ACS Publications. Most Trusted. Most Cited. Most Read
Reverse-Phase Protein Microarrays for Overexpressed Escherichia coli Lysates Reveal a Novel Tyrosine Kinase
More

OR SEARCH CITATIONS

My Activity
Publications

Figure 1Loading Img
Download Hi-Res ImageDownload to MS-PowerPointCite This:Anal. Chem. 2024, 96, 21, 8721-8729
  • Open Access
Article

Reverse-Phase Protein Microarrays for Overexpressed Escherichia coli Lysates Reveal a Novel Tyrosine Kinase
Click to copy article linkArticle link copied!

  • Batuhan Birol Keskin
    Batuhan Birol Keskin
    Department of Biotechnology and Bioindustry Sciences, National Cheng Kung University, Tainan 701, Taiwan
    More by Batuhan Birol Keskin
  • Chien-Sheng Chen
    Chien-Sheng Chen
    Department of Food Safety/Hygiene and Risk Management, College of Medicine, National Cheng Kung University, Tainan 701, Taiwan
    Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University, Tainan 701, Taiwan
    More by Chien-Sheng Chen
    Orcidhttps://orcid.org/0000-0002-2372-324X
  • Pei-Shan Tsai
    Pei-Shan Tsai
    Department of Biotechnology and Bioindustry Sciences, National Cheng Kung University, Tainan 701, Taiwan
    More by Pei-Shan Tsai
  • Pin-Xian Du
    Pin-Xian Du
    Department of Biotechnology and Bioindustry Sciences, National Cheng Kung University, Tainan 701, Taiwan
    More by Pin-Xian Du
  • John Harvey M. Santos
    John Harvey M. Santos
    Department of Biotechnology and Bioindustry Sciences, National Cheng Kung University, Tainan 701, Taiwan
    Centre for Animal Science, Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, Brisbane, QLD 4072, Australia
    More by John Harvey M. Santos
    Orcidhttps://orcid.org/0000-0002-6514-9565
  • Guan-Da Syu*
    Guan-Da Syu
    Department of Biotechnology and Bioindustry Sciences, National Cheng Kung University, Tainan 701, Taiwan
    International Center for Wound Repair and Regeneration, National Cheng Kung University, Tainan 701, Taiwan
    Medical Device Innovation Center, National Cheng Kung University, Tainan 701, Taiwan
    *Email: [email protected]; Tel.: +886-6-275-7575#58231. Fax: +886-6-276-6490.
    More by Guan-Da Syu
    Orcidhttps://orcid.org/0000-0003-4661-414X
Open PDFSupporting Information (1)

Analytical Chemistry

Cite this: Anal. Chem. 2024, 96, 21, 8721–8729
Click to copy citationCitation copied!
https://doi.org/10.1021/acs.analchem.4c00965
Published April 29, 2024

Copyright © 2024 The Authors. Published by American Chemical Society. This publication is licensed under

CC-BY 4.0 .

Abstract

Click to copy section linkSection link copied!
High Resolution Image
Download MS PowerPoint Slide

Tyrosine phosphorylation is one of the most important posttranslational modifications in bacteria, linked to regulating growth, migration, virulence, secondary metabolites, biofilm formation, and capsule production. Only two tyrosine kinases (yccC (etk) and wzc) have been identified in Escherichia coli. The investigation by similarity has not revealed any novel BY-kinases in silico so far, most probably due to their sequence and structural variability. Here we developed a reverse-phase protein array from 4126 overexpressed E. coli clones, lysed, and printed on coated glass slides. These high-density E. coli lysate arrays (ECLAs) were quality controlled by the reproducibility and immobilization of total lysate proteins and specific overexpressed proteins. ECLAs were used to interrogate the relationship between protein overexpression and tyrosine phosphorylation in the total lysate. We identified 6 protein candidates, including etk and wzc, with elevated phosphotyrosine signals in the total lysates. Among them, we identified a novel kinase nrdD with autophosphorylation and transphosphorylation activities in the lysates. Moreover, the overexpression of nrdD induced biofilm formation. Since nrdD is a novel kinase, we used E. coli proteome microarrays (purified 4,126 E. coli proteins) to perform an in vitro kinase assay and identified 33 potential substrates. Together, this study established a new ECLA platform for interrogating posttranslational modifications and identified a novel kinase that is important in biofilm formation, which will shed some light on bacteria biochemistry and new ways to impede drug resistance.

This publication is licensed under

CC-BY 4.0 .
  • cc licence
  • by licence
Copyright © 2024 The Authors. Published by American Chemical Society

Subjects

what are subjects

Introduction

Click to copy section linkSection link copied!
Protein phosphorylation is one of the most studied posttranslational modifications (PTMs) in bacteria as they have crucial regulatory functions. Protein kinases are essential effectors of protein phosphorylation. Bacterial tyrosine kinases, also known as BY-kinases, are generally involved in the biosynthesis, polymerization, and export of complex polysaccharides necessary for the creation of biofilm or capsules. (1) Until now, yccC (etk) and wzc are the only two identified BY-kinases in Escherichia coli (E. coli). (2,3) The presence of unknown BY-kinase in the E. coli proteome has been suggested, as the Δetk-Δwzc double mutants still have tyrosine-phosphorylated substrates. Nevertheless, structure-based in silico search has not revealed any additional BY-kinases in E. coli. (4)
Identification of kinases in prokaryotes is challenging as they have less specific similarities to their eukaryotic counterparts and are sometimes hidden in the genome. (5) The motifs already identified for protein kinases might be too stringent, as some of the discovered protein kinases lack conserved motifs. (6) High-throughput screening of differentially phosphorylated proteins over the whole proteome is a viable approach to interrogate kinases. Nowadays, phosphoproteomics analysis via mass spectrometry (MS) is a tool to reveal the in vivo substrates and characterization of a kinase. However, MS is laborious and has low sample throughput in parallel. (7,8) Moreover, there are no existing high-throughput tools to identify or screen for novel kinases. Therefore, to be able to perform proteome-wide kinase screening, practical and economical approaches are needed.
Functional screening of each gene can be accomplished by overexpressing each gene. Kitagawa et al. established the ASKA library as a collection of open reading frames cloned into E. coli K-12 and can be used to overexpress His-tagged proteins. (9) By purifying every His-tagged protein, Chen et al. established the E. coli proteome microarray as a high-throughput screening platform. Since kinase needs substrates to perform phosphorylation, (10,11) this type of purified E. coli proteome microarray (forward-phase) cannot be used to identify kinases. Reverse-phase protein array (RPPA) is a microarray that immobilizes hundreds or thousands of unpurified protein mixtures. The RPPA can be used to identify a specific protein expression in all the lysates that are printed on the array. (12) The RPPA has been used to profile the histone modifications in the nuclear extracts or cell lysates. (13,14) Since the cell lysates on the RPPA array can serve as the substrates, we propose that the overexpression of a kinase may increase the phosphorylation of the substrates and thus can be screened for kinases. In our study, we constructed RPPA with lysates of 4126 different overexpressed strains from the ASKA library and named E. coli lysate arrays (ECLAs). We profiled phosphotyrosine levels in ECLAs by antiphosphotyrosine antibodies and obtained 6 BY-kinase candidates. We further identified nrdD as a novel BY-kinase and used the purified E. coli proteome microarray (forward-phase) to report the specific substrates.

