria but also to Proteobacteria and Bacteroidetes. Analysis of MG-RAST annotated genes and mRNA recruitment analysis of two cyanobacterial and three proteobacterial microbial mat members confirmed that homologs of the cyanobacterial circadian clock genes were also found in other bacterial members of the microbial mat community. These results suggest that various microbial mat members other than Cyanobacteria have their own molecular clock, which can be entrained by a cocktail of Zeitgebers such as light, temperature or metabolites from neighboring species. Hence, microbial mats can be compared to a complex organism consisting of multiple sub-systems that have to be entrained in a cooperative way such that the corpus functions optimally.
- 4Spring, S., The Genera Leptothrix and Sphaerotilus. In Prokaryotes: A Handbook on the Biology of Bacteria, 3rd ed.; Rosenberg, E., DeLong, E. F., Lory, S., Stackebrandt, E., Thompson, F., Eds.; Springer-Verlag: Berlin Heidelberg, 2006; Vol. 5, pp 758– 777.
- 5Emerson, D.; Fleming, E. J.; McBeth, J. M. Iron-Oxidizing Bacteria: An Environmental and Genomic Perspective. Annu. Rev. Microbiol. 2010, 64, 561– 583, DOI: 10.1146/annurev.micro.112408.134208[Crossref], [PubMed], [CAS], Google Scholar5https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhsVeisL3E&md5=0a8e70640fc422838233db5728ec68edIron-oxidizing bacteria: An environmental and genomic perspectiveEmerson, David; Fleming, Emily J.; McBeth, Joyce M.Annual Review of Microbiology (2010), 64 (), 561-583CODEN: ARMIAZ; ISSN:0066-4227. (Annual Reviews Inc.)A review. In the 1830s, iron bacteria were among the first groups of microbes to be recognized for carrying out a fundamental geol. process, namely the oxidn. of iron. Due to lingering questions about their metab., coupled with difficulties in culturing important community members, studies of Fe-oxidizing bacteria (FeOB) have lagged behind those of other important microbial lithotrophic metabs. Research on lithotrophic, oxygen-dependent FeOB that grow at circumneutral pH has accelerated. This work is driven by several factors including the recognition by both microbiologists and geoscientists of the role FeOB play in the biogeochem. of iron and other elements. The isolation of new strains of obligate FeOB allowed a better understanding of their physiol. and phylogeny and the realization that FeOB are abundant at certain deep-sea hydrothermal vents. These ancient microorganisms offer new opportunities to learn about fundamental biol. processes that can be of practical importance.
- 6Krumbein, W. E.; Brehm, U.; Gerdes, G.; Gorbushina, A. A.; Levit, G.; Palinska, K. A., Biofilm, Biodictyon, Biomat Microbialites, Oolites, Stromatolites Geophysiology, Global Mechanism, Parahistology. In Fossil and Recent Biofilms; Krumbein, W. E., Paterson, D. M., Zavarzin, G. A., Eds.; Springer: Dordrecht, 2003; pp 1– 27.
- 7Emerson, D.; Ghiorse, W. C. Isolation, Cultural Maintenance, and Taxonomy of a Sheath-Forming Strain of Leptothrix discophora and Characterization of Manganese-Oxidizing Activity Associated with the Sheath. Appl. Environ. Microbiol. 1992, 58, 4001– 4010[Crossref], [PubMed], [CAS], Google Scholar7https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3sXlsFyguw%253D%253D&md5=be464a22864a00fbe2b96a8722108091Isolation, cultural maintenance, and taxonomy of a sheath-forming strain of Leptothrix dischophora and characterization of manganese-oxidizing activity associated with the sheathEmerson, David; Ghiorse, William C.Applied and Environmental Microbiology (1992), 58 (12), 4001-10CODEN: AEMIDF; ISSN:0099-2240.Leptothrix discophora SP-6 was isolated from the outflow reservoir of an artificial iron seep. Its sheath-forming phenotype was maintained by slow growth in a mineral salts-vitamin-pyruvate medium under minimal aeration at 20 to 25°. A sheathless variant, SP-6(sl), was isolated from smooth colonies that appeared on spread plates after rapid growth of SP-6 in well-aerated cultures. SP-6 and SP-6(sl) are closely related but not identical to the previously studied sheathless strain SS-1 (ATCC 43182). Increasing Mn2+ concns. in the growth medium of SP-6 increased the phase d. of the sheath indicating increased Mn oxide deposition in the sheath. Electron microscopy of cultures grown without added Mn2+ revealed that the sheath consisted of a well-defined inner layer, 30 to 100 nm thick, and a diffuse outer capsular layer of variable thickness. Mn oxides were identified in the sheath by their characteristic ultrastructure, electron d., and x-ray-dispersive energy spectra. In heavily encrusted sheaths, the Mn oxides were evenly distributed in both layers of the sheath. Sheathed cells retained more Mn-oxidizing activity than did sheathless cells after washing with distd., deionized water; the sheath retained some of its activity after an EDTA-lysozyme-detergent treatment which removed the cells. An ultrafiltration-dialysis procedure significantly increased the recovery of activity from spent media of SP-6 over that reported previously for SS-1 (L. F. Adams and W. C. Ghiorse, 1987). A 108-kDa Mn-oxidizing protein was identified in concd. spent media of SP-6 and SP-6(sl), and the activity of the concs. showed stability in detergents comparable to that of SS-1 and patterns of heat inactivation and chem. inhibition similar to those of SS-1.
- 8Vesenka, J.; Havu, J.; Hruby, K.; Emerson, D. A Model for Sheath Formation Coupled to Motility in Leptothrix ochracea. Geomicrobiol. J. 2018, 35, 366– 374, DOI: 10.1080/01490451.2017.1370516
- 9Corstjens, P. L.; de Vrind, J. P.; Westbroek, P.; de Vrind-de Jong, E. W. Enzymatic Iron Oxidation by Leptothrix discophora: Identification of an Iron-Oxidizing Protein. Appl. Environ. Microbiol. 1992, 58, 450– 454[Crossref], [PubMed], [CAS], Google Scholar9https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK38XhtVantrw%253D&md5=cbd2f65d67606c9fb914343a752d918aEnzymic iron oxidation by Leptothrix discophora: identification of an iron-oxidizing proteinCorstjens, P. L. A. M.; De Vrind, J. P. M.; Westbroek, P.; De Vrind-De Jong, E. W.Applied and Environmental Microbiology (1992), 58 (2), 450-4CODEN: AEMIDF; ISSN:0099-2240.An iron-oxidizing factor was identified in the spent culture medium of the Fe- and Mn-oxidizing bacterial strain L. discophora SS-1. It appeared to be a protein, with an apparent mol. wt. of ∼150,000. Its activity could be demonstrated after fractionation of the spent medium by SDS-PAGE. A spontaneous mutant of L. discophora SS-1 was isolated which excreted neither Mn- nor Fe-oxidizing activity, whereas excretion of other proteins seemed to be unaffected. Although the excretion of both metal-oxidizing factors was probably linked, the difference in other properties suggests that Mn and Fe oxidn. represent 2 different pathways. With a dot-blot assay, it was established that different bacterial species have different metal-oxidizing capacities. Whereas L. discophora oxidized both Fe and Mn, Sphaerotilus natans oxidized only Fe and 2 Pseudomonas spp. oxidized only Mn.
- 10Van Veen, W.; Mulder, E.; Deinema, M. H. The Sphaerotilus-Leptothrix Group of Bacteria. Microbiol. Rev. 1978, 42, 329– 356[Crossref], [PubMed], [CAS], Google Scholar10https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaE1cXkslaktLg%253D&md5=6630df28b23eca3a8c6d3b632575b1fbThe Sphaerotilus-Leptothrix group of bacteriaVan Veen, W. L.; Mulder, E. G.; Deinema, Maria H.Microbiological Reviews (1978), 42 (2), 329-56CODEN: MBRED3; ISSN:0146-0749.A review with 111 refs.
- 11Hashimoto, H.; Kobayashi, G.; Sakuma, R.; Fujii, T.; Hayashi, N.; Suzuki, T.; Kanno, R.; Takano, M.; Takada, J. Bacterial Nanometric Amorphous Fe-Based Oxide: A Potential Lithium-Ion Battery Anode Material. ACS Appl. Mater. Interfaces 2014, 6, 5374– 5378, DOI: 10.1021/am500905y[ACS Full Text], [CAS], Google Scholar11https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXltlymsbk%253D&md5=b4a29ef79208bd79a741475375aa7188Bacterial nanometric amorphous Fe-based oxide: a potential lithium-ion battery anode materialHashimoto, Hideki; Kobayashi, Genki; Sakuma, Ryo; Fujii, Tatsuo; Hayashi, Naoaki; Suzuki, Tomoko; Kanno, Ryoji; Takano, Mikio; Takada, JunACS Applied Materials & Interfaces (2014), 6 (8), 5374-5378CODEN: AAMICK; ISSN:1944-8244. (American Chemical Society)Amorphous Fe3+-based oxide nanoparticles produced by Leptothrix ochracea, aquatic bacteria living worldwide, show a potential as an Fe3+/Fe0 conversion anode material for lithium-ion batteries. The presence of minor components, Si and P, in the original nanoparticles leads to a specific electrode architecture with Fe-based electrochem. centers embedded in a Si, P-based amorphous matrix.
- 12Lovley, D. R. Happy Together: Microbial Communities that Hook Up to Swap Electrons. ISME J. 2017, 11, 327– 336, DOI: 10.1038/ismej.2016.136[Crossref], [PubMed], [CAS], Google Scholar12https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhsFGlsLg%253D&md5=c86e142d350ba8648a5cfa9581909deeHappy together: microbial communities that hook up to swap electronsLovley, Derek R.ISME Journal (2017), 11 (2), 327-336CODEN: IJSOCF; ISSN:1751-7362. (Nature Publishing Group)A review. The discovery of direct interspecies electron transfer (DIET) and cable bacteria has demonstrated that microbial cells can exchange electrons over long distances (μm-cm) through elec. connections. For example, in the presence of cable bacteria electrons are rapidly transported over centimeter distances, coupling the oxidn. of reduced sulfur compds. in anoxic sediments to oxygen redn. in overlying surficial sediments. Bacteria and archaea wired for DIET are found in anaerobic methane-producing and methane-consuming communities. Elec. connections between gut microbes and host cells have also been proposed. Iterative environmental and defined culture studies on methanogenic communities revealed the importance of elec. conductive pili and c-type cytochromes in natural elec. grids, and demonstrated that conductive carbon materials and magnetite can substitute for these biol. connectors to facilitate DIET. This understanding has led to strategies to enhance and stabilize anaerobic digestion. Key unknowns warranting further investigation include elucidation of the archaeal elec. connections facilitating DIET-based methane prodn. and consumption; and the mechanisms for long-range electron transfer through cable bacteria. A better understanding of mechanisms for cell-to-cell electron transfer could facilitate the hunt for addnl. elec. connected microbial communities with omics approaches and could advance spin-off applications such as the development of sustainable bioelectronics materials and bioelectrochem. technologies.
- 13Nielsen, L. P.; Risgaard-Petersen, N.; Fossing, H.; Christensen, P. B.; Sayama, M. Electric Currents Couple Spatially Separated Biogeochemical Processes in Marine Sediment. Nature 2010, 463, 1071– 1074, DOI: 10.1038/nature08790[Crossref], [PubMed], [CAS], Google Scholar13https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXitl2gsr8%253D&md5=33967b4fb936e441ecc497ff82d9c811Electric currents couple spatially separated biogeochemical processes in marine sedimentNielsen, Lars Peter; Risgaard-Petersen, Nils; Fossing, Henrik; Christensen, Peter Bondo; Sayama, MikioNature (London, United Kingdom) (2010), 463 (7284), 1071-1074CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)Some bacteria are capable of extracellular electron transfer, thereby enabling them to use electron acceptors and donors without direct cell contact. Beyond the micrometer scale, however, no firm evidence has previously existed that spatially segregated biogeochem. processes can be coupled by elec. currents in nature. Based on studies on sediment samples from Aarhus harbor and bay, Denmark, the authors give evidence that elec. currents running through defaunated sediment couple oxygen consumption at the sediment surface to oxidn. of hydrogen sulfide and org. carbon deep within the sediment. Altering the oxygen concn. in the seawater overlying the sediment resulted in a rapid (<1-h) change in the hydrogen sulfide concn. within the sediment more than 12 mm below the oxic zone, a change explicable by transmission of electrons but not by diffusion of mols. Mass balances indicated that more than 40% of total oxygen consumption in the sediment was driven by electrons conducted from the anoxic zone. A distinct pH peak in the oxic zone could be explained by electrochem. oxygen redn., but not by any conventional sets of aerobic sediment processes. The authors suggest that the elec. current was conducted by bacterial nanowires combined with pyrite, sol. electron shuttles and outer-membrane cytochromes. Elec. communication between distant chem. and biol. processes in nature adds a new dimension to understanding of biogeochem. and microbial ecol.
- 14Pfeffer, C.; Larsen, S.; Song, J.; Dong, M.; Besenbacher, F.; Meyer, R. L.; Kjeldsen, K. U.; Schreiber, L.; Gorby, Y. A.; El-Naggar, M. Y.; Leung, K. M.; Schramm, A.; Risgaard-Petersen, N.; Nielsen, L. P. Filamentous Bacteria Transport Electrons Over Centimetre Distances. Nature 2012, 491, 218– 221, DOI: 10.1038/nature11586[Crossref], [PubMed], [CAS], Google Scholar14https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhsFOmt7fN&md5=53f87c4d3da6ef2fbcaa62459f2753e7Filamentous bacteria transport electrons over centimeter distancesPfeffer, Christian; Larsen, Steffen; Song, Jie; Dong, Mingdong; Besenbacher, Flemming; Meyer, Rikke Louise; Kjeldsen, Kasper Urup; Schreiber, Lars; Gorby, Yuri A.; El-Naggar, Mohamed Y.; Leung, Kar Man; Schramm, Andreas; Risgaard-Petersen, Nils; Nielsen, Lars PeterNature (London, United Kingdom) (2012), 491 (7423), 218-221CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)Oxygen consumption in marine sediments is often coupled to the oxidn. of sulfide generated by degrdn. of org. matter in deeper, oxygen-free layers. Geochem. observations have shown that this coupling can be mediated by elec. currents carried by unidentified electron transporters across centimeter-wide zones. Her, the authors present evidence that the native conductors are long, filamentous bacteria. They abounded in sediment zones with elec. currents and along their length they contained strings with distinct properties in accordance with a function as electron transporters. Living, elec. cables add a new dimension to the understanding of interactions in nature and may find use in technol. development.
