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Research ArticleApril 15, 2014

Trade-offs in Engineering Sugar Utilization Pathways for Titratable Control
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Cite this: ACS Synth. Biol. 2015, 4, 2, 141–149

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https://doi.org/10.1021/sb400162z

Published April 15, 2014

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Abstract

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Titratable systems are common tools in metabolic engineering to tune the levels of enzymes and cellular components as part of pathway optimization. For nonmodel microorganisms with limited genetic tools, inducible sugar utilization pathways offer built-in titratable systems. However, these pathways can exhibit undesirable single-cell behaviors that hamper the uniform and tunable control of gene expression. Here, we applied mathematical modeling and single-cell measurements of l-arabinose utilization in Escherichia coli to systematically explore how sugar utilization pathways can be altered to achieve desirable inducible properties. We found that different pathway alterations, such as the removal of catabolism, constitutive expression of high-affinity or low-affinity transporters, or further deletion of the other transporters, came with trade-offs specific to each alteration. For instance, sugar catabolism improved the uniformity and linearity of the response at the cost of requiring higher sugar concentrations to induce the pathway. Within these alterations, we also found that a uniform and linear response could be achieved with a single alteration: constitutively expressing the high-affinity transporter. Equivalent modifications to the d-xylose utilization pathway yielded similar responses, demonstrating the applicability of our observations. Overall, our findings indicate that there is no ideal set of typical alterations when co-opting natural utilization pathways for titratable control and suggest design rules for manipulating these pathways to advance basic genetic studies and the metabolic engineering of microorganisms for optimized chemical production.

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SPECIAL ISSUE

This article is part of the Circuits in Metabolic Engineering special issue.

Metabolic engineering requires the optimization of native and heterologous pathways in order to maximize the yield of the desired chemical product, prevent the buildup of toxic intermediates, and maintain cell viability. A common strategy is finely tuning the expression of each cellular component using titratable systems. (1-3) These systems rely on exogenous inducer molecules to tune the expression levels of regulated genes; by varying the concentration of the inducer, the expression levels of the gene can be manipulated without additional genetic modifications to the host organism. A number of titratable systems are available that were derived from or inspired by natural processes—including nutrient utilization pathways, (2-5) riboswitches, (6-9) antibiotic resistance cassettes, (10-12) quorum sensing, (13) and light sensing. (14)

Metabolic engineering has been more frequently adopting nonmodel organisms that already possess desired pathways or have adapted to environments that parallel production conditions. (15) Achieving titratable control in these organisms is more challenging because of limited genetic tools as well as growth conditions that perturb the stability and function of the inducers or regulators. However, these organisms often encode multiple sugar utilization pathways that can serve as ready-made titratable systems. (16-25) Sugar utilization pathways are generally composed of transporters that import the sugar into the cytoplasm, catabolic enzymes that shunt the sugar into central metabolism, and regulators that activate the expression of the transporters and the enzymes when bound to the sugar (Figure 1A). Furthermore, pathways often respond to their individual sugar, offering orthogonal systems for the coordinated optimization of gene expression.

Figure 1. Co-opting sugar utilization pathways for titratable control of gene expression. (A) Components of inducible sugar utilization pathways. High affinity (T_H) and low-affinity (T_L) transporters import the sugar into the cell, while catabolic enzymes (E) shunt the sugar into central metabolism. Transcription regulators (R) up-regulate the expression of the transporters and the enzymes, but only in the presence of the sugar. (B) Desirable properties for a titratable response. These properties include a uniform response at all inducer concentrations, a large dynamic range (high δ), low inducer concentrations to induce the pathway (low EC₅₀), and strong linearity (low η).

The drawback of co-opting natural utilization pathways is their potential to exhibit undesirable behaviors. While an ideal titratable response should be uniform and linear (Figure 1B), sugar utilization pathways have been shown to exhibit bimodal, sharp responses that prevent fine-tuning of expression levels in all cells of the population. (26-30) These responses can be attributed to positive feedback within the pathway, which emerges from the sugar inducing the transporters that import more sugar into the cell. (27, 29, 31, 32) The pathways also possess negative feedback from induction of the catabolic enzymes that break down the sugar. (33) While negative feedback would be expected to counterbalance positive feedback, (33, 34) sugar catabolism also degrades the inducing sugar. With all of these contributing factors, a fundamental question is how to modify a given pathway to achieve a desirable titratable response.

A powerful model has been the l-arabinose utilization pathway in Escherichia coli. This pathway encodes one high-affinity transporter (encoded within the araFGH operon) and one low affinity transporter (encoded by the araE gene), is well characterized, and has been regularly used for inducible control. This pathway has also been shown to exhibit a bimodal response, complicating its use as a titratable system. (26) Accordingly, previous efforts sought to eliminate bimodality through the overexpression of one transporter along with multiple alterations to the pathway. Khlebnikov and co-workers demonstrated that heterologously expressing the low-affinity transporter AraE could remove bimodality, which required disrupting both endogenous transporters. (35, 36) Separately, Morgan-Kiss and co-workers demonstrated that heterologously expressing a substrate-relaxed mutant of the lactose transporter could eliminate bimodality in a strain lacking the l-arabinose transporters and catabolic enzymes. (37) As a result, the consensus is that overexpressing a transporter and stripping away almost all native components of the pathway should be performed to achieve titratable control. While these studies laid the groundwork for generating utilization pathways with titratable responses, they called for extensive genetic manipulations (i.e., plasmid-based expression of a transporter, disruption of multiple genetic loci) that may be difficult to achieve in nonmodel organisms and may not reflect ideal configurations for titratable control. In addition, a systematic analysis of all pathway alterations remains to be performed, which could reveal other configurations that require fewer modifications or generate a more desirable titratable response.

Here, we systematically evaluated how natural utilization pathways can be altered to achieve titratable control. Mathematical modeling and experimental probing of the l-arabinose utilization pathway revealed distinct trade-offs when altering the pathway: (i) constitutively expressing the low affinity-transporter yielded the most linear response but required additional modifications to eliminate bimodality, (ii) constitutively expressing one transporter and deleting the other transporter had both positive and negative effects on the response properties specific to the selected transporter, and (iii) sugar catabolism linearized the response and reduced the extent of bimodality at the cost of elevated sugar concentrations to induce the pathway. Within these trade-offs, one of the most desirable responses came from a single alteration: constitutively expressing the high-affinity transporter. Furthermore, maintaining sugar catabolism improved the linearity of the response at higher cell densities, perceivably due to negative feedback through depletion of the exogenous sugar. Similar trends were observed for the d-xylose utilization pathway, suggesting broad applicability of our findings. These insights demonstrate that there is no perfect set of typical alterations when co-opting sugar utilization pathways, wherein the optimal configuration will depend on the specific demands placed on the titratable system.

Results and Discussion

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We considered four metrics to assess the desirability of the response (Figure 1B): the extent of bimodality (the number of peaks in the fluorescence distribution), the dynamic range of the response (δ, measured as the ratio of expression levels at full and no induction), the inducer concentration to achieve half-maximal induction (EC₅₀), and the steepness of the response curve (η, measured as a Hill coefficient). An ideal titratable system would exhibit uniform expression across the cell population at all inducer concentrations, a large dynamic range, a low inducer concentration to achieve half-maximal induction, and a low Hill coefficient reflecting the steepness of the response curve. We used these metrics as the basis to evaluate the predicted or measured behaviors of sugar utilization.

Simple Mathematical Model of Sugar Utilization

We built a simple mathematical model of sugar utilization to qualitatively explore how different alterations to the pathway influence the response properties (see Supporting Information). This model was composed of two transporters that import sugar into the cell, one enzyme that breaks down the sugar, and one regulator that binds the sugar and subsequently activates the expression of the transporters and the enzyme. This general configuration captures the l-arabinose utilization pathway and many other pathways found in microorganisms. (16-25) Parameter values were selected for one high-affinity/low-capacity transporter and one low-affinity/high-capacity transporter at biologically relevant expression strengths and activities to yield a bistable response (Figure 2, see Supporting Information). This configuration parallels the l-arabinose utilization pathway and creates an undesired behavior for titratable control.

Figure 2. Simple mathematical model predicts trade-offs when altering the pathway structure. (A) The model assumes a base pathway comprising a high-affinity/low-capacity transporter (T_H) and a low-affinity/high-capacity transporter (T_L) that import extracellular sugar (S₀) into the cell, a catabolic enzyme (E) that degrades the sugar, and a constitutively expressed regulator that upregulates the expression of the transporters and the enzymes when bound to the sugar. The steady-state expression levels of the enzyme are reported as a function of extracellular sugar concentration. Note that all variables were nondimensionalized as part of the model derivation. Dashed lines indicate bifurcation regions. To alter the pathway, T_H was constitutively expressed (C,D) and the activity of T_L was further eliminated (E,F), T_L was constitutively expressed (G,H) and the activity of T_H was further eliminated (I,J), and the activity of the catabolic enzymes was eliminated (B,D,F,H,J). Three strengths of constitutive expression were selected for T_H and T_L (low, light blue; medium, blue; high, dark blue). See Supporting Information (SI) for more details.

Experimentally Probing l-arabinose Utilization to Explore Model Predictions

We concurrently altered and assessed the l-arabinose utilization pathway in E. coli K-12. This pathway was attractive because of its extensive characterization, (38) its widespread use as a titratable system, (2) and its bimodal response that prevents titratable control. (26, 35, 37) The pathway comprises three enzymes (encoded by araBAD) that shunt l-arabinose into the pentose phosphate pathway, a low-affinity transporter (encoded by araE), a high-affinity transporter (encoded by araFGH), and a dual transcription regulator (encoded by araC) that upregulates the expression of all other pathway components when bound to l-arabinose (SI Figure S1A). A third l-arabinose-responsive gene (araJ) encodes a putative transporter, although this gene appears to contribute negligibly to the pathway response (39, 40) and thus was not considered in our analyses.

To measure the response to l-arabinose, we equipped the wild type (WT) strain of E. coli MG1655 with a low-copy plasmid encoding the araB promoter upstream of the green fluorescent protein (GFP) gene. (41) The associated strain was then grown for 6 h in a defined medium supplemented with different concentrations of l-arabinose to mid log phase (ABS₆₀₀ ∼ 0.4) and subjected to flow cytometry analysis (SI Figure S2). The incubation time was selected such that longer incubation times would yield similar fluorescence distributions (SI Figure S3). Figure 3 displays representative response curves while Table 1 reports the calculated response metrics. The response curves are depicted as dot plots to capture the extent of bimodality within each fluorescence distribution (the relative size of each dot) and the fluorescence of each subpopulation (vertical location of each dot). Following this approach, the WT strain yielded a bimodal response across a wide range of l-arabinose concentrations (Figure 3A, Table 1) similar to previous observations. (35, 37)

Figure 3. Probing alterations to the l-arabinose utilization pathway in *E. coli*. The wild type pathway (A) was subjected to different alterations: *araFGH* was constitutively expressed (P_con-*araFGH*) (C,D) and *araE* was further deleted (Δ*araE*) (E,F), *araE* was constitutively expressed (P_con-*araE*) (G,H) and *araFGH* was further deleted (Δ*araFGH*) (I,J), and *araBAD* was deleted (Δ*araBAD*) (B,D,F,H,J). Each designated strain was back-diluted into M9 minimal medium supplemented with the indicated concentration of l-arabinose and grown for 6 h to ABS₆₀₀ ∼ 0.4 prior to flow cytometry analysis. For unimodal distributions, the resulting mean fluorescence is plotted. For bimodal distributions, two dots are plotted to represent the mean fluorescence and the relative number of cells in the induced (black) and uninduced (white) subpopulations (see SI Figure S2 for more details on the flow cytometry analysis). The diameter of each dot is directly proportional to the fraction of cells in that subpopulation. Gray boxes indicate the limit of detection due to autofluorescence. Each dot plot is representative of at least three independent experiments conducted on separate days. See Table 1 for the response metrics that account for the replicate experiments.

Table 1. Response Metrics for Each Alteration of the l-arabinose Utilization Pathway in E. coli^a

alteration	bimodality (Y/n)	δ	EC₅₀ (μM)	η
none (WT)	Y	958 ± 118	205 ×/÷ 2.08	0.89 ± 0.19
ΔaraBAD	Y	1023 ± 235	9.8 ×/÷ 1.17	1.56 ± 0.14
P_con-araFGH	n	290 ± 114	160 ×/÷ 1.47	0.96 ± 0.06
P_con-araFGH ΔaraBAD	n	900 ± 288	0.54 ×/÷ 1.20	1.44 ± 0.07
P_con-araFGH ΔaraE	n	262 ± 17	56 ×/÷ 1.16	1.18 ± 0.03
P_con-araFGH ΔaraE ΔaraBAD	n	1440 ± 300	0.48 ×/÷ 1.15	1.65 ± 0.03
P_con-araE	Y	828 ± 282	284 ×/÷ 1.02	0.75 ± 0.04
P_con-araE ΔaraBAD	Y	1351 ± 265	0.86 ×/÷ 1.08	3.27 ± 0.30
P_con-araE ΔaraFGH	n	737 ± 323	626 ×/÷ 1.56	0.76 ± 0.02
P_con-araE ΔaraFGH ΔaraBAD	n	728 ± 407	7.5 ×/÷ 1.04	1.67 ± 0.09
P_con-araE ΔaraFGH (ABS₆₀₀ ∼ 0.004)	n	517 ± 141	10.1 ×/÷ 1.13	1.47 ± 0.03
P_con-araE ΔaraFGH ΔaraBAD (ABS₆₀₀ ∼ 0.004)	n	663 ± 128	7.1 ×/÷ 1.10	1.86 ± 0.01
P_con-araE ΔaraFGH (ABS₆₀₀ ∼ 0.04)	n	371 ± 91	21.2 ×/÷ 1.13	1.25 ± 0.10
P_con-araE ΔaraFGH ΔaraBAD (ABS₆₀₀ ∼ 0.04)	n	512 ± 78	8.6 ×/÷ 1.32	1.78 ± 0.08
P_con-araE ΔaraFGH (ABS₆₀₀ ∼ 1.0)	n	1060 ± 161	2050 ×/÷ 1.07	0.73 ± 0.01
P_con-araE ΔaraFGH ΔaraBAD (ABS₆₀₀ ∼ 1.0)	n	2410 ± 590	7.7 ×/÷ 1.19	1.64 ± 0.02

Listed values are the mean and S.E.M. (dynamic range, δ; Hill coefficient, η) or the geometric mean and geometric S.E.M. (effective concentration to achieve 50% induction, EC₅₀) for three independent experiments conducted on separate days. All values reflect cultures grown to ABS₆₀₀ ∼ 0.4 unless indicated otherwise. The dynamic range was calculated as the ratio of the maximal to minimal mean fluorescence of the entire population, with autofluorescence subtracted from each value. The large error associated with the dynamic range can be attributed to basal levels approaching autofluorescence of cells lacking the reporter plasmid.

Constitutively Expressing the High-Affinity Transporter More Readily Generates a Uniform Response

We considered different alterations to sugar utilization pathways that were previously applied: constitutively expressing one of the transporters, deleting the other transporter, and deleting the catabolic genes. (35-37) From a network perspective, each alteration affects feedback within the pathway: constitutively expressing or deleting the transporter genes disrupts positive feedback, whereas deleting the catabolic genes disrupts negative feedback. While differential growth on the sugar could also impart feedback, (42) we found that the addition of l-arabinose did not have a major impact on growth rates for any of the pathway alterations (SI Table S1).

We first explored the impact of constitutively expressing either transporter to partially disrupt positive feedback in the pathway. Within the model, we replaced the regulator-dependent expression term for either transporter with different constant values. We found that all evaluated expression strengths of the high-affinity transporter eliminated the bifurcation region (Figure 2C). In contrast, only the highest expression strength of the low-affinity transporter eliminated the bifurcation region (Figure 2G). In both cases, increasing the promoter strength reduced the EC₅₀ value by allowing greater sugar import at lower extracellular concentrations. Because positive feedback is required for bistability, the model predictions suggest that the high-affinity transporter rather than the low-affinity transporter primarily drives bistability, at least under the selected parameter set.

To experimentally probe model predictions, we replaced the native araF or araE promoter with a medium-strength, synthetic promoter (P_con-araFGH or P_con-araE) and measured the response to l-arabinose (Figure 3C,G). Paralleling model predictions, constitutively expressing the high-affinity transporter eliminated bimodality, whereas constitutively expressing the araE low-affinity transporter reduced but did not eliminate bimodality. These results clearly demonstrate that (i) constitutively expressing the high-affinity transporter araFGH more readily generated a uniform response and (ii) only a single modification was required to fully convert a bimodal response into a uniform response.