Materials and Methods

Click to copy section linkSection link copied!

Strains and Bacterial Culture

ASKA E. coli open reading frame (ORF) library strains (9) and wild-type (WT) E. coli K-12 strain BW25113 were used in the study. (15) 4126 ASKA strains were inoculated from 56 plates that were available to us from the ASKA library and cultured overnight in Luria–Bertani (LB) medium with 30 μg/mL chloramphenicol and grown in an incubator at 37 °C, 250 rpm in 96-deep well plates. All cultures were brought to OD600 0.1, induced by 1 mM isopropyl-b-D-thiogalactopyranoside (IPTG) (no. I6758, Sigma-Aldrich), and cultured for another 4 h to induce protein expression.

Lysate Preparation and ECLA Fabrication

Cells in 96-deep well plates were harvested by centrifugation at 4121×g for 10 min and frozen immediately at −80 °C. Harvested pellets were resuspended in B-PER lysis buffer (no. 78248, Thermo Scientific), 0.5 mg/mL lysozyme (no. 62971, Sigma-Aldrich) phosphatase inhibitor (1 tablet per 10 mL, no. A32957, Thermofisher) and protease inhibitor cocktail (1 tablet per 10 mL, no. #11836153001, Roche). After vortexing for 2 min and incubating for 8 min at room temperature (RT), the mixtures were sent to a centrifuge at 4121×g for 10 min. Lysates were transferred to 384-well plates, supplemented with glycerol, and stored at −80 °C for printing. Each lysate was spotted in duplicates on aldehyde-coated slides using a SmartArrayer 48 microarrayer (CapitalBio). (16) The printed microarrays were stored at −80 °C for further usage.

Quality Control of ECLA

The quality of ECLA was evaluated by the amount of lysate immobilized, the reproducibility between different batches, and the amount of protein immobilized. To monitor the immobilization of the lysates on the ECLAs, arrays were incubated with 1.66 μg/mL DyLight 650 NHS Ester (no. 62265, Thermo Fisher Scientific) in phosphate-buffered saline with 0.1% Tween 20 (PBST) and shaken for 1 h at 50 rpm. After washing 3 times with PBST, slides were spin-dried and scanned by a laser scanner (#Spinscan HC-BS01, CADUCEUS Biotechnology). The fluorescence intensities were equivalent to the protein amounts which can be used to visualize the immobilization status of lysates on arrays. (17) To determine the protein overexpression in the lysates, ECLAs were blocked with 3% bovine serum albumin (BSA) in Tris-buffered saline with 0.1% Tween 20 (TBST) and incubated with 1 μg/mL of Cy3-labeled anti-6xHis antibody (#200-304-382, Rockland) in 1% BSA in TBST for 1 h with 50 rpm shaking. After 3 washes, the slides were dried and scanned.

Tyrosine Phosphorylation Profiling on ECLA

ECLAs were blocked for 1 h with 50 rpm shaking and incubated with 100x diluted phosphotyrosine mouse mAb (P-Tyr-100, #9411, Cell Signaling) in 1% BSA in TBST for another hour. Arrays were washed three times and then incubated with fluorescence-labeled goat antimouse secondary antibody (0.25 μg/mL, #84540, Thermo Scientific) for 1 h with shaking. Another antiphosphotyrosine antibody (100× dilution, #ab10321, Abcam) was used with similar assay procedures except the concentration of the secondary antibody changed to 0.5 μg/mL. After three final washes, the arrays were dried and scanned. Control experiments were performed using the same procedures without adding the primary antibodies. The phosphotyrosine signals were calculated based on foreground minus background. The three selection criteria for the outstanding phosphotyrosine spots were p < 0.05 between experiment and control groups, 1.5 standard deviations (SDs) differences compared to WT lysates and overlapped with two antiphosphotyrosine antibodies.