- 15Chan, C. S.; Fakra, S. C.; Edwards, D. C.; Emerson, D.; Banfield, J. F. Iron Oxyhydroxide Mineralization on Microbial Extracellular Polysaccharides. Geochim. Cosmochim. Acta 2009, 73, 3807– 3818, DOI: 10.1016/j.gca.2009.02.036[Crossref], [CAS], Google Scholar15https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXmvVyktbo%253D&md5=0631525cf1a01cabfed340b2767843a5Iron oxyhydroxide mineralization on microbial extracellular polysaccharidesChan, Clara S.; Fakra, Sirine C.; Edwards, David C.; Emerson, David; Banfield, Jillian F.Geochimica et Cosmochimica Acta (2009), 73 (13), 3807-3818CODEN: GCACAK; ISSN:0016-7037. (Elsevier B.V.)Iron biominerals can form in neutral pH microaerophilic environments where microbes both catalyze iron oxidn. and create polymers that localize mineral pptn. To classify the microbial polymers that influence FeOOH mineralogy, the org. and mineral components of biominerals were studied using scanning transmission X-ray microscopy (STXM), micro X-ray fluorescence (μXRF) microscopy, and high-resoln. transmission electron microscopy (HRTEM). Focus was on iron microbial mat samples from a creek and abandoned mine; these samples are dominated by iron oxyhydroxide-coated structures with sheath, stalk, and filament morphologies. In addn., the mineralized products of an iron-oxidizing, stalk-forming bacterial culture isolated from the mine were characterized. In both natural and cultured samples, microbial polymers were found to be acidic polysaccharides with carboxyl functional groups, strongly spatially correlated with iron oxyhydroxide distribution patterns. Org. fibrils collect FeOOH and control its recrystn., in some cases resulting in oriented crystals with high aspect ratios. The impact of polymers is particularly pronounced as the materials age. Synthesis expts. designed to mimic the biomineralization processes show that the polysaccharide carboxyl groups bind dissolved iron strongly but release it as mineralization proceeds. Results suggest that carboxyl groups of acidic polysaccharides are produced by different microorganisms to create a wide range of iron oxyhydroxide biomineral structures. The intimate and potentially long-term assocn. controls the crystal growth, phase, and reactivity of iron oxyhydroxide nanoparticles in natural systems.
- 16Kunoh, T.; Hashimoto, H.; McFarlane, I. R.; Hayashi, N.; Suzuki, T.; Taketa, E.; Tamura, K.; Takano, M.; El-Naggar, M. Y.; Kunoh, H.; Takada, J. Abiotic Deposition of Fe Complexes onto Leptothrix Sheaths. Biology 2016, 5, 26, DOI: 10.3390/biology5020026[Crossref], [CAS], Google Scholar16https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXmsFSgsr0%253D&md5=e674a73d86b1d9a277bbc21a3068bcddAbiotic deposition of Fe complexes onto Leptothrix sheathsKunoh, Tatsuki; Hashimoto, Hideki; McFarlane, Ian R.; Hayashi, Naoaki; Suzuki, Tomoko; Taketa, Eisuke; Tamura, Katsunori; Takano, Mikio; El-Naggar, Mohamed Y.; Kunoh, Hitoshi; Takada, JunBiology (Basel, Switzerland) (2016), 5 (2), 26-42CODEN: BBSIBX; ISSN:2079-7737. (MDPI AG)Bacteria classified in species of the genus Leptothrix produce extracellular, microtubular, Fe-encrusted sheaths. The encrustation has been previously linked to bacterial Fe oxidases, which oxidize Fe(II) to Fe(III) and/or active groups of bacterial exopolymers within sheaths to attract and bind aq.-phase inorgs. When L. cholodnii SP-6 cells were cultured in media amended with high Fe(II) concns., Fe(III) ppts. visibly formed immediately after addn. of Fe(II) to the medium, suggesting prompt abiotic oxidn. of Fe(II) to Fe(III). Intriguingly, these ppts. were deposited onto the sheath surface of bacterial cells as the population was actively growing. When Fe(III) was added to the medium, similar ppts. formed in the medium first and were abiotically deposited onto the sheath surfaces. The ppts. in the Fe(II) medium were composed of assemblies of globular, amorphous particles (ca. 50 nm diam.), while those in the Fe(III) medium were composed of large, aggregated particles (≤3 μm diam.) with a similar amorphous structure. These ppts. also adhered to cell-free sheaths. We thus concluded that direct abiotic deposition of Fe complexes onto the sheath surface occurs independently of cellular activity in liq. media contg. Fe salts, although it remains unclear how this deposition is assocd. with the previously proposed mechanisms (oxidn. enzyme- and/or active group of org. components-involved) of Fe encrustation of the Leptothrix sheaths.
- 17Kunoh, T.; Hashimoto, H.; Suzuki, T.; Hayashi, N.; Tamura, K.; Takano, M.; Kunoh, H.; Takada, J. Direct Adherence of Fe (III) Particles Onto Sheaths of Leptothrix sp. Strain OUMS1 in Culture. Minerals 2016, 6, 4, DOI: 10.3390/min6010004[Crossref], [CAS], Google Scholar17https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXmt1yjurs%253D&md5=ce8920347e470172e944a7d66fa5fea4Direct adherence of Fe(III) particles onto sheaths of Leptothrix sp. strain OUMS1 in cultureKunoh, Tatsuki; Hashimoto, Hideki; Suzuki, Tomoko; Hayashi, Naoyuki; Tamura, Katsunori; Takano, Mikio; Kunoh, Hitoshi; Takada, JunMinerals (Basel, Switzerland) (2016), 6 (1), 4/1-4/15CODEN: MBSIBI; ISSN:2075-163X. (MDPI AG)Leptothrix species, one of the Fe/Mn-oxidizing bacteria, oxidize Fe(II) and produce extracellular, microtubuar, Fe-encrusted sheaths. Since protein(s) involved in Fe(II) oxidn. is excreted from Leptothrix cells, the oxidn. from Fe(II) to Fe(III) and subsequent Fe(III) deposition to sheaths have been thought to occur in the vicinity or within the sheaths. Previously, Fe(III) particles generated in MSVP medium amended with Fe(II) salts by abiotic oxidn. were directly recruited onto cell-encasing and/or -free sheaths of L. cholodnii SP-6. In this study, whether this direct Fe(III) adherence to sheaths also occurs in silicon-glucose-peptone (SGP) medium amended with Fe(0) (SGP + Fe) was investigated using another strain of Leptothrix sp., OUMS1. Prepn. of SGP + Fe with Fe powder caused turbidity within a few hours due to abiotic generation of Fe(III) particles via Fe(II), and the medium remained turbid until day 8. When OUMS1 was added to SGP + Fe, the turbidity of the medium cleared within 35 h as Fe(III) particles adhered to sheaths. When primitive sheaths, cell-killed, cell-free, or lysozyme/EDTA/SDS- and proteinase K-treated sheath remnants were mixed with Fe(III) particles, the particles immediately adhered to each. Thus, vital activity of cells was not required for the direct Fe(III) particle deposition onto sheaths regardless of Leptothrix strains.
- 18Kunoh, T.; Matsumoto, S.; Nagaoka, N.; Kanashima, S.; Hino, K.; Uchida, T.; Tamura, K.; Kunoh, H.; Takada, J. Amino Group in Leptothrix Sheath Skeleton Is Responsible for Direct Deposition of Fe(III) Minerals onto the Sheaths. Sci. Rep. 2017, 7, 6498, DOI: 10.1038/s41598-017-06644-8[Crossref], [PubMed], [CAS], Google Scholar18https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC1cfhsFelug%253D%253D&md5=3c69b73ea390d9adf54b549afb24d81dAmino group in Leptothrix sheath skeleton is responsible for direct deposition of Fe(III) minerals onto the sheathsKunoh Tatsuki; Matsumoto Syuji; Tamura Katsunori; Kunoh Hitoshi; Takada Jun; Kunoh Tatsuki; Matsumoto Syuji; Uchida Tetsuya; Tamura Katsunori; Kunoh Hitoshi; Takada Jun; Nagaoka Noriyuki; Kanashima Shoko; Hino KatsuhikoScientific reports (2017), 7 (1), 6498 ISSN:.Leptothrix species produce microtubular organic-inorganic materials that encase the bacterial cells. The skeleton of an immature sheath, consisting of organic exopolymer fibrils of bacterial origin, is formed first, then the sheath becomes encrusted with inorganic material. Functional carboxyl groups of polysaccharides in these fibrils are considered to attract and bind metal cations, including Fe(III) and Fe(III)-mineral phases onto the fibrils, but the detailed mechanism remains elusive. Here we show that NH2 of the amino-sugar-enriched exopolymer fibrils is involved in interactions with abiotically generated Fe(III) minerals. NH2-specific staining of L. cholodnii OUMS1 detected a terminal NH2 on its sheath skeleton. Masking NH2 with specific reagents abrogated deposition of Fe(III) minerals onto fibrils. Fe(III) minerals were adsorbed on chitosan and NH2-coated polystyrene beads but not on cellulose and beads coated with an acetamide group. X-ray photoelectron spectroscopy at the N1s edge revealed that the terminal NH2 of OUMS1 sheaths, chitosan and NH2-coated beads binds to Fe(III)-mineral phases, indicating interaction between the Fe(III) minerals and terminal NH2. Thus, the terminal NH2 in the exopolymer fibrils seems critical for Fe encrustation of Leptothrix sheaths. These insights should inform artificial synthesis of highly reactive NH2-rich polymers for use as absorbents, catalysts and so on.
- 19Takeda, M.; Kondo, K.; Yamada, M.; Koizumi, J.; Mashima, T.; Matsugami, A.; Katahira, M. Solubilization and Structural Determination of a Glycoconjugate Which Is Assembled into the Sheath of Leptothrix cholodnii. Int. J. Biol. Macromol. 2010, 46, 206– 211, DOI: 10.1016/j.ijbiomac.2009.12.006[Crossref], [PubMed], [CAS], Google Scholar19https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhs1ygurw%253D&md5=64fecc7ed10706358ed22f2c160f8192Solubilization and structural determination of a glycoconjugate which is assembled into the sheath of Leptothrix cholodniiTakeda, Minoru; Kondo, Keiko; Yamada, Mina; Koizumi, Jun-ichi; Mashima, Tsukasa; Matsugami, Akimasa; Katahira, MasatoInternational Journal of Biological Macromolecules (2010), 46 (2), 206-211CODEN: IJBMDR; ISSN:0141-8130. (Elsevier B.V.)The sheath of Leptothrix cholodnii is constructed from a structural glycoconjugate, a straight-chained amphoteric heteropolysaccharide modified with glycine and cysteine. Though the structure of the glycan core is already detd., its modifications with amino acids and other mols. are not fully resolved. In this study, we aimed to det. the chem. structure of the glycoconjugate as a whole. Enantiomeric detn. of cysteine in the sheath was performed and as a result, -cysteine was detected. NMR spectroscopy was endeavored to det. overall structure of the glycoconjugate. Prior to NMR anal., solubilization of the glycoconjugate was attempted by adding denaturing reagents or by derivatization. As far as tested, sulfonation by performic acid oxidn. was suitable for solubilization, but further improvement was achieved by N-acetylation. The approx. mol. wt. of the deriv. was estd. to be 4.5 × 104 by size-exclusion chromatog. The NMR studies for the sulfonated glycoconjugate and its N-acetylated deriv. revealed that the sheath glycoconjugate is a glycosaminoglycan consisting of a pentasaccharide repeating unit which is substoichiometrically esterified with 3-hydroxypropionic acid and stoichiometrically amidated with acetic acid and glycyl-L-cysteine.
- 20Kunoh, T.; Nakanishi, M.; Kusano, Y.; Itadani, A.; Ando, K.; Matsumoto, S.; Tamura, K.; Kunoh, H.; Takada, J. Biosorption of Metal Elements by Exopolymer Nanofibrils Excreted From Leptothrix Cells. Water Res. 2017, 122, 139– 147, DOI: 10.1016/j.watres.2017.05.003[Crossref], [PubMed], [CAS], Google Scholar20https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXps1KntLs%253D&md5=a7f57920037f6ca10e103f90e3bcf8a0Biosorption of metal elements by exopolymer nanofibrils excreted from Leptothrix cellsKunoh, Tatsuki; Nakanishi, Makoto; Kusano, Yoshihiro; Itadani, Atsushi; Ando, Kota; Matsumoto, Syuji; Tamura, Katsunori; Kunoh, Hitoshi; Takada, JunWater Research (2017), 122 (), 139-147CODEN: WATRAG; ISSN:0043-1354. (Elsevier Ltd.)Leptothrix species, aquatic Fe-oxidizing bacteria, excrete nano-scaled exopolymer fibrils. Once excreted, the fibrils weave together and coalesce to form extracellular, microtubular, immature sheaths encasing catenulate cells of Leptothrix. The immature sheaths, composed of aggregated nanofibrils with a homogeneous-looking matrix, attract and bind aq.-phase inorgs., esp. Fe, P, and Si, to form seemingly solid, mature sheaths of a hybrid org.-inorg. nature. To verify our assumption that the org. skeleton of the sheaths might sorb a broad range of other metallic and nonmetallic elements, we examd. the sorption potential of chem. and enzymically prepd. protein-free org. sheath remnants for 47 available elements. The sheath remnants were found by XRF to sorb each of the 47 elements, although their sorption degree varied among the elements: >35% at. percentages for Ti, Y, Zr, Ru, Rh, Ag, and Au. Electron microscopy, energy dispersive x-ray spectroscopy, electron and x-ray diffractions, and Fourier transform IR spectroscopy analyses of sheath remnants that had sorbed Ag, Cu, and Pt revealed that (i) the sheath remnants comprised a 5-10 nm thick aggregation of fibrils, (ii) the test elements were distributed almost homogeneously throughout the fibrillar aggregate, (iii) the nanofibril matrix sorbing the elements was nearly amorphous, and (iv) these elements plausibly were bound to the matrix by ionic binding, esp. via OH. The present results show that the constitutive protein-free exopolymer nanofibrils of the sheaths can contribute to creating novel filtering materials for recovering and recycling useful and/or hazardous elements from the environment.