Trade-offs when Deleting One Transporter

We next explored the impact of deleting one of the transporters when the other transporter was constitutively expressed. Within the model, the second transporter was removed by setting its maximal activity (α_H, α_L) equal to zero. The resulting impact on the predicted response was mixed and specific to the transporter (Figure 2E,I). Removing the high-affinity transporter from the pathway with a constitutively expressed low-affinity transporter eliminated the bifurcation region but also shifted the curves to higher extracellular sugar concentrations (Figure 2I). This effect may be attributed to the high-affinity transporter allowing sugar import at lower sugar concentrations while driving positive feedback within the pathway. In support of model predictions, deletion of the araFGH operon (ΔaraFGH) from the l-arabinose pathway with constitutively expressed araE resulted in a uniform response with a modest but statistically significant increase in the EC₅₀ value (p-value = 0.019) (Figure 3I, Table 1).

The model predicted that removing the low-affinity transporter from the pathway constitutively expressing the high-affinity transporter had a negligible effect (compare parts C and E in Figure 2). While this may suggest that the low-affinity transporter is poorly induced for our parameter set, the low-affinity transporter alone could mediate full induction of the pathway at high extracellular sugar concentrations (SI Figure S4). Deletion of araE (ΔaraE) from the l-arabinose utilization pathway constitutively expressing araFGH also maintained the uniform response (Figure 3E). However, there was a significant decrease in the EC₅₀ value (p-value = 0.008) and a significant increase in the Hill coefficient (p-value = 0.003). These changes would suggest that the low-affinity transporter contributes more than that predicted by the model by extending the concentration range that titrates the response. These changes also suggest that deletion of the low-affinity transporter poses a distinct trade-off: a lower EC₅₀ value at the cost of a sharper response.

Trade-offs when Eliminating Sugar Catabolism

We next explored the impact of removing sugar catabolism. Previous work concluded that catabolism posed a barrier to a titratable response by breaking down the inducing molecule. (37) Separately, sugar catabolism was predicted to entirely eliminate bistability, (33) suggesting conflicting contributions. To examine the impact of removing catabolism in the model, we set the activity of the enzyme equal to zero (α_E = 0) and evaluated the impact on the response metrics for all pathway configurations (Figure 2B, D, F, H, J). The model predicted both positive and negative effects of removing catabolism. As a positive effect, removing sugar catabolism lowered the apparent EC₅₀ value by preventing the intracellular breakdown of the sugar. As two negative effects, removing sugar catabolism enhanced the propensity for bistability and sharpened the response. Both negative effects can be attributed to sugar catabolism conferring negative feedback on the pathway similar to negative feedback in transcriptional circuits. (34, 43, 44)

To test these predictions, we deleted the araBAD operon (ΔaraBAD) from all strains and measured the single-cell response curves (Figure 3B, D, F, H, J; Table 1). For all pathways (Figure 3), the response metrics aligned with model predictions. In particular, removing l-arabinose catabolism significantly lowered the EC₅₀ value (p-values = 1 × 10^–6 to 0.008), with over 100-fold differences in some cases. Furthermore, removing l-arabinose catabolism sharpened the response (p-values = 3 × 10^–5 to 0.005) and increased the propensity for bimodality. Finally, removing l-arabinose catabolism did not have a consistent impact on the dynamic range, where a statistically significant increase was observed for only two strains (P_con-araFGH, p-value = 0.027; P_con-araFGH ΔaraE, p-value = 0.010) (Table 1). Thus, we conclude that removing l-arabinose catabolism poses a major trade-off between the amount of sugar necessary to induce the pathway and the sharpness of the response.

Breakdown of the Sugar Can Help Linearize the Response

Catabolism can deplete the reserve of sugar in the medium, which has been viewed as a barrier to the generation of titratable systems. (37) However, such depletion would form negative feedback by depleting the available sugar to induce the pathway, potentially benefiting the response. To explore these potential effects, we first modified the simple model to allow for the depletion of the extracellular sugar through catabolism (see Supporting Information). The resulting model was then employed to predict the response in the presence of catabolism. As part of the model, we could specify the relative volume of the cells to the medium (ν), which dictates in part the rate of depletion. We specifically focused on the pathway with the constitutively expressed low-affinity transporter (T_L = 0.02) and the deleted high-affinity transporter (α_H = 0) that yielded a graded response in the presence or absence of sugar catabolism. The model predicted that depletion of the extracellular sugar flattened the curve and elevated the EC₅₀ value (Figure 4A). High depletion (ν = 0.01) was detrimental based on the sharp response close to saturating sugar concentrations, although intermediate depletion (ν < 0.01) improved the overall linearity of the response.

Figure 4. Effect of cell density in the presence or absence of sugar catabolism. (A) Model predictions for the pathway with a constitutively expressed low-affinity transporter (T_L = 0.2) and the deleted high-affinity transporter (α_H = 0) when accounting for depletion of extracellular sugar through catabolism. Each simulation was conducted to τ = 10. The different curves reflect the relative volume of the cells to the medium (ν). Note that all variables were nondimensionalized as part of the model derivation. See Supporting Information for more details. (B) Growth curves for the P_con-*araE* Δ*araFGH* strain with or without (Δ*araBAD*) sugar catabolism in defined medium with or without 10 mM l-arabinose. Each value represents the mean of three independent experiments. The SEM for each measurement was smaller than the symbol. (C) Representative dot plots for both strains in log phase grown to the indicated final cell densities. See Figure 3 for more information on the dot plots. Each dot plot is representative of at least three experiments conducted from independent colonies. See Table 1 for the response metrics that account for the replicate experiments.

To test these predictions, we cultured the P_con-araFGH ΔaraE and P_con-araFGH ΔaraE ΔaraBAD strains for 6 h to final cell densities ranging from ABS₆₀₀ ∼ 0.004 to ABS₆₀₀ ∼ 1.0 (Figure 4B). Our reasoning was that extracellular l-arabinose would be more depleted at higher densities, but only in the strain with catabolism. For this strain, we observed higher EC₅₀ values and lower Hill coefficients with increasing cell densities, paralleling model predictions (Figure 4C, Table 1). For the strain lacking catabolism, we observed similar EC₅₀ values and Hill coefficients for all cell densities. These findings provide further support for a surprising benefit of sugar catabolism at higher densities and offer a separate form of negative feedback in sugar utilization that may generate more desirable titratable responses. A notable downside to this strategy is that the extent of induction peaks and then decreases over time at higher cell densities (SI Figure S5), potentially complicating the tuning of expression levels.

Similar Trends When Linearizing the Response to d-xylose

To extend the generality of our insights, we focused on the d-xylose utilization pathway in E. coli. Similar to the l-arabinose utilization pathway, the d-xylose utilization pathway encodes a high-affinity transporter and a low-affinity transporter, a transcriptional activator that recognizes d-xylose, and enzymes that shunt d-xylose into the pentose phosphate pathway (Figure S1B). Also paralleling the l-arabinose utilization pathway, the d-xylose utilization pathway exhibits a bimodal response when tracking the activity of the xylA promoter (Figure 5A).

Figure 5. Linearizing the response to d-xylose. The wild type *E. coli* strain (A), the strain constitutively expressing the high-affinity transporter *xylFGH* (B), and the strain constitutively expressing the high-affinity transporter *xylFGH* and lacking the catabolic operon *xylAB* (C) each harbored the reporter plasmid pUA66-pxylA. The designated strain was back-diluted into M9 minimal medium supplemented with the indicated concentration of d-xylose and grown for 6 h to ABS₆₀₀ ∼ 0.4 prior to flow cytometry analysis. See Figure 3 for more information on the dot plots. Each dot plot is representative of at least three experiments conducted from independent colonies.

We first asked how constitutively expressing the native high-affinity transporter (xylFGH) impacts the response. Paralleling the l-arabinose utilization pathway, constitutively expressing the endogenous copy of xylFGH resulted in a graded response at all examined d-xylose concentrations (Figure 5B). We next deleted the catabolic operon (xylAB) to evaluate the impact of removing d-xylose catabolism. Further paralleling the l-arabinose utilization pathway, deletion of the catabolic operon significantly reduced the EC₅₀ value (99.8 μM ×/÷ 2.20 to 0.033 μM ×/÷ 1.10, p-value = 0.0014) at the cost of an increased Hill coefficient (1.00 ± 0.08 to 1.58 ± 0.24, p-value = 0.019) (Figure 5B). Collectively, we were able to generate a linear response to d-xylose through a single alteration and observed similar trade-offs when further deleting sugar catabolism, all matching our observations for the l-arabinose utilization pathway.

Design Rules to Engineer Sugar Utilization Pathways for Titratable Control

Overall, our combination of mathematical modeling and experimental probing of l-arabinose and d-xylose utilization suggest general design rules when co-opting natural sugar utilization pathways as titratable systems. The principal rule is that there is no single, perfect set of alterations within the commonly used set that we investigated. Instead, each alteration comes with specific trade-offs beyond the required number of genomic alterations. Accordingly, the best set of alterations will depend on the demands of the desired titratable system. For instance, systems that rely on expensive inducers would opt for modifications that minimize the EC₅₀ value, whereas systems that require fine-tuning would minimize the Hill coefficient. The trade-offs principally applied to the EC₅₀ value and the Hill coefficient, whereas alterations did not predictably impact the dynamic range.

The next rule pertains to the need for a uniform response free of bimodality. A uniform response can be most readily achieved by constitutively expressing the high-affinity transporter rather than the low-affinity transporter. Theoretically, sufficient expression of either transporter can eliminate bimodality, although the high-affinity transporter could be expressed at much lower levels. The advantage of this strategy is that lower expression conserves cellular resources and limits the toxicity of overexpressing membrane proteins.

The third rule relates to trade-offs associated with removing one transporter when the other is constitutively expressed. Removing the high-affinity transporter can help eliminate bimodality at the potential cost of a higher EC₅₀ value, whereas removing the low-affinity transporter reduces the EC₅₀ value at the cost of a sharper response. An alternative would be constitutively expressing the second transporter, which could be explored in future studies.

A final rule pertains to sugar catabolism. Aside from requiring higher sugar concentrations to induce the pathway, sugar catabolism helps reduce the extent of bimodality and linearizes the response. Linearization may be further enhanced when cultures are grown to higher cell densities. Achieving consistent densities between experiments may be challenging and entry into stationary phase may alter pathway activity (SI Figure S5). Furthermore, sugar catabolism may perturb central metabolism as part of metabolic engineering that complicates the results. However, the enhanced linearity would offer finely tuned control over gene expression that may be essential when precise pathway optimization is needed.

The design rules described above were extracted from a simple model and probing of l-arabinose utilization. The rules also applied to the d-xylose utilization pathway, suggesting broader applicability. However, many utilization pathways exhibit variations on the simple pathway, such as anabolic pathways (45) or pathways that are induced by metabolic intermediates. (46) Further analysis of these pathways will offer insights into the desirability of their natural responses and any required manipulations. Separately, we relied on a more restrained parameter set to probe alterations. Further interrogating parameter space may reveal other parameters and design rules tailored to the behavior of the native pathway. With additional insights, a set of more detailed design principles could be developed. The end result will be reliable means to convert the myriad of sugar utilization pathways in the microbial world into versatile titratable systems for fundamental genetic studies and for the engineering of microorganisms with optimized metabolic processes.

Methods

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Bacterial Strains and Plasmids

All strains used in this work were derived from Escherichia coli K-12 substrain MG1655 and are listed in SI Table S2. With the exception of [ΔxylAB P_xylF]::[cat P_con], all genome modifications were achieved by PCR amplifying the bla or cat resistance cassette from pKD3 and recombineering the linear DNA into NM400 through mini-λ-mediated recombination. (47) The corresponding oligonucleotides are listed in SI Table S4. To insert the constitutive promoters, the promoter sequence (J23110 from the Registry of Standard Biological Parts) was included in one of the primers used to amplify the resistance cassette followed by a second set of primers to add the homology arms for recombination. Successful recombinants were verified by PCR and by sequencing. The genomic locus with the resistance cassette was then transferred to MG1655 by P1 transduction. In the case of ΔaraBAD::cat, the cat cassette was excised with the pCP20 plasmid to allow for the subsequent insertion of the cat cassette as part of the replacement of the promoters for araE or for araFGH. (48) All P1 transductions were verified by PCR. In the case of [ΔxylAB P_xylF]::[cat P_con], the amplified cat cassette was recombineered directly into MG1655 using pDK46.

Once generated, each strain was transformed with the pUA66-ParaB or pUA66-PxylA reporter plasmid encoding the low-copy sc101 origin-of-replication, the promoter region of the araBAD operon or the xylAB operon, respectively, and a downstream copy of gfpmut2 with a strong ribosome-binding site sequence. (41) The reporter plasmids along with the cloning and recombination plasmids are listed in SI Table S3. Each reporter plasmid offers a balance between maximizing the fluorescence for high sensitivity and minimizing the copy number to limit titration of the transcription regulators AraC and XylR. (49)

Growth Conditions and Media

Strains harboring the reporter plasmid were streaked out from glycerol stocks onto LB plates supplemented with appropriate antibiotics. Single colonies were then inoculated in 2 mL of M9 minimal medium (1X M9 salts, 10 μg/mL thiamine, 2 mM MgSO₄, 0.1 mM CaCl₂ supplemented with 0.4% glycerol, 0.2% casamino acids, and 0.25 μg/mL kanamycin) and grown overnight at 250 rpm and 37 °C. The overnight cultures were then back-diluted into 2 mL of the same medium with varying concentrations of the indicated sugar and grown under the same conditions for 6 h to a final ABS₆₀₀ of ∼ 0.4 unless indicated otherwise. Cell density measurements were performed on a Nanodrop 2000c (Thermo Scientific). For the time-course experiments (SI Figure S3), cultures were grown for the indicated time to the same final ABS₆₀₀ of ∼ 0.4.

Flow Cytometry Analysis

Cells were diluted 1:100 in 1× PBS and run on an Accuri C6 flow cytometer (Becton Dickinson) equipped with CFlow plate sampler, a 488 nm laser, and a 530 ± 15 nm bandpass filter. Cells were gated based on forward scatter (FSC-H) and side scatter (SSC-H) using a gate set based on experiments with DRAQ5 dye (Thermo Scientific). A lower cutoff of 11 500 au for FSC-H and 500 au for SSC-H was used to eliminate the appearance of excessive noise. At least 20 000 cells were collected for each sample. See SI Figure S2 for more information on the analysis.

Curve Fitting to Extract Performance Metrics

The Hill equation (Y = a*Xⁿ/(Kⁿ + Xⁿ) + b, where a, K, and n are fit constants and b is the fluorescence in the absence of the inducing sugar) was used to fit the mean fluorescence values (Y) across the different applied concentrations of applied sugar (X). Curve fitting was performed using the least-squares approach with the natural log of the measured and predicted mean fluorescence values. Of the fit values, n is the Hill coefficient (η), K is the EC₅₀ value, and (a + b – AF)/(b – AF) is the dynamic range (δ) (where AF is autofluorescence of the cells harboring the pUA66 plasmid).

Mathematical Modeling

All simulations were conducted in MATLAB. The ordinary differential equations were integrated using the Euler method. The stable and unstable steady-states were calculated by implementing the arc-length continuation method. The details of the model can be found in Supporting Information, including its derivation and the selected values for the model parameters.

Statistical Analyses

P-values were calculated using the student t test with unequal variance when comparing all parameter metrics. The test used one tail for expected changes between metrics (e.g., an increase in the Hill coefficient) and two tails when no changes were expected. EC₅₀ values were assumed to follow a log-normal distribution.

Supporting Information

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Figures S1–S5, Tables S1–S4, and a detailed description of the simple mathematical model. This material is available free of charge via the Internet at http://pubs.acs.org.

sb400162z_si_001.pdf (1.03 MB)

Terms & Conditions

Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.

Author Information

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Corresponding Author
- Chase L. Beisel - Department of Chemical and Biomolecular Engineering North Carolina State University Raleigh, North Carolina 27695, United States; Email: [email protected]
Authors
- Taliman Afroz - Department of Chemical and Biomolecular Engineering North Carolina State University Raleigh, North Carolina 27695, United States
- Konstantinos Biliouris - Department of Chemical Engineering and Materials Science University of Minnesota Minneapolis, Minnesota 55455, United States
- Kelsey E. Boykin - Department of Chemical and Biomolecular Engineering North Carolina State University Raleigh, North Carolina 27695, United States
- Yiannis Kaznessis - Department of Chemical Engineering and Materials Science University of Minnesota Minneapolis, Minnesota 55455, United States
Author Contributions
C.L.B. and T.A. conceived of the study; T.A. and K.E.B. performed the experiments; C.L.B. and T.A. designed and interpreted the experiments; K.B. performed and interpreted the modeling; K.B., Y.K., and C.L.B. designed the model; and C.L.B., T.A. and K.B. wrote the manuscript.
Notes
The authors declare no competing financial interest.

Acknowledgment

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This work was supported by an Undergraduate Research Grant (to K.E.B.), start-up funds from North Carolina State University (to C.L.B.), a mentoring mini-grant from the ADVANCE D-3 program (to C.L.B.), a Doctoral Dissertation Fellowship awarded to K.B., and grants from the National Institutes of Health (GM086865), the National Science Foundation (CBET-0425882, CBET-0644792) and the Minnesota Supercomputing Institute. This work used the Extreme Science and Engineering Discovery Environment (XSEDE), which is supported by National Science Foundation grant number OCI-1053575.