In Vitro Kinase Assays

His6-tagged proteins were IPTG-induced, lysed, and purified with Ni-NTA resin as described earlier. (18) The purified protein concentration was calculated using BSA standards on SDS-PAGE. Autophosphorylation assay is carried out in kinase buffer (no. 9802, Cell Signaling) with 0.4 mM ATP (no. 9804, Cell Signaling) with purified proteins at 30 °C for several time points. The reaction was terminated by adding SDS loading buffer (5× dilution, 10% SDS, 50% Glycerol, 0.05 M DTT, 0.01 EDTA, 0.05% bromophenol blue, and 0.125 M Tris–HCl, pH: 6.8). The substrate phosphorylation was performed with 4 μg of ssuD and 4 μg nrdD. A total reaction volume of 20 μL was prepared with and without 0.4 mM ATP, and with and without nrdD in kinase buffer at 30 °C for 1 h. The reaction was terminated by adding an SDS loading buffer. In vitro kinase assays were also performed by adding purified kinases into WT lysates with 0.4 mM ATP in kinase buffer at 30 °C for 1 h.
For the solid capture experiment, 1 μg of ssuD and 100 μL of carbonate/bicarbonate buffer (pH: 9.6) were adsorbed to each 96-well overnight at 4 °C with 200 rpm shaking. The wells were washed with TBST 4 times. The kinase reaction was performed by adding 8 μg of nrdD with and without 0.4 mM ATP in kinase buffer at 30 °C for 1 h. The wells were washed by TBST 4 times and blocked for 1 h in RT by shaking at 50 rpm for 1 h in 3% BSA in TBST. Wells were incubated with 200 μL of phosphotyrosine mouse mAb (500× dilution, P-Tyr-100) with 1% BSA in TBST. After incubating for 1 h at room temperature with shaking, wells were washed with TBST and then incubated with 200 μL of peroxidase-conjugated goat anti-mouse IgG (2000× dilution, #115-035-003, Jackson Immunoresearch). After the final washes, TMB substrates (#421101, BioLegend) were added followed by a stop solution (2 N H2SO4). The absorbance was recorded at the OD450 by a microplate reader.

Western Blot Assays

Protein samples or lysate mixtures were run on 4–12% gradient gels (#M00654, GenScript) and then transferred to a PVDF membrane at 200 mA for 1 h. Ponceau S staining was performed sometimes. For Pro-Q Diamond staining (#P33356, Thermo Fisher Scientific), membranes were stained, washed, and imaged according to the manufacturer’s protocol. For total protein Coomassie brilliant blue (CBB) staining was performed for 2 min, destained with the destaining solution for 5 min 2 times, and imaged. Phos-tag staining (#BTL-111S1, NARD Institute) was performed based on the manufacturer’s protocol. (19) Briefly, membranes were blocked, washed, incubated with Phos-tag, washed, incubated with streptavidin-conjugated horseradish peroxidase streptavidin, washed, incubated with substrates, and then imaged. For antibody detections, membranes were blocked in 3% BSA for 1 h, washed, incubated with phosphotyrosine mouse mAb (3000×, P-Tyr-100) in 1% BSA for 1 h, washed, incubated with peroxidase-conjugated goat anti-mouse IgG (6000× dilution) for 1 h, washed, incubated with substrates (#WBKL50500, Merck), and imaged by Chemidoc system (#12003153, BIO-RAD). Similar procedures were used in other antibodies, except the concentration of primary antibodies, e.g., phosphoserine/threonine antibody (3000× dilution, #PM3801, ECM Biosciences) and antiphosphohistidine antibody (3000× dilution, #MABS1351, Sigma-Aldrich).

Phosphorylation Profiling on E. coli Proteome Microarrays

E. coli proteome microarrays were prepared and fabricated as previously described. (20) The arrays were blocked in 3% BSA in TBST for 1 h at RT. The kinase reactions were prepared by adding 20 μg nrdD in 1% BSA kinase buffer with or without 0.2 mM ATP. Reactions were then applied to each array in duplicates under the coverslips and incubated in a humidified chamber at 30 °C for 1 h. The arrays were washed twice, probed with 250 μL phosphotyrosine mouse mAb (1000× dilution) in 1% BSA in TBST for 1 h, washed three times, and then probed with 0.125 μg/mL of fluorescence-labeled goat antimouse antibody for 1 h. After three final washes, arrays were dried and scanned. The substrate selection criteria for the outstanding phosphotyrosine spots were p < 0.05 and 1 SD difference between with and without ATP.

Biofilm Formation Assay

Overnight grown nrdD strains in 30 μg/mL chloramphenicol-supplemented LB were brought to 0.1 OD600 and grown until OD600 0.6. The nrdD strains were transferred into a 96-well microplate with 100× dilution in 200 μL LB in five replicates with and without 0.2 mM IPTG. The biofilm assay was performed by adapting the microplate method described previously. (21) After growth for 18 h at 37 °C, OD600 was recorded to normalize the cell counts. Plates were washed twice with RO water, and 300 μL of 0.1% crystal violet was applied for 20 min in RT to stain the remaining E. coli. After 2 washes, 300 μL 95% ethanol was added to the wells and incubated for 20 min in RT. The OD was measured at 540 nm absorbance. The biofilm formation is calculated as OD540/OD600 value.

Data Analysis and Bioinformatics

Foreground and background fluorescence was obtained after spot alignment with GenePix Pro 6.0 software (Molecular Devices). Fluorescence intensities were calculated based on the foreground minus the background. The student’s t-test was used to calculate the p-value between groups. P < 0.05 was defined as significant. All data were presented as mean ± SD nrdD homologous sequences were obtained with a protein BLAST search from the UniProt database. The sequences were aligned by the M-Coffee tool from the T-Coffee alignment server. (22) Maximum likelihood analysis was performed with IQ-Tree web server. (23) The phylogenetic tree was visualized by iTOL online tool. (24) Percent identity and E values were obtained from NCBI BLAST protein alignments.

Results

Click to copy section linkSection link copied!

Fabrication of ECLA and Quality Control

To develop the ECLAs, we cultured 4126 E. coli ORF clones and induced them with IPTG to overexpress proteins. Cell cultures were collected by centrifugation and lysed by lysis buffer with phosphatase and protease inhibitors. After lysis, the clarified supernatants were harvested by centrifuge. All the culturing, induction, lysis, and clarification steps were carried out in the 96-well format. The clarified lysates were transferred into 384-well plates and printed onto aldehyde-coated slides in duplicate (Figure 1A). As the spots were prone to merge due to detergent in the lysis buffer, we optimized the spot distance and adjusted glycerol concentration to prevent merging during the printing.