- 21Kunoh, T.; Kusano, Y.; Takeda, M.; Nakanishi, M.; Matsumoto, S.; Suzuki, I.; Takano, M.; Kunoh, H.; Takada, J. Formation of Gold Particles via Thiol Groups on Glycoconjugates Comprising the Sheath Skeleton of Leptothrix. Geomicrobiol. J. 2019, 36, 251– 260, DOI: 10.1080/01490451.2018.1550127[Crossref], [CAS], Google Scholar21https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhvVChtbo%253D&md5=caad0348dc25b013f66b9aaaf56c8a45Formation of Gold Particles via Thiol Groups on Glycoconjugates Comprising the Sheath Skeleton of LeptothrixKunoh, Tatsuki; Kusano, Yoshihiro; Takeda, Minoru; Nakanishi, Makoto; Matsumoto, Syuji; Suzuki, Ichiro; Takano, Mikio; Kunoh, Hitoshi; Takada, JunGeomicrobiology Journal (2019), 36 (3), 251-260CODEN: GEJODG; ISSN:0149-0451. (Taylor & Francis, Inc.)Leptothrix, iron-oxidizing bacterium, produces microtubular sheaths that surround the catenulate cells. Org. nanofibrils excreted from the cell surfaces interweave and coalesce to form immature sheaths, which attract aq.-phase inorgs. to eventually form mature org.-inorg. sheaths. Such inorg. encrustation of the sheaths results from interactions between functional groups in the sheath skeleton and inorgs. Based on our previous findings that Leptothrix sheath skeleton sorbed 47 inorgs. (Au was one of the most abundant adsorbates), we examd. the sorption status of Au cations on cell-enclosing sheaths and their protein-free remnants and found that nano to sub-micron Au particles (AuNPs and AuSMPs, resp.) formed on the sheath-forming polymer consisting of a glycoconjugate (an amphoteric glycan modified with cysteine, glycine, and 3-hydroxypropionic acid). When the purified polymer was incubated in HAuCl4 soln., AuNPs and AuSMPs formed on the polymer surfaces. Both particles formed also on cell-enclosing sheaths and protein-free sheath remnants incubated in HAuCl4 soln. When SH groups in the cell-enclosing sheaths were masked with a fluorescent protein, Au particles did not form after incubation in HAuCl4 soln. Results implicate that SH groups are at least partially involved in the redn. of Au cations to metallic Au and eventual formation of Au particles.
- 22Ema, T.; Miyazaki, Y.; Taniguchi, T.; Takada, J. Robust Porphyrin Catalysts Immobilized on Biogenous Iron Oxide for the Repetitive Conversions of Epoxides and CO2 Into Cyclic Carbonates. Green Chem. 2013, 15, 2485– 2492, DOI: 10.1039/c3gc41055b[Crossref], [CAS], Google Scholar22https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXht1Oqtr%252FJ&md5=c8f9dd55c74bc1db2de970b5a6708f61Robust porphyrin catalysts immobilized on biogenous iron oxide for the repetitive conversions of epoxides and CO2 into cyclic carbonatesEma, Tadashi; Miyazaki, Yuki; Taniguchi, Tomoya; Takada, JunGreen Chemistry (2013), 15 (9), 2485-2492CODEN: GRCHFJ; ISSN:1463-9262. (Royal Society of Chemistry)Porphyrin magnesium and zinc complexes I [R = (HO)3Si(CH2)3NMe2+(CH2)6 Br-; R1 = H, R; M = Mg, Zn] bound to biogenic iron oxide derived from the iron-oxidizing bacterium Leptothrix ochracea were prepd. and used as supported catalysts for the ring-expansion of primary epoxides II [R2 = Bu, Me, Me(CH2)7, Me(CH2)11, Ph, MeOCH2, PhOCH2, ClCH2] to yield dioxolanones III [R2 = Bu, Me, Me(CH2)7, Me(CH2)11, Ph, MeOCH2, PhOCH2, ClCH2]. In particular, 0.1 mol% I [R = R1 = (HO)3Si(CH2)3NMe2+(CH2)6 Br-; M = Zn] was an effective catalyst for the ring-expansion of II [R2 = Bu, Me, Me(CH2)7, Me(CH2)11, Ph, MeOCH2, PhOCH2, ClCH2] with carbon dioxide at 120° and 17 atm to give III [R2 = Bu, Me, Me(CH2)7, Me(CH2)11, Ph, MeOCH2, PhOCH2, ClCH2] in 41-93% yields under solvent-free conditions. Ring expansion of a deuterated epoxide indicated that initial ring opening occurred at the least hindered epoxide carbon.
- 23Hashimoto, H.; Asaoka, H.; Nakano, T.; Kusano, Y.; Ishihara, H.; Ikeda, Y.; Nakanishi, M.; Fujii, T.; Yokoyama, T.; Horiishi, N.; Nanba, T.; Takada, J. Preparation, Microstructure, and Color Tone of Microtubule Material Composed of Hematite/Amorphous-Silicate Nanocomposite from Iron Oxide of Bacterial Origin. Dyes Pigm. 2012, 95, 639– 643, DOI: 10.1016/j.dyepig.2012.06.024[Crossref], [CAS], Google Scholar23https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhtlWlurvI&md5=629e52be853e2928fb1b407275376c2fPreparation, microstructure, and color tone of microtubule material composed of hematite/amorphous-silicate nanocomposite from iron oxide of bacterial originHashimoto, Hideki; Asaoka, Hiroshi; Nakano, Takuya; Kusano, Yoshihiro; Ishihara, Hiromichi; Ikeda, Yasunori; Nakanishi, Makoto; Fujii, Tatsuo; Yokoyama, Tadanori; Horiishi, Nanao; Nanba, Tokuro; Takada, JunDyes and Pigments (2012), 95 (3), 639-643CODEN: DYPIDX; ISSN:0143-7208. (Elsevier Ltd.)By heating an amorphous iron oxide produced by Leptothrix ochracea, an iron-oxidizing bacterium species, at 600-1100 °C in air for 2 h, vivid red-colored powd. materials including α-Fe2O3 (hematite) and amorphous silicate with high thermostability were prepd. which offer potential for use as overglaze enamels on porcelain. The precise color tone of the materials greatly depends on the heat-treatment temp. The most strikingly beautiful sample, heat-treated at 800 °C, is light yellowish-red in color (L* = 47.3, a* = 34.1, and b* = 34.6), has a unique microstructure, and does not fade in color even with reheating at 800 °C, which is the firing temp. for overglaze enamel on porcelain. The sample primarily consists of cryst. hematite particles ∼40 nm in diam. with slightly longer axis unit-cell parameters than those of pure hematite. The particles are covered with amorphous silicate phase ∼5 nm in thickness and are intricately interconnected into microtubules with an av. diam. of 1.26 μm. The attractive color of this material is due to the following structural features: small particle size (∼40 nm), nanocomposite of hematite and amorphous silicate, and a microtubule structure that inhibits aggregation of individual hematite particles and microtubules.
- 24Kunoh, T.; Kunoh, H.; Takada, J. Perspectives on the Biogenesis of Iron Oxide Complexes Produced by Leptothrix, an Iron-Oxidizing Bacterium and Promising Industrial Applications for Their Functions. J. Microb. Biochem. Technol. 2015, 7, 419– 426, DOI: 10.4172/1948-5948.1000249[Crossref], [CAS], Google Scholar24https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXnslers7c%253D&md5=aebc5493dac0b1ad6a7fd7eba9af446aPerspectives on the biogenesis of iron oxide complexes produced by leptothrix, an iron-oxidizing bacterium and promising industrial applications for their functionsKunoh, Tatsuki; Kunoh, Hitoshi; Takada, JunJournal of Microbial & Biochemical Technology (2015), 7 (6), 419-426CODEN: JMBTA9; ISSN:1948-5948. (OMICS Publishing Group)Leptothrix species, one of the Fe-/Mn-oxidizing bacteria, are ubiquitous in aq. environments, esp. at sites characterized by a circumneutral pH, an oxygen gradient and a source of reduced Fe and Mn minerals. Characteristic traits that distinguish the genus Leptothrix from other phylogenetically related species are its filamentous growth and ability to form uniquely shaped microtubular sheaths through the pptn. of copious amts. of oxidized Fe or Mn. The sheath is an ingenious hybrid of org. and inorg. materials produced through the interaction of bacterial exopolymers with aq.-phase inorgs. Intriguingly, we discovered that Leptothrix sheaths have a variety of unexpected functions that are suitable for industrial applications such as material for lithium battery electrode, a catalyst enhancer, pottery pigment among others. This review focuses on the structural and chem. properties of the Leptothrix sheaths and their noteworthy functions that show promise for development of cost-effective, eco-friendly industrial applications.
- 25Fleming, E. J.; Langdon, A. E.; Martinez-Garcia, M.; Stepanauskas, R.; Poulton, N. J.; Masland, E. D. P.; Emerson, D. What’s New Is Old: Resolving the Identity of Leptothrix ochracea Using Single Cell Genomics, Pyrosequencing and FISH. PLoS One 2011, 6, e17769 DOI: 10.1371/journal.pone.0017769[Crossref], [PubMed], [CAS], Google Scholar25https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXktVWhsbo%253D&md5=48ad56f01304dcc6438c19cc040c4a8dWhat's new is old: Resolving the identity of Leptothrix ochracea using single cell genomics, pyrosequencing and FISHFleming, Emily J.; Langdon, Amy E.; Martinez-Garcia, Manuel; Stepanauskas, Ramunas; Poulton, Nicole J.; Masland, E. Dashiell P.; Emerson, DavidPLoS One (2011), 6 (3), e17769CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)Leptothrix ochracea is a common inhabitant of freshwater iron seeps and iron-rich wetlands. Its defining characteristic is copious prodn. of extracellular sheaths encrusted with iron oxyhydroxides. Surprisingly, over 90% of these sheaths are empty; hence, what appears to be an abundant population of iron-oxidizing bacteria consists of relatively few cells. Because L. ochracea has proven difficult to cultivate, its identification is based solely on habitat preference and morphol. The authors utilized cultivation-independent techniques to resolve this long-standing enigma. By selecting the actively growing edge of a Leptothrix-contg. iron mat, a conventional SSU rRNA gene clone library was obtained that had 29 clones (42% of the total library) related to the Leptothrix/Sphaerotilus group (≤96% identical to cultured representatives). A pyrotagged library of the V4 hypervariable region constructed from the bulk mat showed that 7.2% of the total sequences also belonged to the Leptothrix/Sphaerotilus group. Sorting of individual L. ochracea sheaths, followed by whole genome amplification (WGA) and PCR identified a SSU rRNA sequence that clustered closely with the putative Leptothrix clones and pyrotags. Using these data, a fluorescence in-situ hybridization (FISH) probe, Lepto175, was designed that bound to ensheathed cells. Quant. use of this probe demonstrated that up to 35% of microbial cells in an actively accreting iron mat were L. ochracea. The SSU rRNA gene of L. ochracea shares 96% homol. with its closet cultivated relative, L. cholodnii, This establishes that L. ochracea is indeed related to this group of morphol. similar, filamentous, sheathed microorganisms.
- 26Fleming, E.; Woyke, T.; Donatello, R.; Kuypers, M.; Sczyrba, A.; Littmann, S.; Emerson, D. Insights Into the Fundamental Physiology of the Uncultured Fe-Oxidizing Bacterium Leptothrix ochracea. Appl. Environ. Microbiol. 2018, 84, e02239 DOI: 10.1128/AEM.02239-17[Crossref], [PubMed], [CAS], Google Scholar26https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhslGks7bF&md5=83b3ec04c76700fbc73b236017cb1b3cInsights into the fundamental physiology of the uncultured Fe-oxidizing bacterium Leptothrix ochraceaFleming, E. J.; Woyke, T.; Donatello, R. A.; Kuypers, M. M. M.; Sczyrba, A.; Littmann, S.; Emerson, D.Applied and Environmental Microbiology (2018), 84 (9), e02239-17/1-e02239-17/15CODEN: AEMIDF; ISSN:1098-5336. (American Society for Microbiology)Leptothrix ochracea is known for producing large vols. of iron oxyhydroxide sheaths that alter wetland biogeochem. For over a century, these delicate structures have fascinated microbiologists and geoscientists. Because L. ochracea still resists long-term in vitro culture, the debate regarding its metabolic classification dates back to 1885. We developed a novel culturing technique for L. ochracea using in situ natural waters and coupled this with single-cell genomics and nanoscale secondary-ion mass spectrophotometry (nanoSIMS) to probe L. ochracea's physiol. In microslide cultures L. ochracea doubled every 5.7 h and had an abs. growth requirement for ferrous iron, the genomic capacity for iron oxidn., and a branched electron transport chain with cytochromes putatively involved in lithotrophic iron oxidn. Addnl., its genome encoded several electron transport chain proteins, including a molybdopterin alternative complex III (ACIII), a cytochrome bd oxidase reductase, and several terminal oxidase genes. L. ochracea contained two key autotrophic proteins in the Calvin-Benson-Bassham cycle, a form II ribulose bisphosphate carboxylase, and a phosphoribulose kinase. L. ochracea also assimilated bicarbonate, although calcns. suggest that bicarbonate assimilation is a small fraction of its total carbon assimilation. Finally, L. ochracea's fundamental physiol. is a hybrid of those of the chemolithotrophic Gallionella-type ironoxidizing bacteria and the sheathed, heterotrophic filamentous metal-oxidizing bacteria of the Leptothrix-Sphaerotilus genera. This allows L. ochracea to inhabit a unique niche within the neutrophilic iron seeps. IMPORTANCE Leptothrix ochracea was one of three groups of organisms that Sergei Winogradsky used in the 1880s to develop his hypothesis on chemolithotrophy. L. ochracea continues to resist cultivation and appears to have an abs. requirement for org.-rich waters, suggesting that its true physiol. remains unknown. Further, L. ochracea is an ecol. engineer; a few L. ochracea cells can generate prodigious vols. of iron oxyhydroxides, changing the ecosystem's geochem. and ecol. Therefore, to det. L. ochracea's basic physiol., we employed new single-cell techniques to demonstrate that L. ochracea oxidizes iron to generate energy and, despite having predicted genes for autotrophic growth, assimilates a fraction of the total CO2 that autotrophs do. Although not a true chemolithoautotroph, L. ochracea's physiol. strategy allows it to be flexible and to extensively colonize iron-rich wetlands.