References

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This article references 49 other publications.

1
Temme, K., Zhao, D., and Voigt, C. A. (2012) Refactoring the nitrogen fixation gene cluster from Klebsiella oxytoca Proc. Natl. Acad. Sci. U.S.A. 109, 7085– 7090
Google Scholar
1
Refactoring the nitrogen fixation gene cluster from Klebsiella oxytoca
Temme Karsten; Zhao Dehua; Voigt Christopher A
Proceedings of the National Academy of Sciences of the United States of America (2012), 109 (18), 7085-90 ISSN:.
Bacterial genes associated with a single trait are often grouped in a contiguous unit of the genome known as a gene cluster. It is difficult to genetically manipulate many gene clusters because of complex, redundant, and integrated host regulation. We have developed a systematic approach to completely specify the genetics of a gene cluster by rebuilding it from the bottom up using only synthetic, well-characterized parts. This process removes all native regulation, including that which is undiscovered. First, all noncoding DNA, regulatory proteins, and nonessential genes are removed. The codons of essential genes are changed to create a DNA sequence as divergent as possible from the wild-type (WT) gene. Recoded genes are computationally scanned to eliminate internal regulation. They are organized into operons and placed under the control of synthetic parts (promoters, ribosome binding sites, and terminators) that are functionally separated by spacer parts. Finally, a controller consisting of genetic sensors and circuits regulates the conditions and dynamics of gene expression. We applied this approach to an agriculturally relevant gene cluster from Klebsiella oxytoca encoding the nitrogen fixation pathway for converting atmospheric N(2) to ammonia. The native gene cluster consists of 20 genes in seven operons and is encoded in 23.5 kb of DNA. We constructed a "refactored" gene cluster that shares little DNA sequence identity with WT and for which the function of every genetic part is defined. This work demonstrates the potential for synthetic biology tools to rewrite the genetics encoding complex biological functions to facilitate access, engineering, and transferability.
2
Guzman, L. M., Belin, D., Carson, M. J., and Beckwith, J. (1995) Tight regulation, modulation, and high-level expression by vectors containing the arabinose P_BAD promoter J. Bacteriol. 177, 4121– 4130
Google Scholar
2
Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter
Guzman, Luz-Maria; Belin, Dominique; Carson, Michael J.; Beckwith, Jon
Journal of Bacteriology (1995), 177 (14), 4121-30CODEN: JOBAAY; ISSN:0021-9193. (American Society for Microbiology)
We have constructed a series of plasmid vectors (pBAD vectors) contg. the PBAD promoter of the araBAD (arabinose) operon and the gene encoding the pos. and neg. regulator of this promoter, araC. Using the phoA gene and phoA fusions to monitor expression in these vectors, we show that the ratio of induction/repression can be 1,200-fold, compared with 50-fold for PTAC-based vectors. PhoA expression can be modulated over a wide range of inducer (arabinose) concns. and reduced to extremely low levels by the presence of glucose, which represses expression. Also, the kinetics of induction and repression are very rapid and significantly affected by the ara allele in the host strain. Thus, the use of this system which can be efficiently and rapidly turned on and off allows the study of important aspects of bacterial physiol. in a very simple manner and without changes of temp. We have exploited the tight regulation of the PBAD promoter to study the phenotypes of null mutations of essential genes and explored the use of pBAD vectors as an expression system.
3
De Boer, H. A., Comstock, L. J., and Vasser, M. (1983) The tac promoter: A functional hybrid derived from the trp and lac promoters Proc. Natl. Acad. Sci. U.S.A. 80, 21– 25
Google Scholar
3
The tac promoter: a functional hybrid derived from the trp and lac promoters
De Boer, Herman A.; Comstock, Lisa J.; Vasser, Mark
Proceedings of the National Academy of Sciences of the United States of America (1983), 80 (1), 21-5CODEN: PNASA6; ISSN:0027-8424.
Two hybrid promoters that are functional in Escherichia coli were constructed. These hybrid promoters, tacI and tacII, were derived from sequences of the trp and the lac UV5 promoters. In the 1st hybrid promoter (tacI), the DNA upstream of position -20 with respect to the transcriptional start site was derived from the trp promoter. The DNA downstream of position -20 was derived from the lac UV5 promoter. In the 2nd hybrid promoter (tacII), the DNA upstream of position -11 at the HpaI site within the Pribnow box was derived from the trp promoter. The DNA downstream of position -11 is a 46-base-pair synthetic DNA fragment that specifies part of the hybrid Pribnow box and the entire lac operator. It also specifies a Shine-Dalgarno sequence flanked by 2 unique restriction sites (portable Shine-Dalgarno sequence). The tacI and the tacII promoters resp. direct transcription ∼11 and ∼7 times more efficiently than does the derepressed parental lac UV5 promoter and ∼3 and ∼2 times more efficiently than does the trp promoter in the absence of the trp repressor. Both hybrid promoters can be repressed by the lac repressor, and both can be derepressed with isopropyl β-D-thiogalactoside [367-93-1]. Consequently, these hybrid promoters are useful for the controlled expression of foreign genes at high levels in E. coli. In contrast to the trp and the lac UV5 promoters, the tacI promoter has not only a consensus -35 sequence but also a consensus Pribnow box sequence. This may explain the higher efficiency of this hybrid promoter with respect to either one of the parental promoters.
4
Weber, W., Stelling, J., Rimann, M., Keller, B., Daoud-El Baba, M., Weber, C. C., Aubel, D., and Fussenegger, M. (2007) A synthetic time-delay circuit in mammalian cells and mice Proc. Natl. Acad. Sci. U.S.A. 104, 2643– 2648
Google Scholar
4
A synthetic time-delay circuit in mammalian cells and mice
Weber, Wilfried; Stelling, Joerg; Rimann, Markus; Keller, Bettina; Daoud-El Baba, Marie; Weber, Cornelia C.; Aubel, Dominique; Fussenegger, Martin
Proceedings of the National Academy of Sciences of the United States of America (2007), 104 (8), 2643-2648CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
Time-delay circuitries in which a transcription factor processes independent input parameters can modulate NF-κB activation, manage quorum-sensing cross-talk, and control the circadian clock. We have constructed a synthetic mammalian gene network that processes four different input signals to control either immediate or time-delayed transcription of specific target genes. BirA-mediated ligation of biotin to a biotinylation signal-contg. VP16 transactivation domain triggers heterodimerization of chimeric VP16 to a streptavidin-linked tetracycline repressor (TetR). At increasing biotin concns. up to 20 nM, TetR-specific promoters are gradually activated (off to on, input signal 1), are maximally induced at concns. between 20 nM and 10 μM, and are adjustably shut off at biotin levels exceeding 10 μM (on to off, input signal 2). These specific expression characteristics with a discrete biotin concn. window emulate a biotin-triggered bandpass filter. Removal of biotin from the culture environment (input signal 3) results in time-delayed transgene expression until the intracellular biotinylated VP16 pool is degraded. Because the TetR component of the chimeric transactivator retains its tetracycline responsiveness, addn. of this antibiotic (input signal 4) overrides biotin control and immediately shuts off target gene expression. Biotin-responsive immediate, bandpass filter, and time-delay transcription characteristics were predicted by a computational model and have been validated in std. cultivation settings or biopharmaceutical manufg. scenarios using transgenic CHO-K1 cell derivs. and have been confirmed in mice. Synthetic gene circuitries provide insight into structure-function correlations of native signaling networks and foster advances in gene therapy and biopharmaceutical manufg.
5
Hawkins, K. M. and Smolke, C. D. (2006) The regulatory roles of the galactose permease and kinase in the induction response of the GAL network in Saccharomyces cerevisiae J. Biol. Chem. 281, 13485– 13492
Google Scholar
There is no corresponding record for this reference.
6
Topp, S., Reynoso, C. M. K., Seeliger, J. C., Goldlust, I. S., Desai, S. K., Murat, D., Shen, A., Puri, A. W., Komeili, A., Bertozzi, C. R., Scott, J. R., and Gallivan, J. P. (2010) Synthetic riboswitches that induce gene expression in diverse bacterial species Appl. Environ. Microbiol. 76, 7881– 7884
Google Scholar
6
Synthetic riboswitches that induce gene expression in diverse bacterial species
Topp, Shana; Reynoso, Colleen M. K.; Seeliger, Jessica C.; Goldlust, Ian S.; Desai, Shawn K.; Murat, Dorothee; Shen, Aimee; Puri, Aaron W.; Komeili, Arash; Bertozzi, Carolyn R.; Scott, June R.; Gallivan, Justin P.
Applied and Environmental Microbiology (2010), 76 (23), 7881-7884CODEN: AEMIDF; ISSN:0099-2240. (American Society for Microbiology)
We developed a series of ligand-inducible riboswitches that control gene expression in diverse species of Gram-neg. and Gram-pos. bacteria, including human pathogens that have few or no previously reported inducible expression systems. We anticipate that these riboswitches will be useful tools for genetic studies in a wide range of bacteria.
7
Wieland, M., Benz, A., Klauser, B., and Hartig, J. S. (2009) Artificial ribozyme switches containing natural riboswitch aptamer domains Angew. Chem., Int. Ed. Engl. 48, 2715– 2718
Google Scholar
There is no corresponding record for this reference.
8
Suess, B., Hanson, S., Berens, C., Fink, B., Schroeder, R., and Hillen, W. (2003) Conditional gene expression by controlling translation with tetracycline-binding aptamers Nucleic Acids Res. 31, 1853– 1858
Google Scholar
There is no corresponding record for this reference.
9
Sharma, V., Nomura, Y., and Yokobayashi, Y. (2008) Engineering complex riboswitch regulation by dual genetic selection J. Am. Chem. Soc. 130, 16310– 16315
Google Scholar
9
Engineering complex riboswitch regulation by dual genetic selection
Sharma, Vandana; Nomura, Yoko; Yokobayashi, Yohei
Journal of the American Chemical Society (2008), 130 (48), 16310-16315CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
The recent discovery of riboswitches in diverse species of bacteria and few eukaryotes added metabolite-responsive gene regulation to the growing list of RNA functions in biol. The natural riboswitches have inspired several designs of synthetic analogs capable of gene regulation in response to a small mol. trigger. In this work, we describe our efforts to engineer complex riboswitches capable of sensing and responding to two small mols. according to Boolean logics AND and NAND. Two aptamers that recognize theophylline and thiamin pyrophosphate were embedded in tandem in the 5' UTR of bacterial mRNA, and riboswitches that function as logic gates were isolated by dual genetic selection. The diverse phenotype of the engineered logic gates supports the versatility of RNA-based gene regulation which may have preceded the modern protein-based gene regulators. Addnl., our design strategy advances our ability to harness the versatile capacities of RNA to program complex behavior in bacteria without the use of engineered proteins.
10
Weber, W., Fux, C., Daoud-el Baba, M., Keller, B., Weber, C. C., Kramer, B. P., Heinzen, C., Aubel, D., Bailey, J. E., and Fussenegger, M. (2002) Macrolide-based transgene control in mammalian cells and mice Nat. Biotechnol. 20, 901– 907
Google Scholar
There is no corresponding record for this reference.
11
Gossen, M. and Bujard, H. (1992) Tight control of gene expression in mammalian cells by tetracycline-responsive promoters Proc. Natl. Acad. Sci. U.S.A. 89, 5547– 5551
Google Scholar
11
Tight control of gene expression in mammalian cells by tetracycline-responsive promoters
Gossen, Manfred; Bujard, Hermann
Proceedings of the National Academy of Sciences of the United States of America (1992), 89 (12), 5547-51CODEN: PNASA6; ISSN:0027-8424.
Control elements of the tetracycline-resistance operon encoded in Tn10 of Escherichia coli have been utilized to establish a highly efficient regulatory system in mammalian cells. By fusing the tet repressor with the activating domain of virion protein 16 of herpes simplex virus, a tetracycline-controlled transactivator (tTA) was generated that is constitutively expressed in HeLa cells. This transactivator stimulates transcription from a minimal promoter sequence derived from the human cytomegalovirus promoter IE combined with tet operator sequences. Upon integration of a luciferase gene controlled by a tTA-dependent promoter into a tTA-producing HeLa cell line, high levels of luciferase expression were monitored. These activities are sensitive to tetracycline. Depending on the concn. of the antibiotic in the culture medium (0-1 μg/mL), the luciferase activity can be regulated over up to five orders of magnitude. Thus, the system not only allows differential control of the activity of an individual gene in mammalian cells but also is suitable for creation of on/off situations for such genes in a reversible way.
12
Volzing, K., Biliouris, K., and Kaznessis, Y. N. (2011) proTeOn and proTeOff, new protein devices that inducibly activate bacterial gene expression ACS Chem. Biol. 6, 1107– 1116
Google Scholar
There is no corresponding record for this reference.
13
Neddermann, P., Gargioli, C., Muraglia, E., Sambucini, S., Bonelli, F., De Francesco, R., and Cortese, R. (2003) A novel, inducible, eukaryotic gene expression system based on the quorum-sensing transcription factor TraR EMBO Rep. 4, 159– 165
Google Scholar
There is no corresponding record for this reference.
14
Levskaya, A., Chevalier, A. A., Tabor, J. J., Simpson, Z. B., Lavery, L. A., Levy, M., Davidson, E. A., Scouras, A., Ellington, A. D., Marcotte, E. M., and Voigt, C. A. (2005) Synthetic biology: Engineering Escherichia coli to see light Nature 438, 441– 442
Google Scholar
There is no corresponding record for this reference.
15
Alper, H. and Stephanopoulos, G. (2009) Engineering for biofuels: exploiting innate microbial capacity or importing biosynthetic potential? Nat. Rev. Microbiol. 7, 715– 723
Google Scholar
15
Engineering for biofuels: exploiting innate microbial capacity or importing biosynthetic potential?
Alper, Hal; Stephanopoulos, Gregory
Nature Reviews Microbiology (2009), 7 (10), 715-723CODEN: NRMACK; ISSN:1740-1526. (Nature Publishing Group)
A review. The ideal microorganism for biofuel prodn. will possess high substrate utilization and processing capacities, fast and deregulated pathways for sugar transport, good tolerance to inhibitors and product, and high metabolic fluxes and will produce a single fermn. product. It is unclear whether such an organism will be engineered using a native, isolated strain or a recombinant, model organism as the starting point. The choice between engineering natural function and importing biosynthetic capacity is affected by current progress in metabolic engineering and synthetic biol. This review highlights some of the factors influencing the above decision, in light of current advances.
16
Joshua, C. J., Dahl, R., Benke, P. I., and Keasling, J. D. (2011) Absence of diauxie during simultaneous utilization of glucose and xylose by Sulfolobus acidocaldarius J. Bacteriol. 193, 1293– 1301
Google Scholar
16
Absence of diauxie during simultaneous utilization of glucose and xylose by Sulfolobus acidocaldarius
Joshua, Chijioke J.; Dahl, Robert; Benke, Peter I.; Keasling, Jay D.
Journal of Bacteriology (2011), 193 (6), 1293-1301CODEN: JOBAAY; ISSN:0021-9193. (American Society for Microbiology)
Sulfolobus acidocaldarius utilizes glucose and xylose as sole carbon sources, but its ability to metabolize these sugars simultaneously is not known. The absence of diauxie during growth of S. acidocaldarius on glucose and xylose as co-carbon sources is reported. The presence of glucose did not repress xylose utilization. The organism utilized a mixt. of 1 g/L of each sugar simultaneously with a specific growth rate of 0.079 h-1 and showed no preference for the order in which it utilized each sugar. The organism grew faster on 2 g/L xylose (0.074 h-1) as the sole carbon source than on an equal amt. of glucose (0.022 h-1). When grown on a mixt. of the two carbon sources, the growth rate of the organism increased from 0.052 h-1 to 0.085 h-1 as the ratio of xylose to glucose increased from 0.25 to 4. S. acidocaldarius appeared to utilize a mixt. of glucose and xylose at a rate roughly proportional to their concns. in the medium, resulting in complete utilization of both sugars at about the same time. Gene expression in cells grown on xylose alone was very similar to that in cells grown on a mixt. of xylose and glucose and substantially different from that in cells grown on glucose alone. The mechanism by which the organism utilized a mixt. of sugars has yet to be elucidated.