Figure 1

Figure 1. Workflow of BY-Kinase interrogation by the ECLA. The overall workflow of tyrosine phosphorylation profiling of ECLA starts with (A) ECLA fabrication by high-throughput culturing, induction, lysis, clarify, and printing of the 4126 overexpressed ASKA clones onto aldehyde-coated slides. (B) NHS succinyl ester Dylight 650 was used to visualize total protein on ECLA slides (left). Overexpression of 6xHis-tagged protein in the lysate spots was monitored by anti-His fluorescence staining (right). (C) Phosphorylation levels in each lysate clone were monitored by anti-pTyr antibody staining on ECLA. The BY-kinase candidates were selected by overlapping two different anti-pTyr antibodies. (D) BY-kinase candidates were verified by in vivo overexpressing (left) and in vitro addition (right). The level of phosphorylation in the lysates was monitored by Western blot.

High Resolution Image
Download MS PowerPoint Slide
The quality control of the ECLAs was done by total protein staining with amine-reactive NHS ester and overexpressed protein staining with anti-His antibody in duplicate (Figure 1B). The fluorescence intensity for buffer spots was 995.29 in total protein staining. On the ECLAs, 99.39% of lysate spots have more fluorescence intensities than buffer spots in total protein staining (Figure 2A). As IPTG induced overexpression of His-tagged proteins, the overexpression level of lysate spots can be monitored by anti-His staining. As WT lysates did not contain His-tagged proteins, they can be used as the background with 45.82 fluorescence intensity in anti-His staining. On the ECLAs, 93.80% of lysate spots have His-tagged proteins compared to WT lysate spots in anti-His staining (Figure 2B). The control spots were used for alignment, and positive controls or negative controls were printed at the bottom of each block, containing WT lysate, lysis buffer, BSA, poly-l-lysine, Protein A, antihuman antibody, and fluorescence landmark (Figure 2C, D). The reproducibility of ECLAs was R2 = 0.9306 for total protein staining (Figure 2E) and R2 = 0.9363 for anti-His staining (Figure 2F).

Figure 2

Figure 2. Quality control of ECLAs. Quality control of ECLAs was assessed by Dylight 650 NHS Ester for total protein (A,C,E) and anti-His for overexpressed protein (B,D,F). (A) Distribution of total protein from ECLA spots was obtained from two independent assays. Fluorescence intensity from the buffer was indicated with the dotted line. (B) Distribution of 6xHis-tagged protein from ECLA spots was obtained from two independent assays. Fluorescence intensities from WT lysate signals were indicated with the dotted line. (C) Representative image of an ECLA stained by Dylight 650 NHS Ester. (D) Representative image of an ECLA block stained by anti-His. Control spots were indicated in the bottom row. (E) Reproducibility of total protein staining obtained from two independent ECLA assays. (F) Reproducibility of overexpressed protein in the lysates obtained from two independent ECLA assays.

High Resolution Image
Download MS PowerPoint Slide

Profiling the Level of Tyrosine Phosphorylation in Cell Lysates by Using ECLAs

Since the ECLAs immobilized thousands of lysates from overexpressing clones, they are ideal for profiling the phosphorylation networks because they contain both enzymes and substrates. To quantify the tyrosine phosphorylation on ECLAs, we selected two antiphosphotyrosine antibodies, P-Tyr-100 and PY20, to minimize the potential bias from the antibodies. The phosphotyrosine levels were then visualized with Dylight-conjugated antimouse antibodies. After scanning, alignment, and biostatistics, there were 32 significant hits from P-Tyr-100 (Table S1) and 7 significant hits from PY20 (Table S2). The overlapping between two phosphotyrosine antibodies was 6 hits, e.g., etk, wzc, argG, dgoD, yjjJ, and nrdD. The rank of each candidate hit for both antibodies can be found in Table S3. The representative images from the P-Tyr-100 assays (Figure 3A) and PY20 assays (Figure S1) including primary and secondary antibodies or secondary antibodies only were listed. These 6 hits were potential candidates for the BY-kinases because the overexpression resulted in the elevation of phosphotyrosine levels.

Figure 3

Figure 3. Lysate profiles of overexpressed candidates. There were 6 overlapped hits from the two anti-pTyr ECLA assays. (A) Images from the three assays were done with P-Tyr-100 antibody followed by fluorescent-labeled antimouse, and three assays were done with fluorescent-labeled antimouse as blanks. The WT lysate spots were listed as baseline phosphorylation. (B) Each ASKA clone was listed on top, including etk, wzc, argG, dogD, yjjJ, and nrdD. Immunoblots were performed with P-Tyr-100 antibody to show the phosphorylation profiles in the lysates with or without IPTG inductions. (C) Each purified candidate was incubated with or without WT lysates in the kinase buffer supplemented with ATP before analysis by SDS-PAGE. Pro-Q Diamond staining was used to image the phosphorylated levels.

High Resolution Image
Download MS PowerPoint Slide

Profiling Cell Lysates of BY-Kinase Candidates by Immuno-Western Blot

To further elucidate the BY-kinase activities in 6 candidates, we performed Western blots of cell lysates to show the phosphorylation patterns. Lysates from the overexpressed candidates were used to run SDS-PAGE followed by P-Tyr-100 antibody blot (Figure 1D, left). Each clone that contained candidate ORF was included for candidate protein expression with or without IPTG and further analyzed by a P-Tyr-100 antibody blot (Figure 3B). All IPTG-included clone lysates showed distinctive phosphorylated bands at different degrees compared to their non-IPTG controls. Overexpression of etk exhibited highly saturated signals across the whole lane, and overexpression of wzc had a few saturated bands (Figure 3B).
We analyzed the kinase activities by treating the purified candidates with WT lysates as substrates for in vitro kinase assays. To observe the phosphorylation patterns, we performed the kinase assay with ATP and visualized it with Pro-Q Diamond staining for the WT lysate, WT lysate treated with purified candidates, and purified candidates (Figure 3C). Based on the blot images, etk, wzc, yjjJ, and nrdD showed distinct band formations or increased some phosphorylated bands (̃35 kDa for etk, 55 kDa for wzc, 40 kDa for yjjJ, and several bands between 50 and 70 kDa for nrdD). However, argG and dgoD did not show observable differences (Figure 3C). Total protein staining was done with CBB as a loading control as shown in Figure S2.