- 27Furutani, M.; Suzuki, T.; Ishihara, H.; Hashimoto, H.; Kunoh, H.; Takada, J. Initial Assemblage of Bacterial Saccharic Fibrils and Element Deposition to Form an Immature Sheath in Cultured Leptothrix sp Strain OUMS1. Minerals 2011, 1, 157– 166, DOI: 10.3390/min1010157[Crossref], [CAS], Google Scholar27https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XjtlantLw%253D&md5=a845c5b878a15816aa6042a2a080c8d5Initial assemblage of bacterial saccharic fibrils and element deposition to form an immature sheath in cultured Leptothrix sp. strain OUMS1Furutani, Mitsuaki; Suzuki, Tomoko; Ishihara, Hiromichi; Hashimoto, Hideki; Kunoh, Hitoshi; Takada, JunMinerals (Basel, Switzerland) (2011), 1 (1), 157-166CODEN: MBSIBI; ISSN:2075-163X. (MDPI AG)In an aquatic environment, the genus Leptothrix produces an extracellular Fe- or Mn-encrusted tubular sheath composed of a complex hybrid of bacterial exopolymers and aq.-phase inorg. elements. This ultrastructural study investigated initial assemblage of bacterial saccharic fibrils and subsequent deposition of aq.-phase inorg. elements to form the immature sheath skeleton of cultured Leptothrix sp. strain OUMS1. After one day of culture, a globular and/or thread-like secretion was obsd. on the surface of the bacterial cell envelope, and secreted bodies were transported across the intervening space away from the cell to form an immature sheath skeleton comprising assembled and intermingled fibrils. Energy dispersive X-ray microanal. and specific Bi-staining detected a distinguishable level of P, trace Si, and a notable amt. of carbohydrates in the skeleton, but not Fe. By the second day, the skeleton was prominently thickened with an inner layer of almost parallel aligned fibrils, along with low level of Fe deposition, whereas an outer intermingled fibrous layer exhibited heavy deposition of Fe along with significant deposition of P and Si. These results indicate that basic sheath-construction proceeds in two steps under culture conditions: an initial assemblage of bacterial saccharic fibrils originated from the cell envelope and the subsequent deposition of aq.-phase Fe, P, and Si.
- 28Emerson, D.; Ghiorse, W. C. Role of Disulfide Bonds in Maintaining the Structural Integrity of the Sheath of Leptothrix discophora SP-6. J. Bacteriol. 1993, 175, 7819– 7827, DOI: 10.1128/jb.175.24.7819-7827.1993[Crossref], [PubMed], [CAS], Google Scholar28https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2cXkvFOltw%253D%253D&md5=9d7c9282b4acddbd53762aed510f71a4Role of disulfide bonds in maintaining the structural integrity of the sheath of Leptothrix discophora SP-6Emerson, David; Ghiorse, William C.Journal of Bacteriology (1993), 175 (24), 7819-27CODEN: JOBAAY; ISSN:0021-9193.Isolated sheaths of Leptothrix discophora SP-6 (ATCC 51168) were tested for susceptibility to degrdn. by a variety of chem. denaturants and lytic enzymes and found to be resistant to many reagents and enzyme treatments. However, disulfide bond-reducing agents such as dithiothreitol (DTT), β-mercaptoethanol, sodium cyanide, and sodium sulfite degraded the sheath, esp. at elevated pH (pH 9) and temp. (50°). DTT and β-mercaptoethanol caused more rapid degrdn. of the sheath than cyanide or sulfite. Treatment of the sheath with 1N NaOH resulted in rapid breakdown, while treatment with 1N HCl resulted in slow but significant hydrolysis. Transmission electron microscopy showed that the 6.5-nm fibrils previously shown to be an integral structural element of the sheath fabric (D. Emerson and W. C. Ghiorse, 1993) were progressively dissocd. into random masses during DTT-induced degrdn. Quantitation of disulfide bonds with DTT showed that the sheaths contained approx. 2.2 μmol of disulfides per mg of sheath protein. Reaction with 5,5'-dithio-bis-(2-nitrobenzoic acid) showed that sheaths also contained approx. 0.8 μmol of free sulfhydryls per mg of protein. A sulfhydryl-specific fluorescent probe (fluorescein 5-maleimide) showed that the free sulfhydryls in sheathed cell filaments were evenly distributed throughout the sheath. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis autoradiog. of [14C]iodoacetamide-labeled sheaths and DTT-dissocd. sheath fibril suspensions showed that the majority of 14C-labeled sulfhydryls in the sheaths did not enter the gel. However, low-mol.-mass silver-staining bands (14 to 45 kDa) did appear in the gels after iodoacetic acid or iodoacetamide alkylation of the dissocd. fibrils. These bands did not stain with Coomassie blue. Their migration in gels was slightly affected by digestion with pronase. The fibrils contained 20-25% protein. These results confirm that the sheath fibrils consist of high-mol.-wt. heteropolysaccharide-protein complexes. Evidently, proteins in the fibril complexes provide interfibril crosslinking to maintain the structural integrity of the sheath.
- 29Emerson, D.; Ghiorse, W. C. Ultrastructure and Chemical-Composition of the Sheath of Leptothrix discophora SP-6. J. Bacteriol. 1993, 175, 7808– 7818, DOI: 10.1128/jb.175.24.7808-7818.1993[Crossref], [PubMed], [CAS], Google Scholar29https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2cXkvFOltg%253D%253D&md5=1cb9d3e52ef4f2322b6ce15c6acee738Ultrastructure and chemical composition of sheath of Leptothrix discophora SP-6Emerson, David; Ghiorse, William C.Journal of Bacteriology (1993), 175 (24), 7808-18CODEN: JOBAAY; ISSN:0021-9193.Light microscopy and transmission electron microscopy of thin sections and metal-shadowed specimens showed that the sheath of Leptothrix discophora SP-6 (ATCC 51168) is a tube-like extracellular polymeric structure consisting of a condensed fabric of 6.5-nm-diam. fibrils underlying a more diffuse outer capsular layer. In thin sections, outer membrane bridges seen to contact the inner sheath layer suggested that the sheath fabric was attached to the outer layer of the gram-neg. cell wall. The capsular polymers showed an affinity for cationic colloidal iron and polycationic ferritin, indicating that they carry a neg. charge. Cell-free sheaths were isolated by treatment with a mixt. of lysozyme, EDTA, and N-lauroylsarcosine (Sarkosyl) or sodium dodecyl sulfate (SDS). Both Sarkosyl- and SDS-isolated sheaths were indistinguishable in microscopic appearance. However, the Mn-oxidizing activity of Sarkosyl-isolated sheaths was more stable than that of SDS-isolated sheaths. The Sarkosyl-isolated sheaths also contained more 2-keto-3-deoxyoctanoic acid and more outer membrane protein than SDS-isolated sheaths. The oven-dried mass of detergent-isolated sheaths represented approx. 9% of the total oven-dried biomass of SP-6 cultures; the oven-dried sheaths contained 38% C, 6.9% N, 6% H, and 2.1% S and approx. 34 to 35% carbohydrte (polysaccharide), 23 to 25% protein, 8% lipid, and 4% inorg. ash. Gas-liq. chromatog. showed that the polysaccharide was an approx. 1:1 mixt. of uronic acids (glucuronic, galacturonic, and mannuronic acids and at least one other unidentified uronic acid) and an amino sugar (galactosamine). Neutral sugars were not detected. Amino acid anal. showed that sheath proteins were enriched in cysteine (6 mol%). The cysteine residues in the sheath proteins probably provide sulfhydryls for disulfide bonds that play an important role in maintaining the structural integrity of the sheath.
- 30Ishihara, H.; Suzuki, T.; Hashimoto, H.; Kunoh, H.; Takada, J. Initial Parallel Arrangement of Extracellular Fibrils Holds a Key for Sheath Frame Construction by Leptothrix sp. Strain OUMS1. Minerals 2013, 3, 73– 81, DOI: 10.3390/min3010073[Crossref], [CAS], Google Scholar30https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXlsFCrs7w%253D&md5=5e74a7afc074b0c47df1f5d9b887caf5Initial parallel arrangement of extracellular fibrils holds a key for sheath frame construction by Leptothrix sp. strain OUMS1Ishihara, Hiromichi; Suzuki, Tomoko; Hashimoto, Hideki; Kunoh, Hitoshi; Takada, JunMinerals (Basel, Switzerland) (2013), 3 (1), 73-81CODEN: MBSIBI; ISSN:2075-163X. (MDPI AG)Early stages of sheath formation by Leptothrix sp. strain OUMS1 and its deriv. sheathless mutant grown in media with or without Fe were examd. by light and electron microscopy. Results showed that the initial parallel arrangement of fibrils excreted from the cells holds a key for subsequent construction of the sheath frame and that aq.-phase Fe interacts with excreted fibrils whether fibrils are parallel-arranged or simply-intermingled.
- 31Hol, F. J. H.; Dekker, C. Zooming in to See the Bigger Picture: Microfluidic and Nanofabrication Tools to Study Bacteria. Science 2014, 346, 1251821, DOI: 10.1126/science.1251821[Crossref], [PubMed], [CAS], Google Scholar31https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC2M3jtV2ntQ%253D%253D&md5=3ee5fcb84fe48324170050e3f82bb53cZooming in to see the bigger picture: microfluidic and nanofabrication tools to study bacteriaHol Felix J H; Dekker CeesScience (New York, N.Y.) (2014), 346 (6208), 1251821 ISSN:.The spatial structure of natural habitats strongly affects bacterial life, ranging from nanoscale structural features that individual cells exploit for surface attachment, to micro- and millimeter-scale chemical gradients that drive population-level processes. Nanofabrication and microfluidics are ideally suited to manipulate the environment at those scales and have emerged as powerful tools with which to study bacteria. Here, we review the new scientific insights gained by using a diverse set of nanofabrication and microfluidic techniques to study individual bacteria and multispecies communities. This toolbox is beginning to elucidate disparate bacterial phenomena-including aging, electron transport, and quorum sensing-and enables the dissection of environmental communities through single-cell genomics. A more intimate integration of microfluidics, nanofabrication, and microbiology will enable further exploration of bacterial life at the smallest scales.
- 32Nagy, K.; Abraham, A.; Keymer, J. E.; Galajda, P. Application of Microfluidics in Experimental Ecology: The Importance of Being Spatial. Front. Microbiol. 2018, 9, 496, DOI: 10.3389/fmicb.2018.00496[Crossref], [PubMed], [CAS], Google Scholar32https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC1MnoslOmuw%253D%253D&md5=a1e25e8fcde515bb0338578105effcdaApplication of Microfluidics in Experimental Ecology: The Importance of Being SpatialNagy Krisztina; Abraham Agnes; Galajda Peter; Abraham Agnes; Keymer Juan EFrontiers in microbiology (2018), 9 (), 496 ISSN:1664-302X.Microfluidics is an emerging technology that is used more and more in biology experiments. Its capabilities of creating precisely controlled conditions in cellular dimensions make it ideal to explore cell-cell and cell-environment interactions. Thus, a wide spectrum of problems in microbial ecology can be studied using engineered microbial habitats. Moreover, artificial microfluidic ecosystems can serve as model systems to test ecology theories and principles that apply on a higher level in the hierarchy of biological organization. In this mini review we aim to demonstrate the versatility of microfluidics and the diversity of its applications that help the advance of microbiology, and in more general, experimental ecology.
- 33Aleklett, K.; Kiers, E. T.; Ohlsson, P.; Shimizu, T. S.; Caldas, V. E.; Hammer, E. C. Build Your Own Soil: Exploring Microfluidics to Create Microbial Habitat Structures. ISME J. 2018, 12, 312– 319, DOI: 10.1038/ismej.2017.184[Crossref], [PubMed], [CAS], Google Scholar33https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC1M3htVCjsA%253D%253D&md5=83f9848fafa3fe58dda05716c8f2ffc5Build your own soil: exploring microfluidics to create microbial habitat structuresAleklett Kristin; Hammer Edith C; Kiers E Toby; Caldas Victor Ea; Ohlsson Pelle; Shimizu Thomas S; Caldas Victor EaThe ISME journal (2018), 12 (2), 312-319 ISSN:.Soil is likely the most complex ecosystem on earth. Despite the global importance and extraordinary diversity of soils, they have been notoriously challenging to study. We show how pioneering microfluidic techniques provide new ways of studying soil microbial ecology by allowing simulation and manipulation of chemical conditions and physical structures at the microscale in soil model habitats.
- 34Volfson, D.; Cookson, S.; Hasty, J.; Tsimring, L. S. Biomechanical Ordering of Dense Cell Populations. Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 15346– 15351, DOI: 10.1073/pnas.0706805105[Crossref], [PubMed], [CAS], Google Scholar34https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BD1cnkslSitQ%253D%253D&md5=3aa430ec2ae45f77b262314c1bc3cafeBiomechanical ordering of dense cell populationsVolfson Dmitri; Cookson Scott; Hasty Jeff; Tsimring Lev SProceedings of the National Academy of Sciences of the United States of America (2008), 105 (40), 15346-51 ISSN:.The structure of bacterial populations is governed by the interplay of many physical and biological factors, ranging from properties of surrounding aqueous media and substrates to cell-cell communication and gene expression in individual cells. The biomechanical interactions arising from the growth and division of individual cells in confined environments are ubiquitous, yet little work has focused on this fundamental aspect of colony formation. We analyze the spatial organization of Escherichia coli growing in a microfluidic chemostat. We find that growth and expansion of a dense colony of cells leads to a dynamical transition from an isotropic disordered phase to a nematic phase characterized by orientational alignment of rod-like cells. We develop a continuum model of collective cell dynamics based on equations for local cell density, velocity, and the tensor order parameter. We use this model and discrete element simulations to elucidate the mechanism of cell ordering and quantify the relationship between the dynamics of cell proliferation and the spatial structure of the population.
- 35Grunberger, A.; Paczia, N.; Probst, C.; Schendzielorz, G.; Eggeling, L.; Noack, S.; Wiechert, W.; Kohlheyer, D. A Disposable Picolitre Bioreactor for Cultivation and Investigation of Industrially Relevant Bacteria on the Single Cell Level. Lab Chip 2012, 12, 2060– 2068, DOI: 10.1039/c2lc40156h[Crossref], [PubMed], [CAS], Google Scholar35https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC38rlvVCntw%253D%253D&md5=b04291ec1337d92cf5eaf13a44b9cf92A disposable picolitre bioreactor for cultivation and investigation of industrially relevant bacteria on the single cell levelGrunberger Alexander; Paczia Nicole; Probst Christopher; Schendzielorz Georg; Eggeling Lothar; Noack Stephan; Wiechert Wolfgang; Kohlheyer DietrichLab on a chip (2012), 12 (11), 2060-8 ISSN:.In the continuously growing field of industrial biotechnology the scale-up from lab to industrial scale is still a major hurdle to develop competitive bioprocesses. During scale-up the productivity of single cells might be affected by bioreactor inhomogeneity and population heterogeneity. Currently, these complex interactions are difficult to investigate. In this report, design, fabrication and operation of a disposable picolitre cultivation system is described, in which environmental conditions can be well controlled on a short time scale and bacterial microcolony growth experiments can be observed by time-lapse microscopy. Three exemplary investigations will be discussed emphasizing the applicability and versatility of the device. Growth and analysis of industrially relevant bacteria with single cell resolution (in particular Escherichia coli and Corynebacterium glutamicum) starting from one single mother cell to densely packed cultures is demonstrated. Applying the picolitre bioreactor, 1.5-fold increased growth rates of C. glutamicum wild type cells were observed compared to typical 1 litre lab-scale batch cultivation. Moreover, the device was used to analyse and quantify the morphological changes of an industrially relevant l-lysine producer C. glutamicum after artificially inducing starvation conditions. Instead of a one week lab-scale experiment, only 1 h was sufficient to reveal the same information. Furthermore, time lapse microscopy during 24 h picolitre cultivation of an arginine producing strain containing a genetically encoded fluorescence sensor disclosed time dependent single cell productivity and growth, which was not possible with conventional methods.