17
Johnsen, U., Dambeck, M., Zaiss, H., Fuhrer, T., Soppa, J., Sauer, U., and Schönheit, P. (2009) d-xylose degradation pathway in the halophilic archaeon Haloferax volcanii J. Biol. Chem. 284, 27290– 27303
Google Scholar
There is no corresponding record for this reference.
18
Peng, N., Ao, X., Liang, Y. X., and She, Q. (2011) Archaeal promoter architecture and mechanism of gene activation Biochem. Soc. Trans. 39, 99– 103
Google Scholar
18
Archaeal promoter architecture and mechanism of gene activation
Peng, Nan; Ao, Xiang; Liang, Yun Xiang; She, Qunxin
Biochemical Society Transactions (2011), 39 (1), 99-103CODEN: BCSTB5; ISSN:0300-5127. (Portland Press Ltd.)
A review. Sulfolobus solfataricus and Sulfolobus islandicus contain several genes exhibiting D-arabinose-inducible expression and these systems are ideal for studying mechanisms of archaeal gene expression. At sequence level, only two highly conserved cis elements are present on the promoters: a regulatory element named ara box directing arabinose-inducible expression and the basal promoter element TATA, serving as the binding site for the TATA-binding protein. Strikingly, these promoters possess a modular structure that allows an essentially inactive basal promoter to be strongly activated. The invoked mechanisms include TFB (transcription factor B) recruitment by the ara-box-binding factor to activate gene expression and modulation of TFB recruitment efficiency to yield differential gene expression.
19
Cardona, S. T., Mueller, C. L., and Valvano, M. A. (2006) Identification of essential operons with a rhamnose-inducible promoter in Burkholderia cenocepacia Appl. Environ. Microbiol. 72, 2547– 2555
Google Scholar
There is no corresponding record for this reference.
20
Schlegel, A., Böhm, A., Lee, S.-J., Peist, R., Decker, K., and Boos, W. (2002) Network regulation of the Escherichia coli maltose system J. Mol. Microbiol. Biotechnol. 4, 301– 307
Google Scholar
20
Network regulation of the Escherichia coli maltose system
Schlegel, Anja; Bohm, Alex; Lee, Sung-Jae; Peist, Ralf; Decker, Katja; Boos, Winfried
Journal of Molecular Microbiology and Biotechnology (2002), 4 (3), 301-307CODEN: JMMBFF; ISSN:1464-1801. (Horizon Scientific Press)
A review. The genes of the Escherichia coli maltose regulon are controlled by MalT, the specific transcriptional activator which, together with the inducer maltotriose and ATP, is essential for mal gene transcription. Network regulation in this system affects the function of MalT and occurs on two levels. The first concerns the expression of malT. It has long been known that malT is under catabolite repression and thus under the control of the cAMP/CAP complex. In addn., the global regulator MIc is found to be a repressor for malT transcription. The repressor activity of MIc is controlled by the transport status of the glucose-specific enzyme EIICB of the PTS that causes sequestration (and inactivation as a repressor) of MIc when glucose is transported. The second level of MalT regulation affects its activity. MalT is activated by maltotriose which is not only formed when the cells are growing on any maltodextrin but also, in low amts., endogenously when the cells grow on non-maltodextrin carbon sources. Thus, cellular metab., for instance degrdn. of galactose or trehalose, can cause mal gene induction. It was found that unphosphorylated internal glucose takes part in endogenous maltodextrin biosynthesis and is therefore a key element in endogenous mal gene expression. In addn. to the maltotriose-dependent activation, MalT can interact with three different enzymes that lead to its inactivation as a transcriptional activator. The first is MalK, the energy transducing ABC subunit of the maltodextrin transport system. Transport controls the interaction of MalK and MalT thus affecting gene expression. The second enzyme is MalY, a pyridoxal phosphate contg. enzyme exhibiting cystathionase activity. The crystal structure of MalY was established and mutations in MalY that reduce mal gene repression map in a hydrophobic MalT interaction patch on the surface of the enzyme. The last enzyme is a sol. esterase of as yet unknown function. When overproduced, this enzyme specifically reduces mal gene expression and affects the activity of MalT in an in vitro transcription assay.
21
Peri, K. G., Goldie, H., and Waygood, E. B. (1990) Cloning and characterization of the N-acetylglucosamine operon of Escherichia coli Biochem. Cell Biol. 68, 123– 137
Google Scholar
21
Cloning and characterization of the N-acetylglucosamine operon of Escherichia coli
Peri, Krishna G.; Goldie, Hughes; Waygood, E. Bruce
Biochemistry and Cell Biology (1990), 68 (1), 123-37CODEN: BCBIEQ; ISSN:0829-8211.
Three enzymes are required for N-acetylglucosamine (NAG) utilization in E. coli: enzyme IInag (gene nagE), N-acetylglucosamine-6-phosphate deacetylase (gene nagA), and glucosamine-6-phosphate isomerase (gene nagB). The 3 genes are located near 16 min on the E. coli chromosome. A strain of E. coli, KPN9, incapable of utilizing N-acetylglucosamine, was used to screen a genomic library of E. coli for a complementing recombinant colicin E1 plasmid that allowed for growth on N-acetylglucosamine. Plasmid pLC5-21 was found to contain all 3 known nag genes on a 5.7-kilobase (5.7-kb) fragment of DNA. The products of these nag genes were identified by complementation of E. coli strains with mutations in nagA, nagB, and nagE. The gene products from the 5.7-kb fragment were identified by [35S]methionine-labeled maxicells and autoradiog. of SDS-polyacrylamide electrophoresis gels. The gene products had the following relative masses (Mrs: nagE, 62,000; nagA, 45,000; nagB, 29,000. In addn., another product of Mr 44,000 was detected. The genes have been sequenced to reveal an addnl. open reading frame (nagC), a putative catabolite activator protein binding site that may control nagB and nagE, putative rho-independent terminator sites for nagB and nagE, and sequence homologies for RNA polymerase binding sites preceding each of the open reading frames, except for nagA. The calcd. mol. wts. (MWs) of the gene products derived from the sequence are as follows: nagA, 40,954; nagB, 29,657; nagC, 44,664; nagE, 68,356. No role is known for nagC, although a no. of regulatory roles appear to be plausible. No obvious transcriptional termination site distal to nagC was found and another open reading frame begins after nagC. This gene, nagD, was isolated sep. from pLC5-21, and the sequence revealed a protein with a calcd. MW of 27,181. The nagD gene is followed by repetitive extragenic palindromic sequences. The nag genes appear to be organized in an operon: nagD nagC nagA nagB transcribed in one direction and nagE transcribed in the opposite direction.
22
Tong, S., Porco, A., Isturiz, T., and Conway, T. (1996) Cloning and molecular genetic characterization of the Escherichia coli gntR, gntK, and gntU genes of GntI, the main system for gluconate metabolism J. Bacteriol. 178, 3260– 3269
Google Scholar
22
Cloning and molecular genetic characterization of the Escherichia coli gntR, gntK, and gntU genes of GntI, the main system for gluconate metabolism
Tong, Suxiang; Porco, Antonieta; Isturiz, Tomas; Conway, Tyrrell
Journal of Bacteriology (1996), 178 (11), 3260-3269CODEN: JOBAAY; ISSN:0021-9193. (American Society for Microbiology)
Three genes involved in gluconate metab., gntR, gntK, and gntU, which code for a regulatory protein, a gluconate kinase, and a gluconate transporter, resp., were cloned from Escherichia coli K-12 on the basis of their known locations on the genomic restriction map. The gene order is gntU, gntK, and gntR, which are immediately adjacent to asd at 77.0 min, and all three genes are transcribed in the counterclockwise direction. The gntR product is 331 amino acids long, with a helix-turn-helix motif typical of a regulatory protein. The gntK gene encodes a 175-amino-acid polypeptide that has an ATP-binding motif similar to those found in other sugar kinases. While GntK does not show significant sequence similarity to any known sugar kinases, it is 45% identical to a second putative gluconate kinase from E. coli, gntV. The 445-amino-acid sequence encoded by gntU has a secondary structure typical of membrane-spanning transport proteins and is 37% identical to the gntP product from Bacillus subtilis. Kinetic anal. of GntU indicates an apparent Km for gluconate of 212 μM, indicating that this is a low-affinity transporter. Studies demonstrate that the gntR gene is monocistronic, while the gntU and gntK genes, which are sepd. by only 3 bp, form an operon. Expression of gntR is essentially constitutive, while expression of gntKU is induced by gluconate and is subject to fourfold glucose catabolite repression. These results confirm that gntK and gntU, together with another gluconate transport gene, gntT, constitute the GntI system for gluconate utilization, under control of the gntR gene product, which is also responsible for induction of the edd and eda genes of the Entner-Doudoroff pathway.
23
Lubelska, J. M., Jonuscheit, M., Schleper, C., Albers, S.-V., and Driessen, A. J. M. (2006) Regulation of expression of the arabinose and glucose transporter genes in the thermophilic archaeon Sulfolobus solfataricus Extremophiles 10, 383– 391
Google Scholar
There is no corresponding record for this reference.
24
Song, S. and Park, C. (1997) Organization and regulation of the d-xylose operons in Escherichia coli K-12: XylR acts as a transcriptional activator J. Bacteriol. 179, 7025– 7032
Google Scholar
24
Organization and regulation of the D-xylose operons in Escherichia coli K-12: XylR acts as a transcriptional activator
Song, Sukgil; Park, Chankyu
Journal of Bacteriology (1997), 179 (22), 7025-7032CODEN: JOBAAY; ISSN:0021-9193. (American Society for Microbiology)
The metab. of D-xylose in Escherichia coli K-12 is known to be mediated by the xylAB gene. However, the nearby xylFGHR genes were found by genome sequencing and predicted to be responsible for transport and regulation for xylose based on their sequence similarities to other functionally related genes. Here, we investigated transcriptional organization and functions of the xyl genes. An anal. with random transposon insertions revealed that the xyl genes are organized into two major transcriptional units, xylAB and xylFGHR, governed by the promoters PA and PF, resp. However, there is an addnl. weak promoter, PR, which is specific for xylR. Sites of transcription initiation were detd. by primer extension anal. When studied with operon fusions to lacZ, the PA and PF, promoters were activated by D-xylose and repressed by glucose. In contrast, the PR promoter was not regulated by these sugars. A mutation in xylR completely abolished expression from the PA and PF promoters, causing a defect in both growth and transport. Binding of XylR to the xyl promoter was enhanced by the presence of D-xylose, suggesting that transcription was pos. regulated by XylR. In vivo footprinting anal. revealed that XylR binds to at least two DNA regions, IA and IF,, each with a direct repeat. It is very likely that XyIR interacts with IA and IF as a dimer. The presumed binding sites are located just upstream of the promoter consensus sequences (-35), while IA is addnl. flanked by a cAMP receptor protein-binding site on the other side. The proposed structure of xyl promoters is consistent with the regulation of xyl gene expression and with phenotypes of transposon insertions obtained in the promoter regions.
25
Moralejo, P., Egan, S. M., Hidalgo, E., and Aguilar, J. (1993) Sequencing and characterization of a gene cluster encoding the enzymes for l-rhamnose metabolism in Escherichia coli J. Bacteriol. 175, 5585– 5594
Google Scholar
25
Sequencing and characterization of a gene cluster encoding the enzymes for L-rhamnose metabolism in Escherichia coli
Moralejo, Pilar; Egan, Susan M.; Hidalgo, Elena; Aguilar, Juan
Journal of Bacteriology (1993), 175 (17), 5585-94CODEN: JOBAAY; ISSN:0021-9193.
The sequencing of the EcoRI-HindIII fragment complementing mutations in the structural genes of the L-rhamnose regulon of Escherichia coli has permitted identification of the open reading frames corresponding to rhaB, rhaA, and rhaD. The deduced amino acid sequences gave a 425-amino-acid polypeptide corresponding to rhamnulose kinase for rhaB, a 400-amino-acid polypeptide corresponding to rhamnose isomerase for rhaA, and a 274-amino-acid polypeptide corresponding to rhamnulose-1-phosphate aldolase for rhaD. Transcriptional fusions of the three putative promoter regions to lacZ showed that only the rhaB leader region acted as a promoter, as indicated by the high β-galactosidase activity induced by rhamnose, while no significant activity from the rhaA and rhaD constructions was detected. The rhaB transcription start site was mapped to -24 relative to the start of translation. Mutations in the catabolic genes were used to show that L-rhamnose may directly induce rhaBAD transcription.
26
Siegele, D. A. and Hu, J. C. (1997) Gene expression from plasmids containing the araBAD promoter at subsaturating inducer concentrations represents mixed populations Proc. Natl. Acad. Sci. U.S.A. 94, 8168– 8172
Google Scholar
26
Gene expression from plasmids containing the araBAD promoter at subsaturating inducer concentrations represents mixed populations
Siegele, Deborah A.; Hu, James C.
Proceedings of the National Academy of Sciences of the United States of America (1997), 94 (15), 8168-8172CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
Gene expression from plasmids contg. the araBAD promoter can be regulated by the concn. of arabinose in the growth medium. Guzman et al. [Guzman, L.-M., Belin, D., Carson, M. J. & Beckwith, J. (1995) J. Bacteriol. 177, 4121-4130] showed that expression of a cloned gene could be modulated over several orders of magnitude in cultures grown in the presence of subsaturating concns. of arabinose. We constructed plasmids expressing a fast-folding mutant Aequorea victoria green fluorescent protein from the araBAD promoter to examine the distribution of expressed gene products in individual cells at intermediate induction levels. Microscopic examn. of cells grown at low arabinose concns. shows mixts. of brightly fluorescent and dark cells, suggesting that intermediate expression levels in cultures reflect a population av. of induced and uninduced cells. The kinetics of green fluorescent protein induction suggest that this reflects an "autocatalytic" induction mechanism due to accumulation of the inducer by active transport. This mechanism, which is analogous to the induction of the lac operon at subsaturating inducer concns. in lacY+ cells, was described 40 yr ago by Novic and Weiner [Novick, A. & Weiner, M. (1957) Proc. Natl. Acad. Sci. USA 43, 553-566].
27
Novick, A. and Weiner, M. (1957) Enzyme induction as an all-or-none phenomenon Proc. Natl. Acad. Sci. U.S.A. 43, 553– 566
Google Scholar
There is no corresponding record for this reference.
28
Cohn, M. and Horibata, K. (1959) Analysis of the differentiation and of the heterogeneity within a population of Escherichia coli undergoing induced β-galactosidase synthesis J. Bacteriol. 78, 613– 623
Google Scholar
28
Analysis of the differentiation and of the heterogeneity within a population of Escherichia coli undergoing induced β-galactosidase synthesis
Cohn, Melvin; Horibata, Kengo
Journal of Bacteriology (1959), 78 (), 613-23CODEN: JOBAAY; ISSN:0021-9193.
Under conditions where the metabolic activity of a system under induction is essential to the formation of that system, the response of each cell in a population is sharply dependent upon whether it possesses a min. unit of the induced activity. As a result, the formation of enzyme by the population is heterogeneous. Both the formation and nonformation of enzyme is passed on clonically to the descendents. Two induced systems: the galactoside permease and the β-galactosidase (Cohen and Monod, C.A. 52, 497i) of E. coli and the galactozymase of long-term adapting yeast (Spiegelman, C.A. 46, 7178c) can be understood in terms of this concept. The all-or-none distribution of induction cannot as yet be interpreted to reflect a property of the enzyme-forming system itself. 26 references.
29
Ozbudak, E. M., Thattai, M., Lim, H. N., Shraiman, B. I., and van Oudenaarden, A. (2004) Multistability in the lactose utilization network of Escherichia coli Nature 427, 737– 740
Google Scholar
There is no corresponding record for this reference.
30
Acar, M., Becskei, A., and van Oudenaarden, A. (2005) Enhancement of cellular memory by reducing stochastic transitions Nature 435, 228– 232
Google Scholar
There is no corresponding record for this reference.
31
Kuhlman, T., Zhang, Z., Saier, M. H., Jr, and Hwa, T. (2007) Combinatorial transcriptional control of the lactose operon of Escherichia coli Proc. Natl. Acad. Sci. U.S.A. 104, 6043– 6048
Google Scholar
There is no corresponding record for this reference.
32
Setty, Y., Mayo, A. E., Surette, M. G., and Alon, U. (2003) Detailed map of a cis-regulatory input function Proc. Natl. Acad. Sci. U.S.A. 100, 7702– 7707