Autophosphorylation of nrdD In Vitro

Among 4 candidates validated in cell lysates, nrdD previously known as anaerobic ribonucleoside-triphosphate reductase novel kinase showed phosphorylation activities. Since most BY-kinases are autokinases, we analyzed nrdD for the autophosphorylation activities (Figure 4A). The autophosphorylation was observed immediately after ATP addition (0 min), increased more after 10 min and decreased after 30 min. Both Phos-tag and P-Tyr-100 antibody staining showed similar phosphorylation patterns (Figure 4A). The total protein staining was done with Ponceau S as the loading control.

Figure 4

Figure 4. Contribution of nrdD to autophosphorylation and biofilm formation. (A) Purified nrdD was treated with a kinase buffer supplemented with ATP for various time points. The level of phosphorylation was quantified by Phos-tag and P-Tyr-100 antibody with the SDS–PAGE. The same membrane was stained for Ponceau S for loading controls. (B) Biofilms were quantified based on the crystal violet staining in nrdD clones with or without IPTG induction. Data were analyzed from five replicates using an unpaired two-tailed t test, ****p < 0.0001.

High Resolution Image
Download MS PowerPoint Slide

nrdD Involved in Biofilm Formation

BY-kinases are usually involved in biofilm formation in E. coli that is important in drug resistance and bacterial survival under harsh environments. (25) Therefore, we cultured nrdD clones with or without IPTG induction and performed biofilm assays by crystal violet staining. After 18 h of static growth, a significant increase in biofilm was observed in IPTG compared to that in non-IPTG (Figure 4B). The biofilm formation was normalized to the bacterial count = crystal violet at OD540/bacterial concentration at OD600. A similar biofilm assay was done in the WT and showed no differences in IPTG induction (data not shown).

Sequence Similarities of nrdD

Since nrdD was important in biofilm formation, we analyzed the sequence similarity of amino acids in every species, especially in bacteria. Based on the BLASTP search, various bacteria and a T4 phage have proteins similar to nrdD. Most of the species were Gram (−) bacteria, except two Gram (+) bacteria and one T4 phage. The polyphyletic tree, percent identity, and E value for nrdD from each species are shown in Figure S4A. The Alphafold structure of nrdD proteins from E. coli K-12 and other bacterial homologs are listed in Figure S4B–G. UniProt protein IDs of the nrdD in different species were also listed in Table S4. The low E value of the nrdD proteins (E-threshold 1) indicated that nrdD was highly conserved.

E. coli Proteome Microarray Revealed Substrates of nrdD

After we observed that nrdD was an autokinase and phosphorylated several lysate protein bands on Western blot, we planned to interrogate the substrates of nrdD by using an E. coli proteome microarray with thousands of purified E. coli proteins. By treating the E. coli proteome microarray with nrdD and performing the in vitro kinase assay with or without ATP, we may reveal the substrates for nrdD (Figure S5A). After two repeats and statistical analysis, we identified 33 hits with elevated phosphorylation levels in the presence of ATP (Table S5).
Based on the differences with or without ATP, the top-ranked substrate was ssuD on the E. coli proteome microarrays (Figure S5B,C). We also tested the in vitro kinase assay between nrdD and ssuD with ELISA format and displayed similar results (Figure S5D). Finally, we demonstrated ssuD phosphorylation by nrdD with Western blot analysis. The phosphorylation level was only elevated in ssuD treated with both ATP and nrdD (Figure S5E). The quantification of ssuD phosphorylation based on Pro-Q Dimond blot also showed significance in the presence of ATP and nrdD (Figure S5F).

Discussion

Click to copy section linkSection link copied!
In this study, we adapted the single gene overexpression library (ASKA) and established the world’s first E. coli lysate array (ECLA) for high-throughput screening of tyrosine phosphorylation events. Among 6 candidates identified by ECLA, etk and wzc were already known BY-kinases (2,3) and identified again in this study. The successful identification of etk and wzc strongly supports the robustness and usefulness of the ECLA. nrdD is being reported here for the first time as a BY-kinase because it has autophosphorylation activity and transphosphorylates other substrates. We also tested phosphorylation of WT lysates by nrdD with phosphoserine/threonine and phospho-histidine antibodies. However, we observed phosphorylation with phosphotyrosine antibodies only (Figure S3). This also suggests that nrdD mainly exhibited tyrosine phosphorylation. yjjJ is a known serine/threonine kinase; however, it was still found on our candidate BY-kinase list. The reason might be due to its cross-phosphorylation activity on some proteins’ tyrosine residues, which was also reported before. (26) dgoD seemed to induce some change as it increased the intensity of a phosphorylated band in lysates; however, in E. coli proteome microarray profiling, it did not show any of the potential substrates (data not shown). argG was also among our candidates, but we did not observe any phosphorylation activity. One explanation for that might be the nonspecific binding of the phosphotyrosine antibodies to these proteins. Another reason might be explained as these proteins might be highly phosphorylated substrates or an overexpressed protein triggering a downstream mechanism.
Proteome microarray is a useful tool for global analysis of protein phosphorylation in vitro to identify substrates of known kinases. (27) We used the E. coli proteome microarray and found 33 substrates of nrdD by performing the in vitro kinase assay. ssuD was the highest-ranking substrate candidate, which is the alkanesulfonate monooxygenase enzyme in E. coli. The phosphorylation of ssuD by nrdD was verified with Western blot and solid capture assays. The substrate list of nrdD in Table S5 shows various oxidoreductases such as ssuD that were the targets of nrdD. This supports the function that the function of nrdD might also be related to a protective strategy against oxidative stress.
Our in vivo functional studies revealed that nrdD overexpression significantly increased biofilm production in E. coli. Biofilm formation is another importance for BY-kinases in E. coli, as they contribute to the biofilm and exopolysaccharide production. (25) Cendra et al. have shown that ΔnrdD and ΔnrdD ΔnrdE knockout strains have decreased biofilm formation. (28) They also showed that the level of expression of nrdD was elevated in biofilm-forming E. coli. Among the substrates of nrdD (Table S5), we found that exopolysaccharide-associated wcaC, fcl, and kpsE were among nrdD substrates. (29−31) These results also support the idea that nrdD directly contributes to the formation of exopolysaccharides in E. coli.
The multiple protein sequence alignments made with nrdD in other species showed that nrdD is conserved among the bacteria. nrdD is a Class III anaerobic ribonucleoside-triphosphate reductase. It is known for catalyzing the conversion of ribonucleotides into deoxyribonucleotides, which are required for DNA synthesis and repair. (32) Most of the known BY-kinases have the P-loop structure for ATP binding. (33) Unlike these BY-kinases, nrdD has an ATP-cone structure for ATP binding. Structurally, ATP-cone seems to be conserved among nrdD in several bacterial homologs (Figure S4B–G). This domain was also found in kinases such as phosphoglycerate kinase 2 (PGK2). (34) Therefore, our finding suggests that nrdD belongs to a new family of BY-kinases. Its phosphorylation mechanism should be investigated with nrdD from other species to determine whether the kinase activity is also conserved among species.
In conclusion, ECLA works as a practical approach to profile phosphorylation in cell lysates, as it reflects in vivo conditions to interrogate gene functions in high throughput. Our results show that ECLA might be a useful alternative to in silico surveys to expand the kinome of E. coli. One of the limitations of ECLAs might be the inhibition of culture growth due to the overexpressed proteins. If the growth and viability decrease, this might affect the detection of the PTMs in the lysates. However, this might be overcome by optimizing the IPTG induction of the cultures having slow growth. The other limitation is that the interrogation of PTMs on ECLA depends on the antibody quality. ECLA might be a useful tool to apply to different PTMs and discover various novel enzymes in E. coli. These studies are ongoing in our laboratory, and those PTM enzymes will be investigated in the future.