- 36Cho, H.; Jönsson, H.; Campbell, K.; Melke, P.; Williams, J. W.; Jedynak, B.; Stevens, A. M.; Groisman, A.; Levchenko, A. Self-Organization in High-Density Bacterial Colonies: Efficient Crowd Control. PLoS Biol. 2007, 5, e302 DOI: 10.1371/journal.pbio.0050302[Crossref], [PubMed], [CAS], Google Scholar36https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BD2sjgslymsQ%253D%253D&md5=be7321f0d9f65b8992dcc1e4ae988379Self-organization in high-density bacterial colonies: efficient crowd controlCho HoJung; Jonsson Henrik; Campbell Kyle; Melke Pontus; Williams Joshua W; Jedynak Bruno; Stevens Ann M; Groisman Alex; Levchenko AndrePLoS biology (2007), 5 (11), e302 ISSN:.Colonies of bacterial cells can display complex collective dynamics, frequently culminating in the formation of biofilms and other ordered super-structures. Recent studies suggest that to cope with local environmental challenges, bacterial cells can actively seek out small chambers or cavities and assemble there, engaging in quorum sensing behavior. By using a novel microfluidic device, we showed that within chambers of distinct shapes and sizes allowing continuous cell escape, bacterial colonies can gradually self-organize. The directions of orientation of cells, their growth, and collective motion are mutually correlated and dictated by the chamber walls and locations of chamber exits. The ultimate highly organized steady state is conducive to a more-organized escape of cells from the chambers and increased access of nutrients into and evacuation of waste out of the colonies. Using a computational model, we suggest that the lengths of the cells might be optimized to maximize self-organization while minimizing the potential for stampede-like exit blockage. The self-organization described here may be crucial for the early stage of the organization of high-density bacterial colonies populating small, physically confined growth niches. It suggests that this phenomenon can play a critical role in bacterial biofilm initiation and development of other complex multicellular bacterial super-structures, including those implicated in infectious diseases.
- 37Kunoh, T.; Nagaoka, N.; McFarlane, I. R.; Tamura, K.; El-Naggar, M. Y.; Kunoh, H.; Takada, J. Dissociation and Re-Aggregation of Multicell-Ensheathed Fragments Responsible for Rapid Production of Massive Clumps of Leptothrix Sheaths. Biology 2016, 5, 32, DOI: 10.3390/biology5030032[Crossref], [CAS], Google Scholar37https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXms1OitLo%253D&md5=898bf1601d3d9fd1640c63083627058fDissociation and re-aggregation of multicell-ensheathed fragments responsible for rapid production of massive clumps of leptothrix sheathsKunoh, Tatsuki; Nagaoka, Noriyuki; McFarlane, Ian R.; Tamura, Katsunori; El-Naggar, Mohamed Y.; Kunoh, Hitoshi; Takada, JunBiology (Basel, Switzerland) (2016), 5 (3), 32/1-32/9CODEN: BBSIBX; ISSN:2079-7737. (MDPI AG)Species of the Fe/Mn-oxidizing bacteria Leptothrix produce tremendous amts. of microtubular, Fe/Mn-encrusted sheaths within a few days in outwells of groundwater that can rapidly clog water systems. To understand this mode of rapid sheath prodn. and define the timescales involved, behaviors of sheath-forming Leptothrix sp. strain OUMS1 were examd. using time-lapse video at the initial stage of sheath formation. OUMS1 formed clumps of tangled sheaths. Electron microscopy confirmed the presence of a thin layer of bacterial exopolymer fibrils around catenulate cells (corresponding to the immature sheath). In time-lapse videos, numerous sheath filaments that extended from the periphery of sheath clumps repeatedly fragmented at the apex of the same fragment, the fragments then aggregated and again elongated, eventually forming a large sheath clump comprising tangled sheaths within two days. In this study, we found that fast microscopic fragmentation, dissocn., re-aggregation and re-elongation events are the basis of the rapid, massive prodn. of Leptothrix sheaths typically obsd. at macroscopic scales.
- 38Bennett, R. R.; Lee, C. K.; De Anda, J.; Nealson, K. H.; Yildiz, F. H.; O’Toole, G. A.; Wong, G. C.; Golestanian, R. Species-Dependent Hydrodynamics of Flagellum-Tethered Bacteria in Early Biofilm Development. J. R. Soc., Interface 2016, 13, 20150966, DOI: 10.1098/rsif.2015.0966[Crossref], [PubMed], [CAS], Google Scholar38https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC28nps1ShtA%253D%253D&md5=4b84034d8dd578d99d29e79bb1f7d3ceSpecies-dependent hydrodynamics of flagellum-tethered bacteria in early biofilm developmentBennett Rachel R; Lee Calvin K; De Anda Jaime; Wong Gerard C L; Nealson Kenneth H; Yildiz Fitnat H; O'Toole George A; Golestanian RaminJournal of the Royal Society, Interface (2016), 13 (115), 20150966 ISSN:.Monotrichous bacteria on surfaces exhibit complex spinning movements. Such spinning motility is often a part of the surface detachment launch sequence of these cells. To understand the impact of spinning motility on bacterial surface interactions, we develop a hydrodynamic model of a surface-bound bacterium, which reproduces behaviours that we observe in Pseudomonas aeruginosa, Shewanella oneidensis and Vibrio cholerae, and provides a detailed dictionary for connecting observed spinning behaviour to bacteria-surface interactions. Our findings indicate that the fraction of the flagellar filament adhered to the surface, the rotation torque of this appendage, the flexibility of the flagellar hook and the shape of the bacterial cell dictate the likelihood that a microbe will detach and the optimum orientation that it should have during detachment. These findings are important for understanding species-specific reversible attachment, the key transition event between the planktonic and biofilm lifestyle for motile, rod-shaped organisms.
- 39De Anda, J.; Lee, E. Y.; Lee, C. K.; Bennett, R. R.; Ji, X.; Soltani, S.; Harrison, M. C.; Baker, A. E.; Luo, Y.; Chou, T. High-Speed “4D” Computational Microscopy of Bacterial Surface Motility. ACS Nano 2017, 11, 9340– 9351, DOI: 10.1021/acsnano.7b04738[ACS Full Text], [CAS], Google Scholar39https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhtlyhsL%252FM&md5=c0b8c2763726329389fb18c8fb4ff8e9High-Speed "4D" Computational Microscopy of Bacterial Surface Motilityde Anda, Jaime; Lee, Ernest Y.; Lee, Calvin K.; Bennett, Rachel R.; Ji, Xiang; Soltani, Soheil; Harrison, Mark C.; Baker, Amy E.; Luo, Yun; Chou, Tom; O'Toole, George A.; Armani, Andrea M.; Golestanian, Ramin; Wong, Gerard C. L.ACS Nano (2017), 11 (9), 9340-9351CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)Bacteria exhibit surface motility modes that play pivotal roles in early-stage biofilm community development, such as type IV pili-driven "twitching" motility and flagellum-driven "spinning" and "swarming" motility. Appendage-driven motility is controlled by mol. motors, and anal. of surface motility behavior is complicated by its inherently 3D nature, the speed of which is too fast for confocal microscopy to capture. Here, the authors combine electromagnetic field computation and statistical image anal. to generate 3D movies close to a surface at 5 ms time resoln. using conventional inverted microscopes. The authors treat each bacterial cell as a spherocylindrical lens and use finite element modeling to solve Maxwell's equations and compute the diffracted light intensities assocd. with different angular orientations of the bacterium relative to the surface. By performing cross-correlation calcns. between measured 2D microscopy images and a library of computed light intensities, near-surface 3D movies of Pseudomonas aeruginosa translational and rotational motion are possible at high temporal resoln. Comparison between computational reconstructions and detailed hydrodynamic calcns. reveals that P. aeruginosa act like low Reynolds no. spinning tops with unstable orbits, driven by a flagellum motor with a torque output of ∼2 pN μm. The authors' anal. reveals that P. aeruginosa can undergo complex flagellum-driven dynamical behavior, including precession, nutation, and an unexpected taxonomy of surface motility mechanisms, including upright-spinning bacteria that diffuse laterally across the surface, and horizontal bacteria that follow helicoidal trajectories and exhibit superdiffusive movements parallel to the surface.
- 40Adams, L. F.; Ghiorse, W. C. Influence of Manganese on Growth of a Sheathless Strain of Leptothrix discophora. Appl. Environ. Microbiol. 1985, 49, 556– 562[Crossref], [PubMed], [CAS], Google Scholar40https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL2MXhsVGqtrc%253D&md5=44e448f8e54750d18053f7facfb6dc19Influence of manganese on growth of a sheathless strain of Leptothrix discophoraAdams, Lee F.; Ghiorse, William C.Applied and Environmental Microbiology (1985), 49 (3), 556-62CODEN: AEMIDF; ISSN:0099-2240.Mn2+ exerted various effects on the growth of L. discophora strain SS-1 in batch cultures, depending on the concn. added to the medium. Concns. of 0.55-5.5 μM Mn2+, comparable to those in the environment from which SS-1 was isolated, decreased cell yield and prolonged stationary-phase survival, but did not affect growth rate. Elevated concns. of 55-910 μM Mn2+ also decreased cell yield and prolonged survival, but growth rate was decreased as well. The addn. of 1820 μM Mn2+ caused a decline in cell nos., followed by an exponential rise after 80 h of incubation, indicating the development of a population of cells resistant to Mn2+ toxicity. When ≤360 μM Mn2+ was added to growth flasks, Mn2+ was oxidized to Mn oxide (MnOx, where x is ∼2) which appeared as brown particles in the medium. Quantification of Mn oxidn. during growth of cultures to which 55 μM Mn2+ was added showed that nearly all of the Mn2+ was oxidized at the beginning of the stationary phase of growth (15-25 h). Thus, the decrease in cell yield obsd. at low and moderate concns. of Mn2+ was related to the formation of MnOx, which may have bound cationic nutrients essential to the growth of SS-1. The addn. of excess Fe3+ to cultures contg. 55 μM Mn2+ increased cell yield to levels near those found in cultures with no added Mn2+, indicating that Fe deprivation by MnOx was at least partly responsible for the decreased cell yield.
- 41Sauer, K.; Camper, A. K.; Ehrlich, G. D.; Costerton, J. W.; Davies, D. G. Pseudomonas aeruginosa Displays Multiple Phenotypes During Development As a Biofilm. J. Bacteriol. 2002, 184, 1140– 1154, DOI: 10.1128/jb.184.4.1140-1154.2002[Crossref], [PubMed], [CAS], Google Scholar41https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XhtVeisrg%253D&md5=dc6e3a7aeed3d4bce4f507db4690d1e5Pseudomonas aeruginosa displays multiple phenotypes during development as a biofilmSauer, Karin; Camper, Anne K.; Ehrlich, Garth D.; Costerton, J. William; Davies, David G.Journal of Bacteriology (2002), 184 (4), 1140-1154CODEN: JOBAAY; ISSN:0021-9193. (American Society for Microbiology)Complementary approaches were employed to characterize transitional episodes in Pseudomonas aeruginosa biofilm development using direct observation and whole-cell protein anal. Microscopy and in situ reporter gene anal. were used to directly observe changes in biofilm physiol. and to act as signposts to standardize protein collection for two-dimensional electrophoretic anal. and protein identification in chemostat and continuous-culture biofilm-grown populations. Using these approaches, we characterized five stages of biofilm development: (i) reversible attachment, (ii) irreversible attachment, (iii) maturation-1, (iv) maturation-2, and (v) dispersion. Biofilm cells were shown to change regulation of motility, alginate prodn., and quorum sensing during the process of development. The av. difference in detectable protein regulation between each of the five stages of development was 35% (approx. 525 proteins). When planktonic cells were compared with maturation-2 stage biofilm cells, more than 800 proteins were shown to have a sixfold or greater change in expression level (over 50% of the proteome). This difference was higher than when planktonic P. aeruginosa were compared with planktonic cultures of Pseudomonas putida. Las quorum sensing was shown to play no role in early biofilm development but was important in later stages. Biofilm cells in the dispersion stage were more similar to planktonic bacteria than to maturation-2 stage bacteria. These results demonstrate that P. aeruginosa displays multiple phenotypes during biofilm development and that knowledge of stage-specific physiol. may be important in detecting and controlling biofilm growth.
- 42Hinsa, S. M.; Espinosa-Urgel, M.; Ramos, J. L.; O’Toole, G. A. Transition from Reversible to Irreversible Attachment During Biofilm Formation by Pseudomonas fluorescens WCS365 Requires an ABC Transporter and a Large Secreted Protein. Mol. Microbiol. 2003, 49, 905– 918, DOI: 10.1046/j.1365-2958.2003.03615.x[Crossref], [PubMed], [CAS], Google Scholar42https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXmslKltL8%253D&md5=eabbe27d3809bdb88357fb219e6bcff7Transition from reversible to irreversible attachment during biofilm formation by Pseudomonas fluorescens WCS365 requires an ABC transporter and a large secreted proteinHinsa, Shannon M.; Espinosa-Urgel, Manuel; Ramos, Juan L.; O'Toole, George A.Molecular Microbiology (2003), 49 (4), 905-918CODEN: MOMIEE; ISSN:0950-382X. (Blackwell Publishing Ltd.)We report the identification of an ATP-binding cassette (ABC) transporter and an assocd. large cell-surface protein that are required for biofilm formation by Pseudomonas fluorescens WCS365. The genes coding for these proteins are designated lap for large adhesion protein. The LapA protein, with a predicted mol. wt. of ∼900 kDa, is found to be loosely assocd. with the cell surface and present in the culture supernatant. The LapB, LapC and LapE proteins are predicted to be the cytoplasmic membrane-localized ATPase, membrane fusion protein and outer membrane protein component, resp., of an ABC transporter. Consistent with this prediction, LapE, like other members of this family, is localized to the outer membrane. We propose that the lapEBC-encoded ABC transporter participates in the secretion of LapA, as strains with mutations in the lapEBC genes do not have detectable LapA assocd. with the cell surface or in the supernatant. The lap genes are conserved among environmental pseudomonads such as P. putida KT2440, P. fluorescens PfO1 and P. fluorescens WCS365, but are absent from pathogenic pseudomonads such as P. aeruginosa and P. syringae. The wild-type strain of P. fluorescens WCS365 and its lap mutant derivs. were assessed for their biofilm forming ability in static and flow systems. The lap mutant strains are impaired in an early step in biofilm formation and are unable to develop the mature biofilm structure seen for the wild-type bacterium. Time-lapse microscopy studies detd. that the lap mutants are unable to progress from reversible (or transient) attachment to the irreversible attachment stage of biofilm development. The lap mutants were also defective in attachment to quartz sand, an abiotic surface these organisms likely encounter in the environment.