Google Scholar
There is no corresponding record for this reference.
33
Krishna, S., Semsey, S., and Sneppen, K. (2007) Combinatorics of feedback in cellular uptake and metabolism of small molecules Proc. Natl. Acad. Sci. U.S.A. 104, 20815– 20819
Google Scholar
There is no corresponding record for this reference.
34
Tian, X.-J., Zhang, X.-P., Liu, F., and Wang, W. (2009) Interlinking positive and negative feedback loops creates a tunable motif in gene regulatory networks Phys. Rev. E Stat. Nonlin. Soft Matter Phys. 80, 011926
Google Scholar
There is no corresponding record for this reference.
35
Khlebnikov, A., Datsenko, K. A., Skaug, T., Wanner, B. L., and Keasling, J. D. (2001) Homogeneous expression of the P_BAD promoter in Escherichia coli by constitutive expression of the low-affinity high-capacity AraE transporter Microbiology 147, 3241– 3247
Google Scholar
There is no corresponding record for this reference.
36
Khlebnikov, A., Risa, O., Skaug, T., Carrier, T. A., and Keasling, J. D. (2000) Regulatable arabinose-inducible gene expression system with consistent control in all cells of a culture J. Bacteriol. 182, 7029– 7034
Google Scholar
36
Regulatable arabinose-inducible gene expression system with consistent control in all cells of a culture
Khlebnikov, Artem; Risa, Oystein; Skaug, Tove; Carrier, Trent A.; Keasling, J. D.
Journal of Bacteriology (2000), 182 (24), 7029-7034CODEN: JOBAAY; ISSN:0021-9193. (American Society for Microbiology)
The arabinose-inducible promoter PBAD is subject to all-or-none induction, in which intermediate concns. of arabinose give rise to subpopulations of cells that are fully induced and uninduced. To construct a host-vector expression system with regulatable control in a homogeneous population of cells, the araE gene of Escherichia coli was cloned into an RSF1010-derived plasmid under control of the isopropyl-β-D-thiogalactopyranoside-inducible Ptac and Ptaclac promoters. This gene encodes the low-affinity, high-capacity arabinose transport protein and is controlled natively by an arabinose-inducible promoter. To detect the effect of arabinose-independent araE expression on population homogeneity and cell-specific expression, the gfpuv gene was placed under control of the arabinose-inducible araBAD promoter (PBAD) on the pMB1-derived plasmid pBAD24. The transporter and reporter plasmids were transformed into E. coli strains with native arabinose transport systems and strains deficient in one or both of the arabinose transport systems (araE and/or araFGH). The effects of the arabinose concn. and arabinose-independent transport control on population homogeneity were investigated in these strains using flow cytometry. The araE, and araE araFGH mutant strains harboring the transporter and reporter plasmids were uniformly induced across the population at all inducer concns., and the level of gene expression in individual cells varied with arabinose concn. In contrast, the parent strain, which expressed the native araE and araFGH genes and harbored the transporter and reporter plasmids, exhibited all-or-none behavior. This work demonstrates the importance of including a transport gene that is controlled independently of the inducer to achieve regulatable and consistent induction in all cells of the culture.
37
Morgan-Kiss, R. M., Wadler, C., and Cronan, J. E., Jr. (2002) Long-term and homogeneous regulation of the Escherichia coli araBAD promoter by use of a lactose transporter of relaxed specificity Proc. Natl. Acad. Sci. U.S.A. 99, 7373– 7377
Google Scholar
There is no corresponding record for this reference.
38
Schleif, R. (2010) AraC protein, regulation of the l-arabinose operon in Escherichia coli, and the light switch mechanism of AraC action FEMS Microbiol. Rev. 34, 779– 796
Google Scholar
38
AraC protein, regulation of the L-arabinose operon in Escherichia coli, and the light switch mechanism of AraC action
Schleif, Robert
FEMS Microbiology Reviews (2010), 34 (5), 779-796CODEN: FMREE4; ISSN:0168-6445. (Wiley-Blackwell)
A review. This review covers the physiol. aspects of regulation of the arabinose operon in Escherichia coli and the phys. and regulatory properties of the operon's controlling gene, araC. It also describes the light switch mechanism as an explanation for many of the protein's properties. Although many thousands of homologs of AraC exist and regulate many diverse operons in response to many different inducers or physiol. states, homologs that regulate arabinose-catabolizing genes in response to arabinose were identified. The sequence similarities among them are discussed in light of the known structure of the dimerization and DNA-binding domains of AraC.
39
Reeder, T. and Schleif, R. (1991) Mapping, sequence, and apparent lack of function of araJ, a gene of the Escherichia coli arabinose regulon J. Bacteriol. 173, 7765– 7771
Google Scholar
39
Mapping, sequence, and apparent lack of function of araJ, a gene of Escherichia coli arabinose regulon
Reeder, Thadd; Schleif, Robert
Journal of Bacteriology (1991), 173 (24), 7765-71CODEN: JOBAAY; ISSN:0021-9193.
The mapping, sequencing, and study of the physiol. role of the fourth arabinose-inducible operon from E. coli, araJ is reported here. It is located at 9 min on the chromosome and codes for a single 42-kDa protein that shows no significant homol. to other known proteins. Destruction of the chromosomal araJ gene does not detectably affect either of the 2 arabinose transport systems, the ability of cells to grow on arabinose, or the induction kinetics of the araBAD operon, and thus the physiol. role of AraJ, if any, remains unknown. A long open reading frame was also found upstream of araJ. The sequence of this upstream open reading frame was found to be identical to the previously reported sequence of the sbcC gene (Na0om, I.S., et al., 1989). The carboxyl region of SbcC has an amino acid sequence consistent with this region of SbcC forming an extended alpha-helical coiled-coil.
40
Hendrickson, W., Stoner, C., and Schleif, R. (1990) Characterization of the Escherichia coli araFGH and araJ promoters J. Mol. Biol. 215, 497– 510
Google Scholar
There is no corresponding record for this reference.
41
Zaslaver, A., Bren, A., Ronen, M., Itzkovitz, S., Kikoin, I., Shavit, S., Liebermeister, W., Surette, M. G., and Alon, U. (2006) A comprehensive library of fluorescent transcriptional reporters for Escherichia coli Nat. Methods 3, 623– 628
Google Scholar
41
A comprehensive library of fluorescent transcriptional reporters for Escherichia coli
Zaslaver, Alon; Bren, Anat; Ronen, Michal; Itzkovitz, Shalev; Kikoin, Ilya; Shavit, Seagull; Liebermeister, Wolfram; Surette, Michael G.; Alon, Uri
Nature Methods (2006), 3 (8), 623-628CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)
E. coli is widely used for systems biol. research; there exists a need, however, for tools that can be used to accurately and comprehensively measure expression dynamics in individual living cells. To address this the authors present a library of transcriptional fusions of gfp to each of about 2,000 different promoters in E. coli K12, covering the great majority of the promoters in the organism. Each promoter fusion is expressed from a low-copy plasmid. The authors demonstrate that this library can be used to obtain highly accurate dynamic measurements of promoter activity on a genomic scale, in a glucose-lactose diauxic shift expt. The library allowed detection of about 80 previously uncharacterized transcription units in E. coli, including putative internal promoters within previously known operons, such as the lac operon. This library can serve as a tool for accurate, high-resoln. anal. of transcription networks in living E. coli cells.
42
Tan, C., Marguet, P., and You, L. (2009) Emergent bistability by a growth-modulating positive feedback circuit Nat. Chem. Biol. 5, 842– 848
Google Scholar
42
Emergent bistability by a growth-modulating positive feedback circuit
Tan, Cheemeng; Marguet, Philippe; You, Lingchong
Nature Chemical Biology (2009), 5 (11), 842-848CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)
Synthetic gene circuits are often engineered by considering the host cell as an invariable 'chassis'. Circuit activation, however, may modulate host physiol., which in turn can substantially impact circuit behavior. We illustrate this point by a simple circuit consisting of mutant T7 RNA polymerase (T7 RNAP*) that activates its own expression in the bacterium Escherichia coli. Although activation by the T7 RNAP* is noncooperative, the circuit caused bistable gene expression. This counterintuitive observation can be explained by growth retardation caused by circuit activation, which resulted in nonlinear diln. of T7 RNAP* in individual bacteria. Predictions made by models accounting for such effects were verified by further exptl. measurements. Our results reveal a new mechanism of generating bistability and underscore the need to account for host physiol. modulation when engineering gene circuits.
43
Nevozhay, D., Adams, R. M., Murphy, K. F., Josic, K., and Balázsi, G. (2009) Negative autoregulation linearizes the dose-response and suppresses the heterogeneity of gene expression Proc. Natl. Acad. Sci. U.S.A. 106, 5123– 5128
Google Scholar
There is no corresponding record for this reference.
44
Madar, D., Dekel, E., Bren, A., and Alon, U. (2011) Negative auto-regulation increases the input dynamic-range of the arabinose system of Escherichia coli BMC Syst. Biol. 5, 111
Google Scholar
44
Negative auto-regulation increases the input dynamic-range of the arabinose system of Escherichia coli
Madar Daniel; Dekel Erez; Bren Anat; Alon Uri
BMC systems biology (2011), 5 (), 111 ISSN:.
BACKGROUND: Gene regulation networks are made of recurring regulatory patterns, called network motifs. One of the most common network motifs is negative auto-regulation, in which a transcription factor represses its own production. Negative auto-regulation has several potential functions: it can shorten the response time (time to reach halfway to steady-state), stabilize expression against noise, and linearize the gene's input-output response curve. This latter function of negative auto-regulation, which increases the range of input signals over which downstream genes respond, has been studied by theory and synthetic gene circuits. Here we ask whether negative auto-regulation preserves this function also in the context of a natural system, where it is embedded within many additional interactions. To address this, we studied the negative auto-regulation motif in the arabinose utilization system of Escherichia coli, in which negative auto-regulation is part of a complex regulatory network. RESULTS: We find that when negative auto-regulation is disrupted by placing the regulator araC under constitutive expression, the input dynamic range of the arabinose system is reduced by 10-fold. The apparent Hill coefficient of the induction curve changes from about n = 1 with negative auto-regulation, to about n = 2 when it is disrupted. We present a mathematical model that describes how negative auto-regulation can increase input dynamic-range, by coupling the transcription factor protein level to the input signal. CONCLUSIONS: Here we demonstrate that the negative auto-regulation motif in the native arabinose system of Escherichia coli increases the range of arabinose signals over which the system can respond. In this way, negative auto-regulation may help to increase the input dynamic-range while maintaining the specificity of cooperative regulatory systems. This function may contribute to explaining the common occurrence of negative auto-regulation in biological systems.
45
Weickert, M. J. and Adhya, S. (1993) The galactose regulon of Escherichia coli Mol. Microbiol. 10, 245– 251
Google Scholar
45
The galactose regulon of Escherichia coli
Weickert, Michael J.; Adhya, Sankar
Molecular Microbiology (1993), 10 (2), 245-51CODEN: MOMIEE; ISSN:0950-382X.
A review with 59 refs. Galactose transport and metab. in Escherichia coli involves a multicomponent amphibolic pathway. Galactose transport is accomplished by 2 different galactose-specific transport systems. At least 4 of the genes and operons involved in galactose transport and metab. have promoters contg. similar regulatory sequences. These sequences are recognized by at least 3 regulators, Gal repressor (GalR), Gal isorepressor (GalS) and cAMP receptor protein (CRP), which modulate transcription from these promoters. The neg. regulators, GalR and GalS, discriminate between utilization of the high-affinity (regulated by GalS) and low-affinity (regulated by GalR) transport systems, and modulate the expression of genes for galactose metab. in an overlapping fashion. GalS is itself autogenously regulated and CRP dependent, while the gene for GalR is constitutive. The gal operon encoding the enzymes for galactose metab. has 2 promoters regulated by CRP in opposite ways; one (P1) is stimulated and the other (P2) inhibited by GalR but weakly by GalS. All but one of the constituent promoters of the gal regulon have 2 operators. The gal regulon has the potential to coordinate galactose metab. and transport in a highly efficient manner, under a wide variety of conditions of galactose availability.
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Yildirim, N. and Mackey, M. C. (2003) Feedback regulation in the lactose operon: a mathematical modeling study and comparison with experimental data Biophys. J. 84, 2841– 2851
Google Scholar
There is no corresponding record for this reference.
47
Court, D. L., Swaminathan, S., Yu, D., Wilson, H., Baker, T., Bubunenko, M., Sawitzke, J., and Sharan, S. K. (2003) Mini-lambda: a tractable system for chromosome and BAC engineering Gene 315, 63– 69
Google Scholar
There is no corresponding record for this reference.
48
Cherepanov, P. P. and Wackernagel, W. (1995) Gene disruption in Escherichia coli: Tc^R and Km^R cassettes with the option of Flp-catalyzed excision of the antibiotic-resistance determinant Gene 158, 9– 14
Google Scholar
48
Gene disruption in Escherichia coli: TcR and KmR cassettes with the option of FlP-catalyzed excision of the antibiotic-resistance determinant
Cherepanov, Peter P.; Wackernagel, Wilfried
Gene (1995), 158 (1), 9-14CODEN: GENED6; ISSN:0378-1119. (Elsevier)
Two cassettes with tetracycline-resistance (TcR) and kanamycin-resistance (KmR) determinants have been developed for the construction of insertion and deletion mutants of cloned genes in Escherichia coli. In both cassettes, the resistance determinants are flanked by the short direct repeats (FRT sites) required for site-specific recombination mediated by the yeast Flp recombinase. In addn., a plasmid with temp.-sensitive replication for temporal prodn. of the Flp enzyme in E. coli has been constructed. After a gene disruption or deletion mutation is constructed in vitro by insertion of one of the cassettes into a given gene, the mutated gene is transferred to the E. coli chromosome by homologous recombination and selection for the antibiotic resistance provided by the cassette. If desired, the resistance determinant can subsequently be removed from the chromosome in vivo by Flp action, leaving behind a short nucleotide sequence with one FRT site and with no polar effect on downstream genes. This system was applied in the construction of an E. coli endA deletion mutation which can be transduced by P1 to the genetic background of interest using TcR as a marker. The transductant can then be freed of the TcR if required.
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Del Vecchio, D., Ninfa, A. J., and Sontag, E. D. (2008) Modular cell biology: Retroactivity and insulation Mol. Syst. Biol. 4, 161
Google Scholar
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Modular cell biology: retroactivity and insulation
Del Vecchio Domitilla; Ninfa Alexander J; Sontag Eduardo D
Molecular systems biology (2008), 4 (), 161 ISSN:.
Modularity plays a fundamental role in the prediction of the behavior of a system from the behavior of its components, guaranteeing that the properties of individual components do not change upon interconnection. Just as electrical, hydraulic, and other physical systems often do not display modularity, nor do many biochemical systems, and specifically, genetic networks. Here, we study the effect of interconnections on the input-output dynamic characteristics of transcriptional components, focusing on a property, which we call 'retroactivity', that plays a role analogous to non-zero output impedance in electrical systems. In transcriptional networks, retroactivity is large when the amount of transcription factor is comparable to, or smaller than, the amount of promoter-binding sites, or when the affinity of such binding sites is high. To attenuate the effect of retroactivity, we propose a feedback mechanism inspired by the design of amplifiers in electronics. We introduce, in particular, a mechanism based on a phosphorylation-dephosphorylation cycle. This mechanism enjoys a remarkable insulation property, due to the fast timescales of the phosphorylation and dephosphorylation reactions.