Data Availability

Click to copy section linkSection link copied!

The data sets generated in this study are available from the corresponding author upon reasonable request.

Supporting Information

Click to copy section linkSection link copied!

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.analchem.4c00965.

  • List of ECLA spots with elevated tyrosine phosphorylation ranking, phylogenetic tree, list and figures of nrdD protein from other bacteria, list of nrdD substrates obtained from in vitro kinase assay on E. coli proteome microarray, in vitro kinase assay results on E. coli proteome array to verify ssuD as a substrate, and additional supporting Western blot images (PDF)

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

Click to copy section linkSection link copied!
  • Corresponding Author
    • Guan-Da Syu - Department of Biotechnology and Bioindustry Sciences, National Cheng Kung University, Tainan 701, Taiwan;  International Center for Wound Repair and Regeneration, National Cheng Kung University, Tainan 701, Taiwan;  Medical Device Innovation Center, National Cheng Kung University, Tainan 701, Taiwan;  Orcidhttps://orcid.org/0000-0003-4661-414X;  Email: [email protected]
  • Authors
    • Batuhan Birol Keskin - Department of Biotechnology and Bioindustry Sciences, National Cheng Kung University, Tainan 701, Taiwan
    • Chien-Sheng Chen - Department of Food Safety/Hygiene and Risk Management, College of Medicine, National Cheng Kung University, Tainan 701, Taiwan;  Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University, Tainan 701, Taiwan;  Orcidhttps://orcid.org/0000-0002-2372-324X
    • Pei-Shan Tsai - Department of Biotechnology and Bioindustry Sciences, National Cheng Kung University, Tainan 701, Taiwan
    • Pin-Xian Du - Department of Biotechnology and Bioindustry Sciences, National Cheng Kung University, Tainan 701, Taiwan
    • John Harvey M. Santos - Department of Biotechnology and Bioindustry Sciences, National Cheng Kung University, Tainan 701, Taiwan;  Centre for Animal Science, Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, Brisbane, QLD 4072, Australia;  Orcidhttps://orcid.org/0000-0002-6514-9565
  • Author Contributions

    B.B.K. is the first author. B.B.K., C.-S.C., P.-S.T., P.-X.D., and J.H.S. performed the experimental work. B.B.K. and G.-D.S. contributed to the manuscript preparation. G.-D.S. contributed expertise and supervision to the entire project.

  • Notes
    The authors declare no competing financial interest.

Acknowledgments

Click to copy section linkSection link copied!

This work was supported in part by the National Science and Technology Council, Grants NSTC 113-2321-B-006-007-, NSTC 113-2327-B-006-002-, NSTC 112-2321-B-006-008-, NSTC 112-2628-B-006-004-, NSTC 112-2622-8-182A-001-IE, NSTC 112-2320-B-006-050-MY3, NSTC 111-2320-B-006 -041-MY3, and Hsinchu Science Park Bureau NSTC B11301. We are grateful for the support from the University Center for Bioscience and Biotechnology, National Cheng Kung University, and Headquarters of University Advancement at the National Cheng Kung University, Ministry of Education, Taiwan. We thank Prof. Hirotada Mori for providing the ASKA library. The funders had no role in study design, data collection, analysis, publication decision, or manuscript preparation.

References

Click to copy section linkSection link copied!

This article references 34 other publications.