- 43Tuson, H. H.; Weibel, D. B. Bacteria–Surface Interactions. Soft Matter 2013, 9, 4368– 4380, DOI: 10.1039/c3sm27705d[Crossref], [PubMed], [CAS], Google Scholar43https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXltVOrurg%253D&md5=d0757729a24782a2faa7d4b022be150dBacteria-surface interactionsTuson, Hannah H.; Weibel, Douglas B.Soft Matter (2013), 9 (17), 4368-4380CODEN: SMOABF; ISSN:1744-683X. (Royal Society of Chemistry)A review. The interaction of bacteria with surfaces has important implications in a range of areas, including bioenergy, biofouling, biofilm formation, and the infection of plants and animals. Many of the interactions of bacteria with surfaces produce changes in the expression of genes that influence cell morphol. and behavior, including genes essential for motility and surface attachment. Despite the attention that these phenotypes have garnered, the bacterial systems used for sensing and responding to surfaces are still not well understood. An understanding of these mechanisms will guide the development of new classes of materials that inhibit and promote cell growth, and complement studies of the physiol. of bacteria in contact with surfaces. Recent studies from a range of fields in science and engineering are poised to guide future investigations in this area. This review summarizes recent studies on bacteria-surface interactions, discusses mechanisms of surface sensing and consequences of cell attachment, provides an overview of surfaces that have been used in bacterial studies, and highlights unanswered questions in this field.
- 44Brangwynne, C. P.; MacKintosh, F. C.; Kumar, S.; Geisse, N. A.; Talbot, J.; Mahadevan, L.; Parker, K. K.; Ingber, D. E.; Weitz, D. A. Microtubules Can Bear Enhanced Compressive Loads in Living Cells Because of Lateral Reinforcement. J. Cell Biol. 2006, 173, 733– 741, DOI: 10.1083/jcb.200601060[Crossref], [PubMed], [CAS], Google Scholar44https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28Xls1Shsrc%253D&md5=0119caf6de99e66dce86a304946d4053Microtubules can bear enhanced compressive loads in living cells because of lateral reinforcementBrangwynne, Clifford P.; MacKintosh, Frederick C.; Kumar, Sanjay; Geisse, Nicholas A.; Talbot, Jennifer; Mahadevan, L.; Parker, Kevin K.; Ingber, Donald E.; Weitz, David A.Journal of Cell Biology (2006), 173 (5), 733-741CODEN: JCLBA3; ISSN:0021-9525. (Rockefeller University Press)Cytoskeletal microtubules have been proposed to influence cell shape and mechanics based on their ability to resist large-scale compressive forces exerted by the surrounding contractile cytoskeleton. Consistent with this, cytoplasmic microtubules are often highly curved and appear buckled because of compressive loads. However, the results of in vitro studies suggest that microtubules should buckle at much larger length scales, withstanding only exceedingly small compressive forces. This discrepancy calls into question the structural role of microtubules, and highlights our lack of quant. knowledge of the magnitude of the forces they experience and can withstand in living cells. We show that intracellular microtubules do bear large-scale compressive loads from a variety of physiol. forces, but their buckling wavelength is reduced significantly because of mech. coupling to the surrounding elastic cytoskeleton. We quant. explain this behavior, and show that this coupling dramatically increases the compressive forces that microtubules can sustain, suggesting they can make a more significant structural contribution to the mech. behavior of the cell than previously thought possible.
- 45Duvernoy, M.-C.; Mora, T.; Ardré, M.; Croquette, V.; Bensimon, D.; Quilliet, C.; Ghigo, J.-M.; Balland, M.; Beloin, C.; Lecuyer, S.; Desprat, N. Asymmetric Adhesion of Rod-Shaped Bacteria Controls Microcolony Morphogenesis. Nat. Commun. 2018, 9, 1120, DOI: 10.1038/s41467-018-03446-y[Crossref], [PubMed], [CAS], Google Scholar45https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC1MnitFersw%253D%253D&md5=1d0011ddb7e7089b6091609ccd6a26c7Asymmetric adhesion of rod-shaped bacteria controls microcolony morphogenesisDuvernoy Marie-Cecilia; Mora Thierry; Ardre Maxime; Croquette Vincent; Bensimon David; Desprat Nicolas; Duvernoy Marie-Cecilia; Quilliet Catherine; Balland Martial; Lecuyer Sigolene; Duvernoy Marie-Cecilia; Ardre Maxime; Croquette Vincent; Bensimon David; Desprat Nicolas; Bensimon David; Ghigo Jean-Marc; Beloin Christophe; Desprat NicolasNature communications (2018), 9 (1), 1120 ISSN:.Surface colonization underpins microbial ecology on terrestrial environments. Although factors that mediate bacteria-substrate adhesion have been extensively studied, their spatiotemporal dynamics during the establishment of microcolonies remains largely unexplored. Here, we use laser ablation and force microscopy to monitor single-cell adhesion during the course of microcolony formation. We find that adhesion forces of the rod-shaped bacteria Escherichia coli and Pseudomonas aeruginosa are polar. This asymmetry induces mechanical tension, and drives daughter cell rearrangements, which eventually determine the shape of the microcolonies. Informed by experimental data, we develop a quantitative model of microcolony morphogenesis that enables the prediction of bacterial adhesion strength from simple time-lapse measurements. Our results demonstrate how patterns of surface colonization derive from the spatial distribution of adhesive factors on the cell envelope.
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- 47Amir, A.; Babaeipour, F.; McIntosh, D. B.; Nelson, D. R.; Jun, S. Bending Forces Plastically Deform Growing Bacterial Cell Walls. Proc. Natl. Acad. Sci. U. S. A. 2014, 111, 5778– 5783, DOI: 10.1073/pnas.1317497111[Crossref], [PubMed], [CAS], Google Scholar47https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXmtlWntLo%253D&md5=04c501958cff07e96b03df45712bde40Bending forces plastically deform growing bacterial cell wallsAmir, Ariel; Babaeipour, Farinaz; McIntosh, Dustin B.; Nelson, David R.; Jun, SuckjoonProceedings of the National Academy of Sciences of the United States of America (2014), 111 (16), 5778-5783CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Cell walls define a cell's shape in bacteria. The walls are rigid to resist large internal pressures, but remarkably plastic to adapt to a wide range of external forces and geometric constraints. Currently, it is unknown how bacteria maintain their shape. In this paper, we develop exptl. and theor. approaches and show that mech. stresses regulate bacterial cell wall growth. By applying a precisely controllable hydrodynamic force to growing rod-shaped Escherichia coli and Bacillus subtilis cells, we demonstrate that the cells can exhibit two fundamentally different modes of deformation. The cells behave like elastic rods when subjected to transient forces, but deform plastically when significant cell wall synthesis occurs while the force is applied. The deformed cells always recover their shape. The exptl. results are in quant. agreement with the predictions of the theory of dislocation-mediated growth. In particular, we find that a single dimensionless parameter, which depends on a combination of independently measured phys. properties of the cell, can describe the cell's responses under various exptl. conditions. These findings provide insight into how living cells robustly maintain their shape under varying phys. environments.
- 48Wang, S.; Arellano-Santoyo, H.; Combs, P. A.; Shaevitz, J. W. Actin-Like Cytoskeleton Filaments Contribute to Cell Mechanics in Bacteria. Proc. Natl. Acad. Sci. U. S. A. 2010, 107, 9182– 9185, DOI: 10.1073/pnas.0911517107[Crossref], [PubMed], [CAS], Google Scholar48https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXmslGrt70%253D&md5=d65bddef3d6e8c44fc09719c3b90861aActin-like cytoskeleton filaments contribute to cell mechanics in bacteriaWang, Siyuan; Arellano-Santoyo, Hugo; Combs, Peter A.; Shaevitz, Joshua W.Proceedings of the National Academy of Sciences of the United States of America (2010), 107 (20), 9182-9185, S9182/1-S9182/6CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)A filamentous cytoskeleton largely governs the phys. shape and mech. properties of eukaryotic cells. In bacteria, proteins homologous to all three classes of eukaryotic cytoskeletal filaments have recently been discovered. These proteins are essential for the maintenance of bacterial cell shape and have been shown to guide the localization of key cell-wall-modifying enzymes. However, whether the bacterial cytoskeleton is stiff enough to affect the overall mech. rigidity of a cell has not been probed. Here, we used an optical trap to measure the bending rigidity of live Escherichia coli cells. We find that the actin-homolog MreB contributes nearly as much to the stiffness of a cell as the peptidoglycan cell wall. By quant. modeling these measurements, our data indicate that the MreB is rigidly linked to the cell wall, increasing the mech. stiffness of the overall system. These data are the first evidence that the bacterial cytoskeleton contributes to the mech. integrity of a cell in much the same way as it does in eukaryotes.
- 49Kawasaki, Y.; Kurosaki, K.; Kan, D.; Borges, I. K.; Atagui, A. S.; Sato, M.; Kondo, K.; Katahira, M.; Suzuki, I.; Takeda, M. Identification and Characterization of the S-Layer Formed on the Sheath of Thiothrix nivea. Arch. Microbiol. 2018, 200, 1257– 1265, DOI: 10.1007/s00203-018-1543-x[Crossref], [PubMed], [CAS], Google Scholar49https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXht1Smsb3O&md5=fe1da3fb23ad7254ce10edfb970738ecIdentification and characterization of the S-layer formed on the sheath of Thiothrix niveaKawasaki, Yuta; Kurosaki, Kaishi; Kan, Daisuke; Borges, Isabele Kazahaya; Atagui, Ayumi Satake; Sato, Michio; Kondo, Keiko; Katahira, Masato; Suzuki, Ichiro; Takeda, MinoruArchives of Microbiology (2018), 200 (8), 1257-1265CODEN: AMICCW; ISSN:0302-8933. (Springer)Thiothrix nivea is a filamentous sulfur-oxidizing bacterium common in activated sludge and its filament is covered with a polysaccharide layer called sheath. In this study, we found that T. nivea aggregates under acidic conditions. A hexagonal lattice pattern, a typical morphol. feature of proteinaceous S-layers, was newly obsd. on the surface of the sheath by transmission electron microscopy. The pattern and the acid-dependent aggregation were not obsd. in T. fructosivorans, a relative sheath-forming bacterium of T. nivea. The putative S-layer of T. nivea was detached by washing with unbuffered tris(hydroxymethyl)aminomethane base (Tris) soln. and a protein of 160 kDa was detected by electrophoresis. Based on partial amino acid sequences of the protein, its structural gene was identified. The gene encodes an acidic protein which has a putative secretion signal and a Ca2+-binding domain. The protein was solubilized with urea followed by dialysis in the presence of calcium. A hexagonal lattice pattern was obsd. in the aggregates formed during dialysis, revealing that the protein is responsible for S-layer formation. Biosorption ability of copper, zinc, and cadmium onto the T. nivea filament decreased upon pretreatment with Tris, demonstrating that the S-layer was involved in metal adsorption. Moreover, aggregation of Escherichia coli was promoted by acidification in the presence of the S-layer protein, suggesting that the protein is potentially applicable as an acid-driven flocculant for other bacteria.
- 50Suzuki, T.; Kanagawa, T.; Kamagata, Y. Identification of a Gene Essential for Sheathed Structure Formation in Sphaerotilus natans, a Filamentous Sheathed Bacterium. Appl. Environ. Microbiol. 2002, 68, 365– 371, DOI: 10.1128/AEM.68.1.365-371.2002[Crossref], [PubMed], [CAS], Google Scholar50https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38Xjt1Wlsw%253D%253D&md5=03f0274274fafb3c2a7108e3de9f8bfeIdentification of a gene essential for sheathed structure formation in Sphaerotilus natans, a filamentous sheathed bacteriumSuzuki, Toshihiko; Kanagawa, Takahiro; Kamagata, YoichiApplied and Environmental Microbiology (2002), 68 (1), 365-371CODEN: AEMIDF; ISSN:0099-2240. (American Society for Microbiology)Sphaerotilus natans, a filamentous bacterium that causes bulking in activated sludge processes, can assume two distinct morphologies, depending on the substrate concn. for growth; in substrate-rich media it grows as single rod-shaped cells, whereas in substrate-limited media it grows as filaments. To identify genes responsible for sheath formation, we carried out transposon Tn5 mutagenesis. Of the approx. 20,000 mutants obtained, 7 did not form sheathed structures. Sequencing of the Tn5-flanking regions showed that five of the seven Tn5 insertions converged at the same open reading frame, designated sthA. The deduced amino acids encoded by sthA were found to be homologous to glycosyltransferase, which is known to be involved in linking sugars to lipid carriers during bacterial exopolysaccharide biosynthesis. Disruption of the gene of the wild-type strain by inserting a kanamycin resistance gene cassette also resulted in sheathless growth under either type of nutrient condition. These findings indicate that sthA is a crucial component responsible for sheath formation.