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Abstract
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Figure 1
Figure 1. Co-opting sugar utilization pathways for titratable control of gene expression. (A) Components of inducible sugar utilization pathways. High affinity (T_H) and low-affinity (T_L) transporters import the sugar into the cell, while catabolic enzymes (E) shunt the sugar into central metabolism. Transcription regulators (R) up-regulate the expression of the transporters and the enzymes, but only in the presence of the sugar. (B) Desirable properties for a titratable response. These properties include a uniform response at all inducer concentrations, a large dynamic range (high δ), low inducer concentrations to induce the pathway (low EC₅₀), and strong linearity (low η).
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Figure 2
Figure 2. Simple mathematical model predicts trade-offs when altering the pathway structure. (A) The model assumes a base pathway comprising a high-affinity/low-capacity transporter (T_H) and a low-affinity/high-capacity transporter (T_L) that import extracellular sugar (S₀) into the cell, a catabolic enzyme (E) that degrades the sugar, and a constitutively expressed regulator that upregulates the expression of the transporters and the enzymes when bound to the sugar. The steady-state expression levels of the enzyme are reported as a function of extracellular sugar concentration. Note that all variables were nondimensionalized as part of the model derivation. Dashed lines indicate bifurcation regions. To alter the pathway, T_H was constitutively expressed (C,D) and the activity of T_L was further eliminated (E,F), T_L was constitutively expressed (G,H) and the activity of T_H was further eliminated (I,J), and the activity of the catabolic enzymes was eliminated (B,D,F,H,J). Three strengths of constitutive expression were selected for T_H and T_L (low, light blue; medium, blue; high, dark blue). See Supporting Information (SI) for more details.
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Figure 3
Figure 3. Probing alterations to the l-arabinose utilization pathway in E. coli. The wild type pathway (A) was subjected to different alterations: araFGH was constitutively expressed (P_con-araFGH) (C,D) and araE was further deleted (ΔaraE) (E,F), araE was constitutively expressed (P_con-araE) (G,H) and araFGH was further deleted (ΔaraFGH) (I,J), and araBAD was deleted (ΔaraBAD) (B,D,F,H,J). Each designated strain was back-diluted into M9 minimal medium supplemented with the indicated concentration of l-arabinose and grown for 6 h to ABS₆₀₀ ∼ 0.4 prior to flow cytometry analysis. For unimodal distributions, the resulting mean fluorescence is plotted. For bimodal distributions, two dots are plotted to represent the mean fluorescence and the relative number of cells in the induced (black) and uninduced (white) subpopulations (see SI Figure S2 for more details on the flow cytometry analysis). The diameter of each dot is directly proportional to the fraction of cells in that subpopulation. Gray boxes indicate the limit of detection due to autofluorescence. Each dot plot is representative of at least three independent experiments conducted on separate days. See Table 1 for the response metrics that account for the replicate experiments.
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Figure 4
Figure 4. Effect of cell density in the presence or absence of sugar catabolism. (A) Model predictions for the pathway with a constitutively expressed low-affinity transporter (T_L = 0.2) and the deleted high-affinity transporter (α_H = 0) when accounting for depletion of extracellular sugar through catabolism. Each simulation was conducted to τ = 10. The different curves reflect the relative volume of the cells to the medium (ν). Note that all variables were nondimensionalized as part of the model derivation. See Supporting Information for more details. (B) Growth curves for the P_con-araE ΔaraFGH strain with or without (ΔaraBAD) sugar catabolism in defined medium with or without 10 mM l-arabinose. Each value represents the mean of three independent experiments. The SEM for each measurement was smaller than the symbol. (C) Representative dot plots for both strains in log phase grown to the indicated final cell densities. See Figure 3 for more information on the dot plots. Each dot plot is representative of at least three experiments conducted from independent colonies. See Table 1 for the response metrics that account for the replicate experiments.
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Figure 5
Figure 5. Linearizing the response to d-xylose. The wild type E. coli strain (A), the strain constitutively expressing the high-affinity transporter xylFGH (B), and the strain constitutively expressing the high-affinity transporter xylFGH and lacking the catabolic operon xylAB (C) each harbored the reporter plasmid pUA66-pxylA. The designated strain was back-diluted into M9 minimal medium supplemented with the indicated concentration of d-xylose and grown for 6 h to ABS₆₀₀ ∼ 0.4 prior to flow cytometry analysis. See Figure 3 for more information on the dot plots. Each dot plot is representative of at least three experiments conducted from independent colonies.
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References
This article references 49 other publications.
1. 1
  Temme, K., Zhao, D., and Voigt, C. A. (2012) Refactoring the nitrogen fixation gene cluster from Klebsiella oxytoca Proc. Natl. Acad. Sci. U.S.A. 109, 7085– 7090
  1
  Refactoring the nitrogen fixation gene cluster from Klebsiella oxytoca
  Temme Karsten; Zhao Dehua; Voigt Christopher A
  Proceedings of the National Academy of Sciences of the United States of America (2012), 109 (18), 7085-90 ISSN:.
  Bacterial genes associated with a single trait are often grouped in a contiguous unit of the genome known as a gene cluster. It is difficult to genetically manipulate many gene clusters because of complex, redundant, and integrated host regulation. We have developed a systematic approach to completely specify the genetics of a gene cluster by rebuilding it from the bottom up using only synthetic, well-characterized parts. This process removes all native regulation, including that which is undiscovered. First, all noncoding DNA, regulatory proteins, and nonessential genes are removed. The codons of essential genes are changed to create a DNA sequence as divergent as possible from the wild-type (WT) gene. Recoded genes are computationally scanned to eliminate internal regulation. They are organized into operons and placed under the control of synthetic parts (promoters, ribosome binding sites, and terminators) that are functionally separated by spacer parts. Finally, a controller consisting of genetic sensors and circuits regulates the conditions and dynamics of gene expression. We applied this approach to an agriculturally relevant gene cluster from Klebsiella oxytoca encoding the nitrogen fixation pathway for converting atmospheric N(2) to ammonia. The native gene cluster consists of 20 genes in seven operons and is encoded in 23.5 kb of DNA. We constructed a "refactored" gene cluster that shares little DNA sequence identity with WT and for which the function of every genetic part is defined. This work demonstrates the potential for synthetic biology tools to rewrite the genetics encoding complex biological functions to facilitate access, engineering, and transferability.
2. 2
  Guzman, L. M., Belin, D., Carson, M. J., and Beckwith, J. (1995) Tight regulation, modulation, and high-level expression by vectors containing the arabinose P_BAD promoter J. Bacteriol. 177, 4121– 4130
  2
  Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter
  Guzman, Luz-Maria; Belin, Dominique; Carson, Michael J.; Beckwith, Jon
  Journal of Bacteriology (1995), 177 (14), 4121-30CODEN: JOBAAY; ISSN:0021-9193. (American Society for Microbiology)
  We have constructed a series of plasmid vectors (pBAD vectors) contg. the PBAD promoter of the araBAD (arabinose) operon and the gene encoding the pos. and neg. regulator of this promoter, araC. Using the phoA gene and phoA fusions to monitor expression in these vectors, we show that the ratio of induction/repression can be 1,200-fold, compared with 50-fold for PTAC-based vectors. PhoA expression can be modulated over a wide range of inducer (arabinose) concns. and reduced to extremely low levels by the presence of glucose, which represses expression. Also, the kinetics of induction and repression are very rapid and significantly affected by the ara allele in the host strain. Thus, the use of this system which can be efficiently and rapidly turned on and off allows the study of important aspects of bacterial physiol. in a very simple manner and without changes of temp. We have exploited the tight regulation of the PBAD promoter to study the phenotypes of null mutations of essential genes and explored the use of pBAD vectors as an expression system.
3. 3
  De Boer, H. A., Comstock, L. J., and Vasser, M. (1983) The tac promoter: A functional hybrid derived from the trp and lac promoters Proc. Natl. Acad. Sci. U.S.A. 80, 21– 25
  3
  The tac promoter: a functional hybrid derived from the trp and lac promoters
  De Boer, Herman A.; Comstock, Lisa J.; Vasser, Mark
  Proceedings of the National Academy of Sciences of the United States of America (1983), 80 (1), 21-5CODEN: PNASA6; ISSN:0027-8424.
  Two hybrid promoters that are functional in Escherichia coli were constructed. These hybrid promoters, tacI and tacII, were derived from sequences of the trp and the lac UV5 promoters. In the 1st hybrid promoter (tacI), the DNA upstream of position -20 with respect to the transcriptional start site was derived from the trp promoter. The DNA downstream of position -20 was derived from the lac UV5 promoter. In the 2nd hybrid promoter (tacII), the DNA upstream of position -11 at the HpaI site within the Pribnow box was derived from the trp promoter. The DNA downstream of position -11 is a 46-base-pair synthetic DNA fragment that specifies part of the hybrid Pribnow box and the entire lac operator. It also specifies a Shine-Dalgarno sequence flanked by 2 unique restriction sites (portable Shine-Dalgarno sequence). The tacI and the tacII promoters resp. direct transcription ∼11 and ∼7 times more efficiently than does the derepressed parental lac UV5 promoter and ∼3 and ∼2 times more efficiently than does the trp promoter in the absence of the trp repressor. Both hybrid promoters can be repressed by the lac repressor, and both can be derepressed with isopropyl β-D-thiogalactoside [367-93-1]. Consequently, these hybrid promoters are useful for the controlled expression of foreign genes at high levels in E. coli. In contrast to the trp and the lac UV5 promoters, the tacI promoter has not only a consensus -35 sequence but also a consensus Pribnow box sequence. This may explain the higher efficiency of this hybrid promoter with respect to either one of the parental promoters.
4. 4
  Weber, W., Stelling, J., Rimann, M., Keller, B., Daoud-El Baba, M., Weber, C. C., Aubel, D., and Fussenegger, M. (2007) A synthetic time-delay circuit in mammalian cells and mice Proc. Natl. Acad. Sci. U.S.A. 104, 2643– 2648
  4
  A synthetic time-delay circuit in mammalian cells and mice
  Weber, Wilfried; Stelling, Joerg; Rimann, Markus; Keller, Bettina; Daoud-El Baba, Marie; Weber, Cornelia C.; Aubel, Dominique; Fussenegger, Martin
  Proceedings of the National Academy of Sciences of the United States of America (2007), 104 (8), 2643-2648CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
  Time-delay circuitries in which a transcription factor processes independent input parameters can modulate NF-κB activation, manage quorum-sensing cross-talk, and control the circadian clock. We have constructed a synthetic mammalian gene network that processes four different input signals to control either immediate or time-delayed transcription of specific target genes. BirA-mediated ligation of biotin to a biotinylation signal-contg. VP16 transactivation domain triggers heterodimerization of chimeric VP16 to a streptavidin-linked tetracycline repressor (TetR). At increasing biotin concns. up to 20 nM, TetR-specific promoters are gradually activated (off to on, input signal 1), are maximally induced at concns. between 20 nM and 10 μM, and are adjustably shut off at biotin levels exceeding 10 μM (on to off, input signal 2). These specific expression characteristics with a discrete biotin concn. window emulate a biotin-triggered bandpass filter. Removal of biotin from the culture environment (input signal 3) results in time-delayed transgene expression until the intracellular biotinylated VP16 pool is degraded. Because the TetR component of the chimeric transactivator retains its tetracycline responsiveness, addn. of this antibiotic (input signal 4) overrides biotin control and immediately shuts off target gene expression. Biotin-responsive immediate, bandpass filter, and time-delay transcription characteristics were predicted by a computational model and have been validated in std. cultivation settings or biopharmaceutical manufg. scenarios using transgenic CHO-K1 cell derivs. and have been confirmed in mice. Synthetic gene circuitries provide insight into structure-function correlations of native signaling networks and foster advances in gene therapy and biopharmaceutical manufg.
5. 5
  Hawkins, K. M. and Smolke, C. D. (2006) The regulatory roles of the galactose permease and kinase in the induction response of the GAL network in Saccharomyces cerevisiae J. Biol. Chem. 281, 13485– 13492
  There is no corresponding record for this reference.
6. 6
  Topp, S., Reynoso, C. M. K., Seeliger, J. C., Goldlust, I. S., Desai, S. K., Murat, D., Shen, A., Puri, A. W., Komeili, A., Bertozzi, C. R., Scott, J. R., and Gallivan, J. P. (2010) Synthetic riboswitches that induce gene expression in diverse bacterial species Appl. Environ. Microbiol. 76, 7881– 7884
  6
  Synthetic riboswitches that induce gene expression in diverse bacterial species
  Topp, Shana; Reynoso, Colleen M. K.; Seeliger, Jessica C.; Goldlust, Ian S.; Desai, Shawn K.; Murat, Dorothee; Shen, Aimee; Puri, Aaron W.; Komeili, Arash; Bertozzi, Carolyn R.; Scott, June R.; Gallivan, Justin P.
  Applied and Environmental Microbiology (2010), 76 (23), 7881-7884CODEN: AEMIDF; ISSN:0099-2240. (American Society for Microbiology)
  We developed a series of ligand-inducible riboswitches that control gene expression in diverse species of Gram-neg. and Gram-pos. bacteria, including human pathogens that have few or no previously reported inducible expression systems. We anticipate that these riboswitches will be useful tools for genetic studies in a wide range of bacteria.
7. 7
  Wieland, M., Benz, A., Klauser, B., and Hartig, J. S. (2009) Artificial ribozyme switches containing natural riboswitch aptamer domains Angew. Chem., Int. Ed. Engl. 48, 2715– 2718
  There is no corresponding record for this reference.
8. 8
  Suess, B., Hanson, S., Berens, C., Fink, B., Schroeder, R., and Hillen, W. (2003) Conditional gene expression by controlling translation with tetracycline-binding aptamers Nucleic Acids Res. 31, 1853– 1858
  There is no corresponding record for this reference.
9. 9
  Sharma, V., Nomura, Y., and Yokobayashi, Y. (2008) Engineering complex riboswitch regulation by dual genetic selection J. Am. Chem. Soc. 130, 16310– 16315
  9
  Engineering complex riboswitch regulation by dual genetic selection
  Sharma, Vandana; Nomura, Yoko; Yokobayashi, Yohei
  Journal of the American Chemical Society (2008), 130 (48), 16310-16315CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
  The recent discovery of riboswitches in diverse species of bacteria and few eukaryotes added metabolite-responsive gene regulation to the growing list of RNA functions in biol. The natural riboswitches have inspired several designs of synthetic analogs capable of gene regulation in response to a small mol. trigger. In this work, we describe our efforts to engineer complex riboswitches capable of sensing and responding to two small mols. according to Boolean logics AND and NAND. Two aptamers that recognize theophylline and thiamin pyrophosphate were embedded in tandem in the 5' UTR of bacterial mRNA, and riboswitches that function as logic gates were isolated by dual genetic selection. The diverse phenotype of the engineered logic gates supports the versatility of RNA-based gene regulation which may have preceded the modern protein-based gene regulators. Addnl., our design strategy advances our ability to harness the versatile capacities of RNA to program complex behavior in bacteria without the use of engineered proteins.
10. 10
  Weber, W., Fux, C., Daoud-el Baba, M., Keller, B., Weber, C. C., Kramer, B. P., Heinzen, C., Aubel, D., Bailey, J. E., and Fussenegger, M. (2002) Macrolide-based transgene control in mammalian cells and mice Nat. Biotechnol. 20, 901– 907
  There is no corresponding record for this reference.
11. 11
  Gossen, M. and Bujard, H. (1992) Tight control of gene expression in mammalian cells by tetracycline-responsive promoters Proc. Natl. Acad. Sci. U.S.A. 89, 5547– 5551
  11
  Tight control of gene expression in mammalian cells by tetracycline-responsive promoters
  Gossen, Manfred; Bujard, Hermann
  Proceedings of the National Academy of Sciences of the United States of America (1992), 89 (12), 5547-51CODEN: PNASA6; ISSN:0027-8424.
  Control elements of the tetracycline-resistance operon encoded in Tn10 of Escherichia coli have been utilized to establish a highly efficient regulatory system in mammalian cells. By fusing the tet repressor with the activating domain of virion protein 16 of herpes simplex virus, a tetracycline-controlled transactivator (tTA) was generated that is constitutively expressed in HeLa cells. This transactivator stimulates transcription from a minimal promoter sequence derived from the human cytomegalovirus promoter IE combined with tet operator sequences. Upon integration of a luciferase gene controlled by a tTA-dependent promoter into a tTA-producing HeLa cell line, high levels of luciferase expression were monitored. These activities are sensitive to tetracycline. Depending on the concn. of the antibiotic in the culture medium (0-1 μg/mL), the luciferase activity can be regulated over up to five orders of magnitude. Thus, the system not only allows differential control of the activity of an individual gene in mammalian cells but also is suitable for creation of on/off situations for such genes in a reversible way.
12. 12
  Volzing, K., Biliouris, K., and Kaznessis, Y. N. (2011) proTeOn and proTeOff, new protein devices that inducibly activate bacterial gene expression ACS Chem. Biol. 6, 1107– 1116
  There is no corresponding record for this reference.
13. 13
  Neddermann, P., Gargioli, C., Muraglia, E., Sambucini, S., Bonelli, F., De Francesco, R., and Cortese, R. (2003) A novel, inducible, eukaryotic gene expression system based on the quorum-sensing transcription factor TraR EMBO Rep. 4, 159– 165
  There is no corresponding record for this reference.
14. 14