  1. 1
    Hajredini, F.; Alphonse, S.; Ghose, R. BY-kinases: Protein tyrosine kinases like no other. J. Biol. Chem. 2023, 299 (1), 102737  DOI: 10.1016/j.jbc.2022.102737
    Google Scholar
    There is no corresponding record for this reference.
  2. 2
    Ilan, O.; Bloch, Y.; Frankel, G.; Ullrich, H.; Geider, K.; Rosenshine, I. Protein tyrosine kinases in bacterial pathogens are associated with virulence and production of exopolysaccharide. EMBO J. 1999, 18 (12), 3241– 3248,  DOI: 10.1093/emboj/18.12.3241
    Google Scholar
    There is no corresponding record for this reference.
  3. 3
    Vincent, C.; Doublet, P.; Grangeasse, C.; Vaganay, E.; Cozzone, A. J.; Duclos, B. Cells of Escherichia coli Contain a Protein-Tyrosine Kinase, Wzc, and a Phosphotyrosine-Protein Phosphatase. Wzb. J. Bacteriol. 1999, 181 (11), 3472– 3477,  DOI: 10.1128/JB.181.11.3472-3477.1999
    Google Scholar
    There is no corresponding record for this reference.
  4. 4
    Hansen, A.-M.; Chaerkady, R.; Sharma, J.; Díaz-mejía, J. J.; Tyagi, N.; Renuse, S.; Jacob, H. K. C.; Pinto, S. M.; Sahasrabuddhe, N. A.; Kim, M.-S. The Escherichia coli Phosphotyrosine Proteome Relates to Core Pathways and Virulence. PLoS Pathol. 2013, 9 (6), e1003403  DOI: 10.1371/journal.ppat.1003403
    Google Scholar
    There is no corresponding record for this reference.
  5. 5
    Schastnaya, E.; Raguz nakic, Z.; Gruber, C. H.; Doubleday, P. F.; Krishnan, A.; Johns, N. I.; Park, J.; Wang, H. H.; Sauer, U. Extensive regulation of enzyme activity by phosphorylation in Escherichia coli. Nat. Commun. 2021, 12 (1), 5650,  DOI: 10.1038/s41467-021-25988-4
    Google Scholar
    There is no corresponding record for this reference.
  6. 6
    Rajagopalan, K.; Dworkin, J. Escherichia coli YegI is a novel Ser/Thr kinase lacking conserved motifs that localizes to the inner membrane. FEBS Lett. 2020, 594 (21), 3530– 3541,  DOI: 10.1002/1873-3468.13920
    Google Scholar
    There is no corresponding record for this reference.
  7. 7
    Dueñas, M. E.; Peltier-heap, R. E.; Leveridge, M.; Annan, R. S.; Büttner, F. H.; Trost, M. Advances in high-throughput mass spectrometry in drug discovery. EMBO Mol. Med. 2023, 15 (1), e14850  DOI: 10.15252/emmm.202114850
    Google Scholar
    7
    Advances in high-throughput mass spectrometry in drug discovery
    Duenas, Maria Emilia; Peltier-Heap, Rachel E.; Leveridge, Melanie; Annan, Roland S.; Buettner, Frank H.; Trost, Matthias
    EMBO Molecular Medicine (2023), 15 (1), e14850CODEN: EMMMAM; ISSN:1757-4684. (Wiley-Blackwell)
    A review. High-throughput (HT) screening drug discovery, during which thousands or millions of compds. are screened, remains the key methodol. for identifying active chem. matter in early drug discovery pipelines. Recent technol. developments in mass spectrometry (MS) and automation have revolutionized the application of MS for use in HT screens. These methods allow the targeting of unlabeled biomols. in HT assays, thereby expanding the breadth of targets for which HT assays can be developed compared to traditional approaches. Moreover, these label-free MS assays are often cheaper, faster, and more physiol. relevant than competing assay technologies. In this review, we will describe current MS techniques used in drug discovery and explain their advantages and disadvantages. We will highlight the power of mass spectrometry in label-free in vitro assays, and its application for setting up multiplexed cellular phenotypic assays, providing an exciting new tool for screening compds. in cell lines, and even primary cells. Finally, we will give an outlook on how technol. advances will increase the future use and the capabilities of mass spectrometry in drug discovery.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XjtFWqtL7I&md5=068329c09fb22018c83791dd0d9b29d6
  8. 8
    Gavriilidou, A. F. M.; Sokratous, K.; Yen, H.-Y.; De Colibus, L. High-Throughput Native Mass Spectrometry Screening in Drug Discovery. Front. Mol. Biosci. 2022, 9, 837901  DOI: 10.3389/fmolb.2022.837901
    Google Scholar
    8
    High-Throughput Native Mass Spectrometry Screening in Drug Discovery
    Gavriilidou Agni F M; Sokratous Kleitos; Yen Hsin-Yung; De Colibus Luigi
    Frontiers in molecular biosciences (2022), 9 (), 837901 ISSN:2296-889X.
    The design of new therapeutic molecules can be significantly informed by studying protein-ligand interactions using biophysical approaches directly after purification of the protein-ligand complex. Well-established techniques utilized in drug discovery include isothermal titration calorimetry, surface plasmon resonance, nuclear magnetic resonance spectroscopy, and structure-based drug discovery which mainly rely on protein crystallography and, more recently, cryo-electron microscopy. Protein-ligand complexes are dynamic, heterogeneous, and challenging systems that are best studied with several complementary techniques. Native mass spectrometry (MS) is a versatile method used to study proteins and their non-covalently driven assemblies in a native-like folded state, providing information on binding thermodynamics and stoichiometry as well as insights on ternary and quaternary protein structure. Here, we discuss the basic principles of native mass spectrometry, the field's recent progress, how native MS is integrated into a drug discovery pipeline, and its future developments in drug discovery.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB2Mrks1Ohuw%253D%253D&md5=80ce8f1d387bace38193aa9c29ce383e
  9. 9
    Kitagawa, M.; Ara, T.; Arifuzzaman, M.; Ioka-nakamichi, T.; Inamoto, E.; Toyonaga, H.; Mori, H. Complete set of ORF clones of Escherichia coli ASKA library (a complete set of E. coli K-12 ORF archive): unique resources for biological research. DNA Res. 2006, 12 (5), 291– 299,  DOI: 10.1093/dnares/dsi012