- 51Kawasaki, Y.; Endo, T.; Fujiwara, A.; Kondo, K.; Katahira, M.; Nittami, T.; Sato, M.; Takeda, M. Elongation Pattern and Fine Structure of the Sheaths Formed by Thiothrix nivea and Thiothrix fructosivorans. Int. J. Biol. Macromol. 2017, 95, 1280– 1288, DOI: 10.1016/j.ijbiomac.2016.11.025[Crossref], [PubMed], [CAS], Google Scholar51https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhvVGgt77I&md5=c40e9a5d8d4a6d7ac0ef74716310b4b8Elongation pattern and fine structure of the sheaths formed by Thiothrix nivea and Thiothrix fructosivoransKawasaki, Yuta; Endo, Tomoyuki; Fujiwara, Atsuo; Kondo, Keiko; Katahira, Masato; Nittami, Tadashi; Sato, Michio; Takeda, MinoruInternational Journal of Biological Macromolecules (2017), 95 (), 1280-1288CODEN: IJBMDR; ISSN:0141-8130. (Elsevier B.V.)Thiothrix strains are filamentous sulfur-oxidizing bacteria common in activated sludge. Some of the members, including Thiothrix nivea and T. fructosivorans, are known to form a microtubular sheath that covers a line of cells. The sheaths are assemblages of [→4)-β-D-GlcN-(1 → 4)-β-D-Glc-(1→]n modified with unusual deoxy sugars. To elucidate the sheath-forming mechanism, the patterns of sheath formation and cell proliferation were detd. Prior to anal., both sheaths were confirmed to be highly de-N-acetylated. Sheaths in viable filaments were N-biotinylated followed by cultivation and then fluorescently immunostained. Epifluorescence microscopy of the filaments revealed ubiquitous elongation of the sheaths. For visualization of the cell proliferation pattern, the cell membrane was fluorescently stained. The epifluorescence images demonstrated that cell proliferation also proceeds ubiquitously, suggesting that sheath elongation proceeds surrounding an elongating cell. In addn., the fine structure of the Thiothrix filaments was analyzed by transmission electron microscopy employing a freeze-substitution technique. The micrographs of freeze-substituted filaments showed that the sheaths were thin and single layered. In contrast, the sheaths in chem. fixed filaments appeared thick and multilayered. Treatment with glutaraldehyde probably caused deformation of the sheaths. Supporting this possibility, the sheaths were found to be deformed or solubilized by N-acetylation.
- 52Kondo, K.; Umezu, T.; Shimura, S.; Narizuka, R.; Koizumi, J.-i.; Mashima, T.; Katahira, M.; Takeda, M. Structure of Perosamine-Containing Polysaccharide, a Component of the Sheath of Thiothrix fructosivorans. Int. J. Biol. Macromol. 2013, 59, 59– 66, DOI: 10.1016/j.ijbiomac.2013.04.013[Crossref], [PubMed], [CAS], Google Scholar52https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXptFOms7w%253D&md5=9cbbc84059ef6889e1dee235484b9990Structure of perosamine-containing polysaccharide, a component of the sheath of Thiothrix fructosivoransKondo, Keiko; Umezu, Takuto; Shimura, Shoichi; Narizuka, Rie; Koizumi, Jun-ichi; Mashima, Tsukasa; Katahira, Masato; Takeda, MinoruInternational Journal of Biological Macromolecules (2013), 59 (), 59-66CODEN: IJBMDR; ISSN:0141-8130. (Elsevier B.V.)A sheath-forming and sulfur-oxidizing bacterium, Thiothrix fructosivorans, was heterotrophically cultured. The sheath, which is an extracellular microtube, was prepd. by selectively removing the cells using lysozyme, sodium dodecyl sulfate, and sodium hydroxide. Solid-state 13C-NMR spectrum revealed that the sheath is assembled from a glycan possessing acetyl and Me groups. When the sheath was deacetylated, the original microtube structure was lost and the sheath became sol. under acidic conditions, revealing the importance of acetyl groups in maintaining the sheath structure. Equimolar D-glucose, D-glucosamine, and L-fucose were detected in the acid hydrolyzate of the sheath by gas liq. chromatog. In addn. to these sugars, β-GlcN-(1 → 4)-Glc and unidentified sugar were detected by analyzing the hydrolyzate using HPLC anal. 1H and 13C NMR spectroscopy was used to identify a disaccharide composed of 4-deoxy-4-aminorhamnose (perosamine, Rha4N) and fucose. N-Acetyl-perosamine prepd. from the disaccharide was polarimetric and exhibited a D-configuration. The previously unidentified disaccharide is α-D-Rhap4N-(1→3)-D-Fuc. According to 1H and 13C NMR analyses, the deacetylated sheath-forming polysaccharide was found to h have a main chain of [(→4)-β-D-GlcpN-(1 → 4)-β-D-Glcp-(1→)]n, to which disaccharide side chains of α-D-Rhap4N-(1→3)-α-L-Fucp-(1 →) were attached at position 3 of Glc.
- 53Stanley, C. E.; Grossmann, G.; Casadevall i Solvas, X.; deMello, A. J. Soil-On-A-Chip: Microfluidic Platforms for Environmental Organismal Studies. Lab Chip 2016, 16, 228– 241, DOI: 10.1039/C5LC01285F[Crossref], [PubMed], [CAS], Google Scholar53https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhvFWlu7vO&md5=064f9995395a44eab18893c8a99f5e01Soil-on-a-Chip: microfluidic platforms for environmental organismal studiesStanley, Claire E.; Grossmann, Guido; Casadevall i Solvas, Xavier; deMello, Andrew J.Lab on a Chip (2016), 16 (2), 228-241CODEN: LCAHAM; ISSN:1473-0189. (Royal Society of Chemistry)Soil is the habitat of countless organisms and encompasses an enormous variety of dynamic environmental conditions. While it is evident that a thorough understanding of how organisms interact with the soil environment may have substantial ecol. and economical impact, current lab.-based methods depend on reductionist approaches that are incapable of simulating natural diversity. The application of Lab-on-a-Chip or microfluidic technologies to organismal studies is an emerging field, where the unique benefits afforded by system miniaturization offer new opportunities for the experimentalist. Indeed, precise spatiotemporal control over the microenvironments of soil organisms in combination with high-resoln. imaging has the potential to provide an unprecedented view of biol. events at the single-organism or single-cell level, which in turn opens up new avenues for environmental and organismal studies. Herein we review some of the most recent and interesting developments in microfluidic technologies for the study of soil organisms and their interactions with the environment. We discuss how so-called "Soil-on-a-Chip" technol. has already contributed significantly to the study of bacteria, nematodes, fungi and plants, as well as inter-organismal interactions, by advancing exptl. access and environmental control. Most crucially, we highlight where distinct advantages over traditional approaches exist and where novel biol. insights will ensue.
- 54Nelson, Y. M.; Lion, L. W.; Shuler, M. L.; Ghiorse, W. C. Effect of Oxide Formation Mechanisms on Lead Adsorption by Biogenic Manganese (Hydr)Oxides, Iron (Hydr)Oxides, and Their Mixtures. Environ. Sci. Technol. 2002, 36, 421– 425, DOI: 10.1021/es010907c[ACS Full Text], [CAS], Google Scholar54https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XhsVWisQ%253D%253D&md5=2afadf2f0552d2d976909c749e3ee3dcEffect of Oxide Formation Mechanisms on Lead Adsorption by Biogenic Manganese (Hydr)oxides, Iron (Hydr)oxides, and Their MixturesNelson, Yarrow M.; Lion, Leonard W.; Shuler, Michael L.; Ghiorse, William C.Environmental Science and Technology (2002), 36 (3), 421-425CODEN: ESTHAG; ISSN:0013-936X. (American Chemical Society)The effects of iron and manganese (hydr)oxide formation processes on the trace metal adsorption properties of these metal (hydr)oxides and their mixts. was investigated by measuring Pb adsorption by iron and manganese (hydr)oxides prepd. by a variety of methods. Amorphous iron (hydr)oxide formed by fast pptn. at pH 7.5 exhibited greater Pb adsorption (Γmax = 50 mmol of Pb/mol of Fe at pH 6.0) than iron (hydr)oxide formed by slow, diffusion-controlled oxidn. of Fe(II) at pH 4.5-7.0 or goethite. Biogenic manganese(III/IV) (hydr)oxide prepd. by enzymic oxidn. of Mn(II) by the bacterium Leptothrix discophora SS-1 adsorbed five times more Pb (per mole of Mn) than an abiotic manganese (hydr)oxide prepd. by oxidn. of Mn(II) with permanganate, and 500-5000 times more Pb than pyrolusite oxides (β-MnO2). X-ray crystallog. indicated that biogenic manganese (hydr)oxide and iron (hydr)oxide were predominantly amorphous or poorly cryst. and their x-ray diffraction patterns were not significantly affected by the presence of the other (hydr)oxide during formation. When iron and manganese (hydr)oxides were mixed after formation, or for Mn biol. oxidized with iron(III) (hydr)oxide present, the obsd. Pb adsorption was similar to that expected for the mixt. based on Langmuir parameters for the individual (hydr)oxides. These results indicate that interactions in iron/manganese (hydr)oxide mixts. related to the formation process and sequence of formation such as site masking, alterations in sp. surface area, or changes in cryst. structure either did not occur or had a negligible effect on Pb adsorption by the mixts.
- 55Katsoyiannis, I. A.; Zouboulis, A. I. Application of Biological Processes for the Removal of Arsenic From Groundwaters. Water Res. 2004, 38, 17– 26, DOI: 10.1016/j.watres.2003.09.011[Crossref], [PubMed], [CAS], Google Scholar55https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BD3srltF2ltg%253D%253D&md5=08fece0d01f1658ff4223d2bf0979bccApplication of biological processes for the removal of arsenic from groundwatersKatsoyiannis Ioannis A; Zouboulis Anastasios IWater research (2004), 38 (1), 17-26 ISSN:0043-1354.Bacteria are widespread, abundant, geochemically reactive components of aquatic environments. In particular, iron-oxidizing bacteria, are involved in the oxidation and subsequent precipitation of ferrous ions. Due to this property, they have been applied in drinking water treatment processes, in order to accelerate the removal of ferrous iron from groundwaters. Iron also exerts a strong influence on arsenic concentrations in groundwater sources, while iron oxides are efficient adsorbents in arsenic removal processes. In the present study, the removal of arsenic (III and V), during biological iron oxidation has been investigated. The results showed that both inorganic forms of arsenic could be efficiently treated, for the concentration range of interest in drinking water (50-200microg/L). In addition, the oxidation of trivalent arsenic was found to be catalyzed by bacteria, leading to enhanced overall arsenic removal, because arsenic in the form of arsenites cannot be efficiently sorbed onto iron oxides. This method comprises a cost competitive technology, which can find application in treatment of groundwaters with elevated concentrations of iron and arsenic.
- 56Ghiorse, W. Biology of Iron- and Manganese-Depositing Bacteria. Annu. Rev. Microbiol. 1984, 38, 515– 550, DOI: 10.1146/annurev.mi.38.100184.002503[Crossref], [PubMed], [CAS], Google Scholar56https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL2cXmtVOht74%253D&md5=358a2be031bfbf201a76ada3ef999287Biology of iron- and manganese-depositing bacteriaGhiorse, W. C.Annual Review of Microbiology (1984), 38 (), 515-50CODEN: ARMIAZ; ISSN:0066-4227.A review with 200 refs.
- 57Rhoads, A.; Beyenal, H.; Lewandowski, Z. Microbial Fuel Cell Using Anaerobic Respiration as an Anodic Reaction and Biomineralized Manganese as a Cathodic Reactant. Environ. Sci. Technol. 2005, 39, 4666– 4671, DOI: 10.1021/es048386r[ACS Full Text], [CAS], Google Scholar57https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXktFWksLY%253D&md5=332aab447fb9215e6bf4947063a01f45Microbial Fuel Cell using Anaerobic Respiration as an Anodic Reaction and Biomineralized Manganese as a Cathodic ReactantRhoads, Allison; Beyenal, Haluk; Lewandowski, ZbigniewEnvironmental Science and Technology (2005), 39 (12), 4666-4671CODEN: ESTHAG; ISSN:0013-936X. (American Chemical Society)The authors have operated a microbial fuel cell in which glucose was oxidized by Klebsiella pneumoniae in the anodic compartment, and biomineralized manganese oxides, deposited by Leptothrix discophora, were electrochem. reduced in the cathodic compartment. In the anodic compartment, to facilitate the electron transfer from glucose to the graphite electrode, the authors added a redox mediator, 2-hydroxy-1,4-naphthoquinone. The authors did not add any redox mediator to the cathodic compartment because the biomineralized manganese oxides were deposited on the surface of a graphite electrode and were reduced directly by electrons from the electrode. Biomineralized manganese oxides are superior to oxygen when used as cathodic reactants in microbial fuel cells. The c.d. delivered by using biomineralized manganese oxides as the cathodic reactant was almost 2 orders of magnitude higher than that delivered using oxygen. Several fuel cells were operated for 500 h, reaching anodic potentials of -441.5 ± 31 mVSCE and cathodic potentials of +384.5 ± 64 mVSCE. When the electrodes were connected by a 50 Ω resistor, the fuel cell delivered the peak power d. of 126.7 ± 31.5 mW/m2.
- 58Yan, X.; Zhou, Q.; Vincent, M.; Deng, Y.; Yu, J.; Xu, J.; Xu, T.; Tang, T.; Bian, L.; Wang, Y.-X. J. Multifunctional Biohybrid Magnetite Microrobots for Imaging-Guided Therapy. Science Robotics 2017, 2, eaaq1155, DOI: 10.1126/scirobotics.aaq1155
- 59Bente, K.; Codutti, A.; Bachmann, F.; Faivre, D. Biohybrid and Bioinspired Magnetic Microswimmers. Small 2018, 14, 1704374, DOI: 10.1002/smll.201704374
- 60Sanchez, S.; Solovev, A. A.; Schulze, S.; Schmidt, O. G. Controlled Manipulation of Multiple Cells Using Catalytic Microbots. Chem. Commun. 2011, 47, 698– 700, DOI: 10.1039/C0CC04126B[Crossref], [PubMed], [CAS], Google Scholar60https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhsFGrtb3J&md5=eb971f1b9123eb8c433fe70e6328fcedControlled manipulation of multiple cells using catalytic microbotsSanchez, Samuel; Solovev, Alexander A.; Schulze, Sabine; Schmidt, Oliver G.Chemical Communications (Cambridge, United Kingdom) (2011), 47 (2), 698-700CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)Self-propelled microjet engines (microbots) can transport multiple cells into specific locations in a fluid. The motion is externally controlled by a magnetic field which allows to selectively load, transport and deliver the cells.