  Levskaya, A., Chevalier, A. A., Tabor, J. J., Simpson, Z. B., Lavery, L. A., Levy, M., Davidson, E. A., Scouras, A., Ellington, A. D., Marcotte, E. M., and Voigt, C. A. (2005) Synthetic biology: Engineering Escherichia coli to see light Nature 438, 441– 442
  There is no corresponding record for this reference.
15. 15
  Alper, H. and Stephanopoulos, G. (2009) Engineering for biofuels: exploiting innate microbial capacity or importing biosynthetic potential? Nat. Rev. Microbiol. 7, 715– 723
  15
  Engineering for biofuels: exploiting innate microbial capacity or importing biosynthetic potential?
  Alper, Hal; Stephanopoulos, Gregory
  Nature Reviews Microbiology (2009), 7 (10), 715-723CODEN: NRMACK; ISSN:1740-1526. (Nature Publishing Group)
  A review. The ideal microorganism for biofuel prodn. will possess high substrate utilization and processing capacities, fast and deregulated pathways for sugar transport, good tolerance to inhibitors and product, and high metabolic fluxes and will produce a single fermn. product. It is unclear whether such an organism will be engineered using a native, isolated strain or a recombinant, model organism as the starting point. The choice between engineering natural function and importing biosynthetic capacity is affected by current progress in metabolic engineering and synthetic biol. This review highlights some of the factors influencing the above decision, in light of current advances.
16. 16
  Joshua, C. J., Dahl, R., Benke, P. I., and Keasling, J. D. (2011) Absence of diauxie during simultaneous utilization of glucose and xylose by Sulfolobus acidocaldarius J. Bacteriol. 193, 1293– 1301
  16
  Absence of diauxie during simultaneous utilization of glucose and xylose by Sulfolobus acidocaldarius
  Joshua, Chijioke J.; Dahl, Robert; Benke, Peter I.; Keasling, Jay D.
  Journal of Bacteriology (2011), 193 (6), 1293-1301CODEN: JOBAAY; ISSN:0021-9193. (American Society for Microbiology)
  Sulfolobus acidocaldarius utilizes glucose and xylose as sole carbon sources, but its ability to metabolize these sugars simultaneously is not known. The absence of diauxie during growth of S. acidocaldarius on glucose and xylose as co-carbon sources is reported. The presence of glucose did not repress xylose utilization. The organism utilized a mixt. of 1 g/L of each sugar simultaneously with a specific growth rate of 0.079 h-1 and showed no preference for the order in which it utilized each sugar. The organism grew faster on 2 g/L xylose (0.074 h-1) as the sole carbon source than on an equal amt. of glucose (0.022 h-1). When grown on a mixt. of the two carbon sources, the growth rate of the organism increased from 0.052 h-1 to 0.085 h-1 as the ratio of xylose to glucose increased from 0.25 to 4. S. acidocaldarius appeared to utilize a mixt. of glucose and xylose at a rate roughly proportional to their concns. in the medium, resulting in complete utilization of both sugars at about the same time. Gene expression in cells grown on xylose alone was very similar to that in cells grown on a mixt. of xylose and glucose and substantially different from that in cells grown on glucose alone. The mechanism by which the organism utilized a mixt. of sugars has yet to be elucidated.
17. 17
  Johnsen, U., Dambeck, M., Zaiss, H., Fuhrer, T., Soppa, J., Sauer, U., and Schönheit, P. (2009) d-xylose degradation pathway in the halophilic archaeon Haloferax volcanii J. Biol. Chem. 284, 27290– 27303
  There is no corresponding record for this reference.
18. 18
  Peng, N., Ao, X., Liang, Y. X., and She, Q. (2011) Archaeal promoter architecture and mechanism of gene activation Biochem. Soc. Trans. 39, 99– 103
  18
  Archaeal promoter architecture and mechanism of gene activation
  Peng, Nan; Ao, Xiang; Liang, Yun Xiang; She, Qunxin
  Biochemical Society Transactions (2011), 39 (1), 99-103CODEN: BCSTB5; ISSN:0300-5127. (Portland Press Ltd.)
  A review. Sulfolobus solfataricus and Sulfolobus islandicus contain several genes exhibiting D-arabinose-inducible expression and these systems are ideal for studying mechanisms of archaeal gene expression. At sequence level, only two highly conserved cis elements are present on the promoters: a regulatory element named ara box directing arabinose-inducible expression and the basal promoter element TATA, serving as the binding site for the TATA-binding protein. Strikingly, these promoters possess a modular structure that allows an essentially inactive basal promoter to be strongly activated. The invoked mechanisms include TFB (transcription factor B) recruitment by the ara-box-binding factor to activate gene expression and modulation of TFB recruitment efficiency to yield differential gene expression.
19. 19
  Cardona, S. T., Mueller, C. L., and Valvano, M. A. (2006) Identification of essential operons with a rhamnose-inducible promoter in Burkholderia cenocepacia Appl. Environ. Microbiol. 72, 2547– 2555
  There is no corresponding record for this reference.
20. 20
  Schlegel, A., Böhm, A., Lee, S.-J., Peist, R., Decker, K., and Boos, W. (2002) Network regulation of the Escherichia coli maltose system J. Mol. Microbiol. Biotechnol. 4, 301– 307
  20
  Network regulation of the Escherichia coli maltose system
  Schlegel, Anja; Bohm, Alex; Lee, Sung-Jae; Peist, Ralf; Decker, Katja; Boos, Winfried
  Journal of Molecular Microbiology and Biotechnology (2002), 4 (3), 301-307CODEN: JMMBFF; ISSN:1464-1801. (Horizon Scientific Press)
  A review. The genes of the Escherichia coli maltose regulon are controlled by MalT, the specific transcriptional activator which, together with the inducer maltotriose and ATP, is essential for mal gene transcription. Network regulation in this system affects the function of MalT and occurs on two levels. The first concerns the expression of malT. It has long been known that malT is under catabolite repression and thus under the control of the cAMP/CAP complex. In addn., the global regulator MIc is found to be a repressor for malT transcription. The repressor activity of MIc is controlled by the transport status of the glucose-specific enzyme EIICB of the PTS that causes sequestration (and inactivation as a repressor) of MIc when glucose is transported. The second level of MalT regulation affects its activity. MalT is activated by maltotriose which is not only formed when the cells are growing on any maltodextrin but also, in low amts., endogenously when the cells grow on non-maltodextrin carbon sources. Thus, cellular metab., for instance degrdn. of galactose or trehalose, can cause mal gene induction. It was found that unphosphorylated internal glucose takes part in endogenous maltodextrin biosynthesis and is therefore a key element in endogenous mal gene expression. In addn. to the maltotriose-dependent activation, MalT can interact with three different enzymes that lead to its inactivation as a transcriptional activator. The first is MalK, the energy transducing ABC subunit of the maltodextrin transport system. Transport controls the interaction of MalK and MalT thus affecting gene expression. The second enzyme is MalY, a pyridoxal phosphate contg. enzyme exhibiting cystathionase activity. The crystal structure of MalY was established and mutations in MalY that reduce mal gene repression map in a hydrophobic MalT interaction patch on the surface of the enzyme. The last enzyme is a sol. esterase of as yet unknown function. When overproduced, this enzyme specifically reduces mal gene expression and affects the activity of MalT in an in vitro transcription assay.
21. 21
  Peri, K. G., Goldie, H., and Waygood, E. B. (1990) Cloning and characterization of the N-acetylglucosamine operon of Escherichia coli Biochem. Cell Biol. 68, 123– 137
  21
  Cloning and characterization of the N-acetylglucosamine operon of Escherichia coli
  Peri, Krishna G.; Goldie, Hughes; Waygood, E. Bruce
  Biochemistry and Cell Biology (1990), 68 (1), 123-37CODEN: BCBIEQ; ISSN:0829-8211.
  Three enzymes are required for N-acetylglucosamine (NAG) utilization in E. coli: enzyme IInag (gene nagE), N-acetylglucosamine-6-phosphate deacetylase (gene nagA), and glucosamine-6-phosphate isomerase (gene nagB). The 3 genes are located near 16 min on the E. coli chromosome. A strain of E. coli, KPN9, incapable of utilizing N-acetylglucosamine, was used to screen a genomic library of E. coli for a complementing recombinant colicin E1 plasmid that allowed for growth on N-acetylglucosamine. Plasmid pLC5-21 was found to contain all 3 known nag genes on a 5.7-kilobase (5.7-kb) fragment of DNA. The products of these nag genes were identified by complementation of E. coli strains with mutations in nagA, nagB, and nagE. The gene products from the 5.7-kb fragment were identified by [35S]methionine-labeled maxicells and autoradiog. of SDS-polyacrylamide electrophoresis gels. The gene products had the following relative masses (Mrs: nagE, 62,000; nagA, 45,000; nagB, 29,000. In addn., another product of Mr 44,000 was detected. The genes have been sequenced to reveal an addnl. open reading frame (nagC), a putative catabolite activator protein binding site that may control nagB and nagE, putative rho-independent terminator sites for nagB and nagE, and sequence homologies for RNA polymerase binding sites preceding each of the open reading frames, except for nagA. The calcd. mol. wts. (MWs) of the gene products derived from the sequence are as follows: nagA, 40,954; nagB, 29,657; nagC, 44,664; nagE, 68,356. No role is known for nagC, although a no. of regulatory roles appear to be plausible. No obvious transcriptional termination site distal to nagC was found and another open reading frame begins after nagC. This gene, nagD, was isolated sep. from pLC5-21, and the sequence revealed a protein with a calcd. MW of 27,181. The nagD gene is followed by repetitive extragenic palindromic sequences. The nag genes appear to be organized in an operon: nagD nagC nagA nagB transcribed in one direction and nagE transcribed in the opposite direction.
22. 22
  Tong, S., Porco, A., Isturiz, T., and Conway, T. (1996) Cloning and molecular genetic characterization of the Escherichia coli gntR, gntK, and gntU genes of GntI, the main system for gluconate metabolism J. Bacteriol. 178, 3260– 3269
  22
  Cloning and molecular genetic characterization of the Escherichia coli gntR, gntK, and gntU genes of GntI, the main system for gluconate metabolism
  Tong, Suxiang; Porco, Antonieta; Isturiz, Tomas; Conway, Tyrrell
  Journal of Bacteriology (1996), 178 (11), 3260-3269CODEN: JOBAAY; ISSN:0021-9193. (American Society for Microbiology)
  Three genes involved in gluconate metab., gntR, gntK, and gntU, which code for a regulatory protein, a gluconate kinase, and a gluconate transporter, resp., were cloned from Escherichia coli K-12 on the basis of their known locations on the genomic restriction map. The gene order is gntU, gntK, and gntR, which are immediately adjacent to asd at 77.0 min, and all three genes are transcribed in the counterclockwise direction. The gntR product is 331 amino acids long, with a helix-turn-helix motif typical of a regulatory protein. The gntK gene encodes a 175-amino-acid polypeptide that has an ATP-binding motif similar to those found in other sugar kinases. While GntK does not show significant sequence similarity to any known sugar kinases, it is 45% identical to a second putative gluconate kinase from E. coli, gntV. The 445-amino-acid sequence encoded by gntU has a secondary structure typical of membrane-spanning transport proteins and is 37% identical to the gntP product from Bacillus subtilis. Kinetic anal. of GntU indicates an apparent Km for gluconate of 212 μM, indicating that this is a low-affinity transporter. Studies demonstrate that the gntR gene is monocistronic, while the gntU and gntK genes, which are sepd. by only 3 bp, form an operon. Expression of gntR is essentially constitutive, while expression of gntKU is induced by gluconate and is subject to fourfold glucose catabolite repression. These results confirm that gntK and gntU, together with another gluconate transport gene, gntT, constitute the GntI system for gluconate utilization, under control of the gntR gene product, which is also responsible for induction of the edd and eda genes of the Entner-Doudoroff pathway.
23. 23
  Lubelska, J. M., Jonuscheit, M., Schleper, C., Albers, S.-V., and Driessen, A. J. M. (2006) Regulation of expression of the arabinose and glucose transporter genes in the thermophilic archaeon Sulfolobus solfataricus Extremophiles 10, 383– 391
  There is no corresponding record for this reference.
24. 24
  Song, S. and Park, C. (1997) Organization and regulation of the d-xylose operons in Escherichia coli K-12: XylR acts as a transcriptional activator J. Bacteriol. 179, 7025– 7032
  24
  Organization and regulation of the D-xylose operons in Escherichia coli K-12: XylR acts as a transcriptional activator
  Song, Sukgil; Park, Chankyu
  Journal of Bacteriology (1997), 179 (22), 7025-7032CODEN: JOBAAY; ISSN:0021-9193. (American Society for Microbiology)
  The metab. of D-xylose in Escherichia coli K-12 is known to be mediated by the xylAB gene. However, the nearby xylFGHR genes were found by genome sequencing and predicted to be responsible for transport and regulation for xylose based on their sequence similarities to other functionally related genes. Here, we investigated transcriptional organization and functions of the xyl genes. An anal. with random transposon insertions revealed that the xyl genes are organized into two major transcriptional units, xylAB and xylFGHR, governed by the promoters PA and PF, resp. However, there is an addnl. weak promoter, PR, which is specific for xylR. Sites of transcription initiation were detd. by primer extension anal. When studied with operon fusions to lacZ, the PA and PF, promoters were activated by D-xylose and repressed by glucose. In contrast, the PR promoter was not regulated by these sugars. A mutation in xylR completely abolished expression from the PA and PF promoters, causing a defect in both growth and transport. Binding of XylR to the xyl promoter was enhanced by the presence of D-xylose, suggesting that transcription was pos. regulated by XylR. In vivo footprinting anal. revealed that XylR binds to at least two DNA regions, IA and IF,, each with a direct repeat. It is very likely that XyIR interacts with IA and IF as a dimer. The presumed binding sites are located just upstream of the promoter consensus sequences (-35), while IA is addnl. flanked by a cAMP receptor protein-binding site on the other side. The proposed structure of xyl promoters is consistent with the regulation of xyl gene expression and with phenotypes of transposon insertions obtained in the promoter regions.
25. 25
  Moralejo, P., Egan, S. M., Hidalgo, E., and Aguilar, J. (1993) Sequencing and characterization of a gene cluster encoding the enzymes for l-rhamnose metabolism in Escherichia coli J. Bacteriol. 175, 5585– 5594
  25
  Sequencing and characterization of a gene cluster encoding the enzymes for L-rhamnose metabolism in Escherichia coli
  Moralejo, Pilar; Egan, Susan M.; Hidalgo, Elena; Aguilar, Juan
  Journal of Bacteriology (1993), 175 (17), 5585-94CODEN: JOBAAY; ISSN:0021-9193.
  The sequencing of the EcoRI-HindIII fragment complementing mutations in the structural genes of the L-rhamnose regulon of Escherichia coli has permitted identification of the open reading frames corresponding to rhaB, rhaA, and rhaD. The deduced amino acid sequences gave a 425-amino-acid polypeptide corresponding to rhamnulose kinase for rhaB, a 400-amino-acid polypeptide corresponding to rhamnose isomerase for rhaA, and a 274-amino-acid polypeptide corresponding to rhamnulose-1-phosphate aldolase for rhaD. Transcriptional fusions of the three putative promoter regions to lacZ showed that only the rhaB leader region acted as a promoter, as indicated by the high β-galactosidase activity induced by rhamnose, while no significant activity from the rhaA and rhaD constructions was detected. The rhaB transcription start site was mapped to -24 relative to the start of translation. Mutations in the catabolic genes were used to show that L-rhamnose may directly induce rhaBAD transcription.
26. 26
  Siegele, D. A. and Hu, J. C. (1997) Gene expression from plasmids containing the araBAD promoter at subsaturating inducer concentrations represents mixed populations Proc. Natl. Acad. Sci. U.S.A. 94, 8168– 8172
  26
  Gene expression from plasmids containing the araBAD promoter at subsaturating inducer concentrations represents mixed populations
  Siegele, Deborah A.; Hu, James C.
  Proceedings of the National Academy of Sciences of the United States of America (1997), 94 (15), 8168-8172CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
  Gene expression from plasmids contg. the araBAD promoter can be regulated by the concn. of arabinose in the growth medium. Guzman et al. [Guzman, L.-M., Belin, D., Carson, M. J. & Beckwith, J. (1995) J. Bacteriol. 177, 4121-4130] showed that expression of a cloned gene could be modulated over several orders of magnitude in cultures grown in the presence of subsaturating concns. of arabinose. We constructed plasmids expressing a fast-folding mutant Aequorea victoria green fluorescent protein from the araBAD promoter to examine the distribution of expressed gene products in individual cells at intermediate induction levels. Microscopic examn. of cells grown at low arabinose concns. shows mixts. of brightly fluorescent and dark cells, suggesting that intermediate expression levels in cultures reflect a population av. of induced and uninduced cells. The kinetics of green fluorescent protein induction suggest that this reflects an "autocatalytic" induction mechanism due to accumulation of the inducer by active transport. This mechanism, which is analogous to the induction of the lac operon at subsaturating inducer concns. in lacY+ cells, was described 40 yr ago by Novic and Weiner [Novick, A. & Weiner, M. (1957) Proc. Natl. Acad. Sci. USA 43, 553-566].
27. 27
  Novick, A. and Weiner, M. (1957) Enzyme induction as an all-or-none phenomenon Proc. Natl. Acad. Sci. U.S.A. 43, 553– 566
  There is no corresponding record for this reference.
28. 28
  Cohn, M. and Horibata, K. (1959) Analysis of the differentiation and of the heterogeneity within a population of Escherichia coli undergoing induced β-galactosidase synthesis J. Bacteriol. 78, 613– 623
  28
  Analysis of the differentiation and of the heterogeneity within a population of Escherichia coli undergoing induced β-galactosidase synthesis
  Cohn, Melvin; Horibata, Kengo
  Journal of Bacteriology (1959), 78 (), 613-23CODEN: JOBAAY; ISSN:0021-9193.
  Under conditions where the metabolic activity of a system under induction is essential to the formation of that system, the response of each cell in a population is sharply dependent upon whether it possesses a min. unit of the induced activity. As a result, the formation of enzyme by the population is heterogeneous. Both the formation and nonformation of enzyme is passed on clonically to the descendents. Two induced systems: the galactoside permease and the β-galactosidase (Cohen and Monod, C.A. 52, 497i) of E. coli and the galactozymase of long-term adapting yeast (Spiegelman, C.A. 46, 7178c) can be understood in terms of this concept. The all-or-none distribution of induction cannot as yet be interpreted to reflect a property of the enzyme-forming system itself. 26 references.