- 61Son, S. J.; Reichel, J.; He, B.; Schuchman, M.; Lee, S. B. Magnetic Nanotubes for Magnetic-Field-Assisted Bioseparation, Biointeraction, and Drug Delivery. J. Am. Chem. Soc. 2005, 127, 7316– 7317, DOI: 10.1021/ja0517365[ACS Full Text], [CAS], Google Scholar61https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXjslKqt78%253D&md5=3443abb4a5a2c85c3c1d299663bc3589Magnetic nanotubes for magnetic-field-assisted bioseparation, biointeraction, and drug deliverySon, Sang Jun; Reichel, Jonathan; He, Bo; Schuchman, Mattan; Lee, Sang BokJournal of the American Chemical Society (2005), 127 (20), 7316-7317CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Tubular structure of nanoparticles is highly attractive due to their structural attributes, such as the distinctive inner and outer surfaces, over conventional spherical nanoparticles. Inner voids can be used for capturing, concg., and releasing species ranging in size from large proteins to small mols. Distinctive outer surfaces can be differentially functionalized with environment-friendly and/or probe mols. to a specific target. Magnetic particles have been extensively studied in the field of biomedical and biotechnol. applications, including drug delivery, biosensors, chem. and biochem. sepn. and concn. of trace amts. of specific targets, and contrast enhancement in magnetic resonance imaging (MRI). Therefore, by combining the attractive tubular structure with magnetic property, the magnetic nanotube (MNT) can be an ideal candidate for the multifunctional nanomaterial toward biomedical applications, such as targeting drug delivery with MRI capability. Here, we successfully synthesized magnetic silica-iron oxide composite nanotubes and demonstrated the magnetic-field-assisted chem. and biochem. sepns., immunobinding, and drug delivery.
- 62Qin, D.; Xia, Y.; Whitesides, G. M. Soft Lithography for Micro- and Nanoscale Patterning. Nat. Protoc. 2010, 5, 491– 502, DOI: 10.1038/nprot.2009.234[Crossref], [PubMed], [CAS], Google Scholar62https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXislOmsbs%253D&md5=ddc10ed2bb9b84f4799c8ec3b59a473eSoft lithography for micro- and nanoscale patterningQin, Dong; Xia, Younan; Whitesides, George M.Nature Protocols (2010), 5 (3), 491-502CODEN: NPARDW; ISSN:1750-2799. (Nature Publishing Group)This protocol provides an introduction to soft lithog.-a collection of techniques based on printing, molding and embossing with an elastomeric stamp. Soft lithog. provides access to three-dimensional and curved structures, tolerates a wide variety of materials, generates well-defined and controllable surface chemistries, and is generally compatible with biol. applications. It is also low in cost, exptl. convenient and has emerged as a technol. useful for a no. of applications that include cell biol., microfluidics, lab-on-a-chip, microelectromech. systems and flexible electronics/photonics. As examples, here we focus on three of the commonly used soft lithog. techniques: (i) microcontact printing of alkanethiols and proteins on gold-coated and glass substrates; (ii) replica molding for fabrication of microfluidic devices in poly(di-Me siloxane), and of nanostructures in polyurethane or epoxy; and (iii) solvent-assisted micromolding of nanostructures in poly(Me methacrylate).
- 63Rotem, A.; Abate, A. R.; Utada, A. S.; Van Steijn, V.; Weitz, D. A. Drop Formation in Non-Planar Microfluidic Devices. Lab Chip 2012, 12, 4263– 4268, DOI: 10.1039/c2lc40546f[Crossref], [PubMed], [CAS], Google Scholar63https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhsVCqu7vM&md5=a8ad19fd65e0af7f14fd6eb13566204dDrop formation in non-planar microfluidic devicesRotem, Assaf; Abate, Adam R.; Utada, Andrew S.; Van Steijn, Volkert; Weitz, David A.Lab on a Chip (2012), 12 (21), 4263-4268CODEN: LCAHAM; ISSN:1473-0189. (Royal Society of Chemistry)Microfluidic devices can be used to produce single or multiple emulsions with remarkably precise control of both the contents and size of the drops. Since each level of a multiple emulsion is formed by a distinct fluid stream, very efficient encapsulation of materials can be achieved. To obtain high throughput, these devices can be fabricated lithog., allowing many devices to operate in parallel. However, to form multiple emulsions using a planar microfluidic device, the wettability of its surface must switch from hydrophobic to hydrophilic on the scale of micrometers where the drops are formed; this makes the fabrication of the devices very difficult. To overcome this constraint, non-planar microfluidic devices are introduced with graduated thicknesses; these can make drops even when their wetting properties do not favor drop formation. Nevertheless, the dependence of drop formation on the device geometry, the flow rates, and the properties of the fluids, particularly in the case of unfavorable wetting, is very complex, making the successful design of these devices more difficult. Here it is shown that there exists a crit. value of flow of the continuous phase above which drop formation occurs; this value decreases by two orders of magnitude as the wetting to the device wall of the continuous phase improves. How this new understanding can be used to optimize device design is demonstrated for efficient prodn. of double or multiple emulsions.
- 64Strathmann, M.; Wingender, J.; Flemming, H.-C. Application of Fluorescently Labelled Lectins for the Visualization and Biochemical Characterization of Polysaccharides in Biofilms of Pseudomonas aeruginosa. J. Microbiol. Methods 2002, 50, 237– 248, DOI: 10.1016/S0167-7012(02)00032-5[Crossref], [PubMed], [CAS], Google Scholar64https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XjvVejtrs%253D&md5=e3b42845df9f2eb4805b8bcc44eedd6dApplication of fluorescently labelled lectins for the visualization and biochemical characterization of polysaccharides in biofilms of Pseudomonas aeruginosaStrathmann, Martin; Wingender, Jost; Flemming, Hans-CurtJournal of Microbiological Methods (2002), 50 (3), 237-248CODEN: JMIMDQ; ISSN:0167-7012. (Elsevier Science B.V.)Fluorescently labeled lectins were used in combination with epifluorescence microscopy and confocal laser scanning microscopy to allow the visualization and characterization of carbohydrate-contg. extracellular polymeric substances (EPS) in biofilms of Pseudomonas aeruginosa. A mucoid strain characterized by an overprodn. of the exopolysaccharide alginate, and an isogenic, non-mucoid strain were used. Model biofilms grown on polycarbonate filters were treated with lectins Con A (ConA) and wheat germ agglutinin (WGA) that were fluorescently labeled with fluorescein isothiocyanate or tetra-Me rhodamine isothiocyanate. Fluorescently labeled ConA yielded cloud-like regions that were heterogeneously distributed within mucoid biofilms, whereas these structures were only rarely present in biofilms of the non-mucoid strain. The bacteria visualized with the fluorochrome SYTO 9 were localized both within and between the ConA-stained regions. In WGA-treated biofilms, the lectin was predominantly assocd. with bacterial cells. Alginate seemed to be involved in the interaction of ConA with the EPS matrix, since (i) pre-treatment of biofilms with an alginate lyase resulted in a loss of ConA biofilm staining, and (ii) using an enzyme-linked lectinsorbent assay (ELLA), ConA was shown to bind to purified alginate, but not to alginate that was degraded by alginate lyase. The application of fluorescently labeled lectins in combination with ELLA was found to be useful for the visualization and characterization of extracellular polysaccharide structures in P. aeruginosa biofilms.
- 65Toda, N.; Doi, A.; Jimbo, A.; Matsumoto, I.; Seno, N. Interaction of Sulfated Glycosaminoglycans with Lectins. J. Biol. Chem. 1981, 256, 5345– 5349[PubMed], [CAS], Google Scholar65https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL3MXkt1aksbw%253D&md5=25de5ab7d967117035a0f6edba455bbeInteraction of sulfated glycosaminoglycans with lectinsToda, Noriko; Doi, Akiko; Jimbo, Akiko; Matsumoto, Isamu; Seno, NobukoJournal of Biological Chemistry (1981), 256 (11), 5345-9CODEN: JBCHA3; ISSN:0021-9258.The sulfated glycosaminoglycans, such as keratan sulfate and chitin sulfate having 3-hydroxyl free N-acetyl-β-D-glucosaminyl residues as constituents, reacted with wheat germ agglutinin and Solanum tuberosum agglutinin by sugar-specific interaction. The glycosaminoglycans showed different inhibitory activities to the hemagglutination reaction of these lectins and keratan sulfate and its modified products formed insol. complexes with both of the lectins at pH 7.0 in physiol. saline soln. (0.15M NaCl). S. tuberosum Agglutinin was pptd. within a particularly narrow concn. range of keratan sulfate, and the formation of a sol. complex was obsd. by gel chromatog. These interactions were specifically inhibited by N,N'-diacetylchitobiose but not by 2M NaCl. The specific interactions of the glycosaminoglycans with S. tuberosum agglutinin were confirmed by their UV difference spectra with 2 peaks at 285 and 293 nm attributable to the tryptophan residues in the binding site of the agglutinin. S. tuberosum Agglutinin and wheat germ agglutinin have different binding specificities. The presence of sulfate groups in either keratan sulfate or chitin sulfate did not interfere with their specific interactions with S. tuberosum agglutinin as strongly as with wheat germ agglutinin. The N-acetylneuraminic acid residues in keratan sulfate were receptor sites for wheat germ agglutinin but not for S. tuberosum agglutinin.
- 66Takeda, M.; Makita, H.; Ohno, K.; Nakahara, Y.; Koizumi, J.-i. Structural Analysis of the Sheath of a Sheathed Bacterium, Leptothrix cholodnii. Int. J. Biol. Macromol. 2005, 37, 92– 98, DOI: 10.1016/j.ijbiomac.2005.09.002[Crossref], [PubMed], [CAS], Google Scholar66https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXhtFKnsbvP&md5=8e6b7d175ea42bf3bb5022cca65551e3Structural analysis of the sheath of a sheathed bacterium, Leptothrix cholodniiTakeda, Minoru; Makita, Hiroko; Ohno, Katsutoshi; Nakahara, Yuichi; Koizumi, Jun-ichiInternational Journal of Biological Macromolecules (2005), 37 (1-2), 92-98CODEN: IJBMDR; ISSN:0141-8130. (Elsevier B.V.)L. cholodnii is an aerobic sheath-forming bacterium often found in oligotrophic and metal-rich aquatic environments. The sheath of this bacterium was isolated by selectively lysing the cells. Glycine and cysteine were the major amino acids of the sheath. The sheath was readily dissolved in hydrazine, and a polysaccharide substituted with cysteine was recovered from the soln. Galactosamine, glucosamine and galacturonic acid were detected in the hydrazinolyzate by gas liq. chromatog. anal. FAB-MS anal. of the hydrazinolyzate suggested a sugar sequence of HexN-GalA-HexN-HexN. Methylation linkage anal. revealed the presence of 4-linked GalA, 3-linked HexN and 4-linked HexN. The sulfhydryl groups of the sheath were used for labeling with the fluorogenic reagent 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (ABD-F). The labeled sheath (ABD-sheath) was partially hydrolyzed and 3 fluorescent fragments were purified by HPLC. One of them was identified as ABD-cysteine. The 2nd was the ABD-cysteine tetramer. Another fragment was indicated to be a pentasaccharide substituted with ABD-cysteine by NMR anal. It can be assumed that the polysaccharide and peptide moieties of the sheath are connected by a cysteine residue. NMR anal. of the hydrazinolyzate revealed that the polysaccharide moiety of the sheath was constructed from a pentasaccharide repeating unit contg. 2-amino-2-deoxygalacturonic acid (GalNA): →4)-α-GalNA-(1→4)-α-D-GalN(p)-(1→4)-α-D-GalA(p)-(1→4)-β-D-GlcN(p)-(1→3)-β-D-GalN(p)-(1→.
- 67Sugimoto, S.; Okuda, K.; Miyakawa, R.; Sato, M.; Arita-Morioka, K.; Chiba, A.; Yamanaka, K.; Ogura, T.; Mizunoe, Y.; Sato, C. Imaging of Bacterial Multicellular Behaviour in Biofilms in Liquid by Atmospheric Scanning Electron Microscopy. Sci. Rep. 2016, 6, 25889, DOI: 10.1038/srep25889[Crossref], [PubMed], [CAS], Google Scholar67https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XotFOiu7s%253D&md5=a8394ff0a1cdec458a2e84413c140fceImaging of bacterial multicellular behaviour in biofilms in liquid by atmospheric scanning electron microscopySugimoto, Shinya; Okuda, Ken-ichi; Miyakawa, Reina; Sato, Mari; Arita-Morioka, Ken-ichi; Chiba, Akio; Yamanaka, Kunitoshi; Ogura, Teru; Mizunoe, Yoshimitsu; Sato, ChikaraScientific Reports (2016), 6 (), 25889CODEN: SRCEC3; ISSN:2045-2322. (Nature Publishing Group)Biofilms are complex communities of microbes that attach to biotic or abiotic surfaces causing chronic infectious diseases. Within a biofilm, microbes are embedded in a self-produced soft extracellular matrix (ECM), which protects them from the host immune system and antibiotics. The nanoscale visualisation of delicate biofilms in liq. is challenging. Here, we develop atm. SEM (ASEM) to visualise Gram-pos. and -neg. bacterial biofilms immersed in aq. soln. Biofilms cultured on electron-transparent film were directly imaged from below using the inverted SEM, allowing the formation of the region near the substrate to be studied at high resoln. We visualised intercellular nanostructures and the exocytosis of membrane vesicles, and linked the latter to the trafficking of cargos, including cytoplasmic proteins and the toxins hemolysin and coagulase. A thick dendritic nanotube network was obsd. between microbes, suggesting multicellular communication in biofilms. A universal immuno-labeling system was developed for biofilms and tested on various examples, including S. aureus biofilms. In the ECM, fine DNA and protein networks were visualised and the precise distribution of protein complexes was detd. (e.g., straight curli, flagella, and excreted cytoplasmic mol. chaperones). Our observations provide structural insights into bacteria-substratum interactions, biofilm development and the internal microbe community.
Supporting Information
ARTICLE SECTIONSThis material is available free of charge via the Internet at: . The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsnano.9b04663.
Figure S1: Microscopic images of DNA-stained cells in large aggregates cultured in a batch-culture. Figure S2: A schematic of the quasi-2D microfluidic chamber. Figure S3: Kymograph of an elongating filament. Figure S4: Fluorescent staining of NH2 groups in the nanofibrils. Figure S5: Fluorescent co-staining of sugars and NH2 groups in the nanofibrils. Figure S6: Additional ASEM images of SP-6 and SP-6SL cells. Figure S7: The radius of curvature measured as a function of measured for collisions between filaments and obstacles. Figure S8: Fluorescent staining of cytoplasmic membrane of SP-6 cells (PDF)
Movie S1: A representative time lapse video of SP-6 cells (AVI)
Movie S2: Tracking the centroid of SP-6 cells at the bottom of a glass bottom dish (AVI)
Movie S3: A representative time lapse video of SP-6 SL cells (AVI)
Movie S4: The time lapse video of a bending cell filaments (AVI)
Movie S5: The time lapse video of a reversing filaments (AVI)
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