29. 29
  Ozbudak, E. M., Thattai, M., Lim, H. N., Shraiman, B. I., and van Oudenaarden, A. (2004) Multistability in the lactose utilization network of Escherichia coli Nature 427, 737– 740
  There is no corresponding record for this reference.
30. 30
  Acar, M., Becskei, A., and van Oudenaarden, A. (2005) Enhancement of cellular memory by reducing stochastic transitions Nature 435, 228– 232
  There is no corresponding record for this reference.
31. 31
  Kuhlman, T., Zhang, Z., Saier, M. H., Jr, and Hwa, T. (2007) Combinatorial transcriptional control of the lactose operon of Escherichia coli Proc. Natl. Acad. Sci. U.S.A. 104, 6043– 6048
  There is no corresponding record for this reference.
32. 32
  Setty, Y., Mayo, A. E., Surette, M. G., and Alon, U. (2003) Detailed map of a cis-regulatory input function Proc. Natl. Acad. Sci. U.S.A. 100, 7702– 7707
  There is no corresponding record for this reference.
33. 33
  Krishna, S., Semsey, S., and Sneppen, K. (2007) Combinatorics of feedback in cellular uptake and metabolism of small molecules Proc. Natl. Acad. Sci. U.S.A. 104, 20815– 20819
  There is no corresponding record for this reference.
34. 34
  Tian, X.-J., Zhang, X.-P., Liu, F., and Wang, W. (2009) Interlinking positive and negative feedback loops creates a tunable motif in gene regulatory networks Phys. Rev. E Stat. Nonlin. Soft Matter Phys. 80, 011926
  There is no corresponding record for this reference.
35. 35
  Khlebnikov, A., Datsenko, K. A., Skaug, T., Wanner, B. L., and Keasling, J. D. (2001) Homogeneous expression of the P_BAD promoter in Escherichia coli by constitutive expression of the low-affinity high-capacity AraE transporter Microbiology 147, 3241– 3247
  There is no corresponding record for this reference.
36. 36
  Khlebnikov, A., Risa, O., Skaug, T., Carrier, T. A., and Keasling, J. D. (2000) Regulatable arabinose-inducible gene expression system with consistent control in all cells of a culture J. Bacteriol. 182, 7029– 7034
  36
  Regulatable arabinose-inducible gene expression system with consistent control in all cells of a culture
  Khlebnikov, Artem; Risa, Oystein; Skaug, Tove; Carrier, Trent A.; Keasling, J. D.
  Journal of Bacteriology (2000), 182 (24), 7029-7034CODEN: JOBAAY; ISSN:0021-9193. (American Society for Microbiology)
  The arabinose-inducible promoter PBAD is subject to all-or-none induction, in which intermediate concns. of arabinose give rise to subpopulations of cells that are fully induced and uninduced. To construct a host-vector expression system with regulatable control in a homogeneous population of cells, the araE gene of Escherichia coli was cloned into an RSF1010-derived plasmid under control of the isopropyl-β-D-thiogalactopyranoside-inducible Ptac and Ptaclac promoters. This gene encodes the low-affinity, high-capacity arabinose transport protein and is controlled natively by an arabinose-inducible promoter. To detect the effect of arabinose-independent araE expression on population homogeneity and cell-specific expression, the gfpuv gene was placed under control of the arabinose-inducible araBAD promoter (PBAD) on the pMB1-derived plasmid pBAD24. The transporter and reporter plasmids were transformed into E. coli strains with native arabinose transport systems and strains deficient in one or both of the arabinose transport systems (araE and/or araFGH). The effects of the arabinose concn. and arabinose-independent transport control on population homogeneity were investigated in these strains using flow cytometry. The araE, and araE araFGH mutant strains harboring the transporter and reporter plasmids were uniformly induced across the population at all inducer concns., and the level of gene expression in individual cells varied with arabinose concn. In contrast, the parent strain, which expressed the native araE and araFGH genes and harbored the transporter and reporter plasmids, exhibited all-or-none behavior. This work demonstrates the importance of including a transport gene that is controlled independently of the inducer to achieve regulatable and consistent induction in all cells of the culture.
37. 37
  Morgan-Kiss, R. M., Wadler, C., and Cronan, J. E., Jr. (2002) Long-term and homogeneous regulation of the Escherichia coli araBAD promoter by use of a lactose transporter of relaxed specificity Proc. Natl. Acad. Sci. U.S.A. 99, 7373– 7377
  There is no corresponding record for this reference.
38. 38
  Schleif, R. (2010) AraC protein, regulation of the l-arabinose operon in Escherichia coli, and the light switch mechanism of AraC action FEMS Microbiol. Rev. 34, 779– 796
  38
  AraC protein, regulation of the L-arabinose operon in Escherichia coli, and the light switch mechanism of AraC action
  Schleif, Robert
  FEMS Microbiology Reviews (2010), 34 (5), 779-796CODEN: FMREE4; ISSN:0168-6445. (Wiley-Blackwell)
  A review. This review covers the physiol. aspects of regulation of the arabinose operon in Escherichia coli and the phys. and regulatory properties of the operon's controlling gene, araC. It also describes the light switch mechanism as an explanation for many of the protein's properties. Although many thousands of homologs of AraC exist and regulate many diverse operons in response to many different inducers or physiol. states, homologs that regulate arabinose-catabolizing genes in response to arabinose were identified. The sequence similarities among them are discussed in light of the known structure of the dimerization and DNA-binding domains of AraC.
39. 39
  Reeder, T. and Schleif, R. (1991) Mapping, sequence, and apparent lack of function of araJ, a gene of the Escherichia coli arabinose regulon J. Bacteriol. 173, 7765– 7771
  39
  Mapping, sequence, and apparent lack of function of araJ, a gene of Escherichia coli arabinose regulon
  Reeder, Thadd; Schleif, Robert
  Journal of Bacteriology (1991), 173 (24), 7765-71CODEN: JOBAAY; ISSN:0021-9193.
  The mapping, sequencing, and study of the physiol. role of the fourth arabinose-inducible operon from E. coli, araJ is reported here. It is located at 9 min on the chromosome and codes for a single 42-kDa protein that shows no significant homol. to other known proteins. Destruction of the chromosomal araJ gene does not detectably affect either of the 2 arabinose transport systems, the ability of cells to grow on arabinose, or the induction kinetics of the araBAD operon, and thus the physiol. role of AraJ, if any, remains unknown. A long open reading frame was also found upstream of araJ. The sequence of this upstream open reading frame was found to be identical to the previously reported sequence of the sbcC gene (Na0om, I.S., et al., 1989). The carboxyl region of SbcC has an amino acid sequence consistent with this region of SbcC forming an extended alpha-helical coiled-coil.
40. 40
  Hendrickson, W., Stoner, C., and Schleif, R. (1990) Characterization of the Escherichia coli araFGH and araJ promoters J. Mol. Biol. 215, 497– 510
  There is no corresponding record for this reference.
41. 41
  Zaslaver, A., Bren, A., Ronen, M., Itzkovitz, S., Kikoin, I., Shavit, S., Liebermeister, W., Surette, M. G., and Alon, U. (2006) A comprehensive library of fluorescent transcriptional reporters for Escherichia coli Nat. Methods 3, 623– 628
  41
  A comprehensive library of fluorescent transcriptional reporters for Escherichia coli
  Zaslaver, Alon; Bren, Anat; Ronen, Michal; Itzkovitz, Shalev; Kikoin, Ilya; Shavit, Seagull; Liebermeister, Wolfram; Surette, Michael G.; Alon, Uri
  Nature Methods (2006), 3 (8), 623-628CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)
  E. coli is widely used for systems biol. research; there exists a need, however, for tools that can be used to accurately and comprehensively measure expression dynamics in individual living cells. To address this the authors present a library of transcriptional fusions of gfp to each of about 2,000 different promoters in E. coli K12, covering the great majority of the promoters in the organism. Each promoter fusion is expressed from a low-copy plasmid. The authors demonstrate that this library can be used to obtain highly accurate dynamic measurements of promoter activity on a genomic scale, in a glucose-lactose diauxic shift expt. The library allowed detection of about 80 previously uncharacterized transcription units in E. coli, including putative internal promoters within previously known operons, such as the lac operon. This library can serve as a tool for accurate, high-resoln. anal. of transcription networks in living E. coli cells.
42. 42
  Tan, C., Marguet, P., and You, L. (2009) Emergent bistability by a growth-modulating positive feedback circuit Nat. Chem. Biol. 5, 842– 848
  42
  Emergent bistability by a growth-modulating positive feedback circuit
  Tan, Cheemeng; Marguet, Philippe; You, Lingchong
  Nature Chemical Biology (2009), 5 (11), 842-848CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)
  Synthetic gene circuits are often engineered by considering the host cell as an invariable 'chassis'. Circuit activation, however, may modulate host physiol., which in turn can substantially impact circuit behavior. We illustrate this point by a simple circuit consisting of mutant T7 RNA polymerase (T7 RNAP*) that activates its own expression in the bacterium Escherichia coli. Although activation by the T7 RNAP* is noncooperative, the circuit caused bistable gene expression. This counterintuitive observation can be explained by growth retardation caused by circuit activation, which resulted in nonlinear diln. of T7 RNAP* in individual bacteria. Predictions made by models accounting for such effects were verified by further exptl. measurements. Our results reveal a new mechanism of generating bistability and underscore the need to account for host physiol. modulation when engineering gene circuits.
43. 43
  Nevozhay, D., Adams, R. M., Murphy, K. F., Josic, K., and Balázsi, G. (2009) Negative autoregulation linearizes the dose-response and suppresses the heterogeneity of gene expression Proc. Natl. Acad. Sci. U.S.A. 106, 5123– 5128
  There is no corresponding record for this reference.
44. 44
  Madar, D., Dekel, E., Bren, A., and Alon, U. (2011) Negative auto-regulation increases the input dynamic-range of the arabinose system of Escherichia coli BMC Syst. Biol. 5, 111
  44
  Negative auto-regulation increases the input dynamic-range of the arabinose system of Escherichia coli
  Madar Daniel; Dekel Erez; Bren Anat; Alon Uri
  BMC systems biology (2011), 5 (), 111 ISSN:.
  BACKGROUND: Gene regulation networks are made of recurring regulatory patterns, called network motifs. One of the most common network motifs is negative auto-regulation, in which a transcription factor represses its own production. Negative auto-regulation has several potential functions: it can shorten the response time (time to reach halfway to steady-state), stabilize expression against noise, and linearize the gene's input-output response curve. This latter function of negative auto-regulation, which increases the range of input signals over which downstream genes respond, has been studied by theory and synthetic gene circuits. Here we ask whether negative auto-regulation preserves this function also in the context of a natural system, where it is embedded within many additional interactions. To address this, we studied the negative auto-regulation motif in the arabinose utilization system of Escherichia coli, in which negative auto-regulation is part of a complex regulatory network. RESULTS: We find that when negative auto-regulation is disrupted by placing the regulator araC under constitutive expression, the input dynamic range of the arabinose system is reduced by 10-fold. The apparent Hill coefficient of the induction curve changes from about n = 1 with negative auto-regulation, to about n = 2 when it is disrupted. We present a mathematical model that describes how negative auto-regulation can increase input dynamic-range, by coupling the transcription factor protein level to the input signal. CONCLUSIONS: Here we demonstrate that the negative auto-regulation motif in the native arabinose system of Escherichia coli increases the range of arabinose signals over which the system can respond. In this way, negative auto-regulation may help to increase the input dynamic-range while maintaining the specificity of cooperative regulatory systems. This function may contribute to explaining the common occurrence of negative auto-regulation in biological systems.
45. 45
  Weickert, M. J. and Adhya, S. (1993) The galactose regulon of Escherichia coli Mol. Microbiol. 10, 245– 251
  45
  The galactose regulon of Escherichia coli
  Weickert, Michael J.; Adhya, Sankar
  Molecular Microbiology (1993), 10 (2), 245-51CODEN: MOMIEE; ISSN:0950-382X.
  A review with 59 refs. Galactose transport and metab. in Escherichia coli involves a multicomponent amphibolic pathway. Galactose transport is accomplished by 2 different galactose-specific transport systems. At least 4 of the genes and operons involved in galactose transport and metab. have promoters contg. similar regulatory sequences. These sequences are recognized by at least 3 regulators, Gal repressor (GalR), Gal isorepressor (GalS) and cAMP receptor protein (CRP), which modulate transcription from these promoters. The neg. regulators, GalR and GalS, discriminate between utilization of the high-affinity (regulated by GalS) and low-affinity (regulated by GalR) transport systems, and modulate the expression of genes for galactose metab. in an overlapping fashion. GalS is itself autogenously regulated and CRP dependent, while the gene for GalR is constitutive. The gal operon encoding the enzymes for galactose metab. has 2 promoters regulated by CRP in opposite ways; one (P1) is stimulated and the other (P2) inhibited by GalR but weakly by GalS. All but one of the constituent promoters of the gal regulon have 2 operators. The gal regulon has the potential to coordinate galactose metab. and transport in a highly efficient manner, under a wide variety of conditions of galactose availability.
46. 46
  Yildirim, N. and Mackey, M. C. (2003) Feedback regulation in the lactose operon: a mathematical modeling study and comparison with experimental data Biophys. J. 84, 2841– 2851
  There is no corresponding record for this reference.
47. 47
  Court, D. L., Swaminathan, S., Yu, D., Wilson, H., Baker, T., Bubunenko, M., Sawitzke, J., and Sharan, S. K. (2003) Mini-lambda: a tractable system for chromosome and BAC engineering Gene 315, 63– 69
  There is no corresponding record for this reference.
48. 48
  Cherepanov, P. P. and Wackernagel, W. (1995) Gene disruption in Escherichia coli: Tc^R and Km^R cassettes with the option of Flp-catalyzed excision of the antibiotic-resistance determinant Gene 158, 9– 14
  48
  Gene disruption in Escherichia coli: TcR and KmR cassettes with the option of FlP-catalyzed excision of the antibiotic-resistance determinant
  Cherepanov, Peter P.; Wackernagel, Wilfried
  Gene (1995), 158 (1), 9-14CODEN: GENED6; ISSN:0378-1119. (Elsevier)
  Two cassettes with tetracycline-resistance (TcR) and kanamycin-resistance (KmR) determinants have been developed for the construction of insertion and deletion mutants of cloned genes in Escherichia coli. In both cassettes, the resistance determinants are flanked by the short direct repeats (FRT sites) required for site-specific recombination mediated by the yeast Flp recombinase. In addn., a plasmid with temp.-sensitive replication for temporal prodn. of the Flp enzyme in E. coli has been constructed. After a gene disruption or deletion mutation is constructed in vitro by insertion of one of the cassettes into a given gene, the mutated gene is transferred to the E. coli chromosome by homologous recombination and selection for the antibiotic resistance provided by the cassette. If desired, the resistance determinant can subsequently be removed from the chromosome in vivo by Flp action, leaving behind a short nucleotide sequence with one FRT site and with no polar effect on downstream genes. This system was applied in the construction of an E. coli endA deletion mutation which can be transduced by P1 to the genetic background of interest using TcR as a marker. The transductant can then be freed of the TcR if required.
49. 49
  Del Vecchio, D., Ninfa, A. J., and Sontag, E. D. (2008) Modular cell biology: Retroactivity and insulation Mol. Syst. Biol. 4, 161
  49
  Modular cell biology: retroactivity and insulation
  Del Vecchio Domitilla; Ninfa Alexander J; Sontag Eduardo D
  Molecular systems biology (2008), 4 (), 161 ISSN:.
  Modularity plays a fundamental role in the prediction of the behavior of a system from the behavior of its components, guaranteeing that the properties of individual components do not change upon interconnection. Just as electrical, hydraulic, and other physical systems often do not display modularity, nor do many biochemical systems, and specifically, genetic networks. Here, we study the effect of interconnections on the input-output dynamic characteristics of transcriptional components, focusing on a property, which we call 'retroactivity', that plays a role analogous to non-zero output impedance in electrical systems. In transcriptional networks, retroactivity is large when the amount of transcription factor is comparable to, or smaller than, the amount of promoter-binding sites, or when the affinity of such binding sites is high. To attenuate the effect of retroactivity, we propose a feedback mechanism inspired by the design of amplifiers in electronics. We introduce, in particular, a mechanism based on a phosphorylation-dephosphorylation cycle. This mechanism enjoys a remarkable insulation property, due to the fast timescales of the phosphorylation and dephosphorylation reactions.
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Trade-offs in Engineering Sugar Utilization Pathways for Titratable ControlClick to copy article linkArticle link copied!

ACS Synthetic Biology

Abstract

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Figure 1

Results and Discussion

Simple Mathematical Model of Sugar Utilization

Figure 2

Experimentally Probing l-arabinose Utilization to Explore Model Predictions

Figure 3

Constitutively Expressing the High-Affinity Transporter More Readily Generates a Uniform Response

Trade-offs when Deleting One Transporter

Trade-offs when Eliminating Sugar Catabolism

Breakdown of the Sugar Can Help Linearize the Response

Figure 4

Similar Trends When Linearizing the Response to d-xylose

Figure 5

Design Rules to Engineer Sugar Utilization Pathways for Titratable Control

Methods

Bacterial Strains and Plasmids

Growth Conditions and Media

Flow Cytometry Analysis

Curve Fitting to Extract Performance Metrics

Mathematical Modeling

Statistical Analyses

Supporting Information

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Acknowledgment

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Trade-offs in Engineering Sugar Utilization Pathways for Titratable Control
Click to copy article linkArticle link copied!