    Google Scholar
    There is no corresponding record for this reference.
  10. 10
    Jehle, S.; Kunowska, N.; Benlasfer, N.; Woodsmith, J.; Weber, G.; Wahl, M. C.; Stelzl, U. A human kinase yeast array for the identification of kinases modulating phosphorylation-dependent protein–protein interactions. Mol. Syst. Biol. 2022, 18 (3), e10820  DOI: 10.15252/msb.202110820
    Google Scholar
    There is no corresponding record for this reference.
  11. 11
    Chen, J.; Bell, J.; Lau, B. T.; Whittaker, T.; Stapleton, D.; Ji, H. P. A functional CRISPR/Cas9 screen identifies kinases that modulate FGFR inhibitor response in gastric cancer. Oncogenesis 2019, 8 (5), 33,  DOI: 10.1038/s41389-019-0145-z
    Google Scholar
    There is no corresponding record for this reference.
  12. 12
    Coarfa, C.; Grimm, S. L.; Rajapakshe, K.; Perera, D.; Lu, H. Y.; Wang, X.; Christensen, K. R.; Mo, Q.; Edwards, D. P.; Huang, S. Reverse-Phase Protein Array: Technology, Application, Data Processing, and Integration. J. Biomol Tech 2021, 32 (1), 15– 29,  DOI: 10.7171/jbt.21-3202-001
    Google Scholar
    12
    Reverse-Phase Protein Array: Technology, Application, Data Processing, and Integration
    Coarfa Cristian; Grimm Sandra L; Rajapakshe Kimal; Edwards Dean P; Huang Shixia; Coarfa Cristian; Mo Qianxing; Edwards Dean P; Huang Shixia; Coarfa Cristian; Perera Dimuthu; Lu Hsin-Yi; Wang Xuan; Christensen Kurt R; Edwards Dean P; Huang Shixia
    Journal of biomolecular techniques : JBT (2021), 32 (1), 15-29 ISSN:.
    Reverse-phase protein array (RPPA) is a high-throughput antibody-based targeted proteomics platform that can quantify hundreds of proteins in thousands of samples derived from tissue or cell lysates, serum, plasma, or other body fluids. Protein samples are robotically arrayed as microspots on nitrocellulose-coated glass slides. Each slide is probed with a specific antibody that can detect levels of total protein expression or post-translational modifications, such as phosphorylation as a measure of protein activity. Here we describe workflow protocols and software tools that we have developed and optimized for RPPA in a core facility setting that includes sample preparation, microarray mapping and printing of protein samples, antibody labeling, slide scanning, image analysis, data normalization and quality control, data reporting, statistical analysis, and management of data. Our RPPA platform currently analyzes ∼240 validated antibodies that primarily detect proteins in signaling pathways and cellular processes that are important in cancer biology. This is a robust technology that has proven to be of value for both validation and discovery proteomic research and integration with other omics data sets.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB2c%252FotVSrtw%253D%253D&md5=9f70d7e3e3a1dd18b42442072e1447da
  13. 13
    Wang, X.; Shi, Z.; Lu, H.-Y.; Kim, J. J.; Bu, W.; Villalobos, J. A.; Perera, D. N.; Jung, S. Y.; Wang, T.; Grimm, S. L. High-throughput profiling of histone post-translational modifications and chromatin modifying proteins by reverse phase protein array. Journal of Proteomics 2022, 262, 104596  DOI: 10.1016/j.jprot.2022.104596
    Google Scholar
    There is no corresponding record for this reference.
  14. 14
    Cheng, L.; Liu, C.-X.; Jiang, S.; Hou, S.; Huang, J.-G.; Chen, Z.-Q.; Sun, Y.-Y.; Qi, H.; Jiang, H.-W.; Wang, J.-F. Cell Lysate Microarray for Mapping the Network of Genetic Regulators for Histone Marks. Molecular & cellular proteomics: MCP 2018, 17 (9), 1720– 1736,  DOI: 10.1074/mcp.RA117.000550
    Google Scholar
    There is no corresponding record for this reference.
  15. 15
    Baba, T.; Ara, T.; Hasegawa, M.; Takai, Y.; Okumura, Y.; Baba, M.; Datsenko, K. A.; Tomita, M.; Wanner, B. L.; Mori, H. Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol. Syst. Biol. 2006, 2, 2006.0008  DOI: 10.1038/msb4100050
    Google Scholar
    There is no corresponding record for this reference.
  16. 16
    Du, P.-X.; Chou, Y.-Y.; Santos, H. M.; Keskin, B. B.; Hsieh, M.-H.; Ho, T.-S.; Wang, J.-Y.; Lin, Y.-L.; Syu, G.-D. Development and Application of Human Coronavirus Protein Microarray for Specificity Analysis. Anal. Chem. 2021, 93 (21), 7690– 7698,  DOI: 10.1021/acs.analchem.1c00614
    Google Scholar
    There is no corresponding record for this reference.
  17. 17
    Jeon, S.; Kim, T.-I.; Jin, H.; Lee, U.; Bae, J.; Bouffard, J.; Kim, Y. Amine-Reactive Activated Esters of meso-CarboxyBODIPY: Fluorogenic Assays and Labeling of Amines, Amino Acids, and Proteins. J. Am. Chem. Soc. 2020, 142 (20), 9231– 9239,  DOI: 10.1021/jacs.9b13982
    Google Scholar
    17
    Amine-Reactive Activated Esters of meso-CarboxyBODIPY: Fluorogenic Assays and Labeling of Amines, Amino Acids, and Proteins
    Jeon, Sungjin; Kim, Tae-Il; Jin, Hanyong; Lee, Uisung; Bae, Jeehyeon; Bouffard, Jean; Kim, Youngmi
    Journal of the American Chemical Society (2020), 142 (20), 9231-9239CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    Fluorescence-based amine-reactive dyes are highly valuable for the sensing of amines and the labeling of biomols. Although it would be highly desirable, large changes in emission spectra and intensity seldom accompany the conjugation of known amine-reactive dyes to their target mols. On the contrary, amide bond formation between amines and the pentafluorophenyl (2-PFP) and succinimidyl (2-NHS) esters of meso-carboxyBODIPY results in significant changes in emission maxima (Δλ: 70-100 nm) and intensity (up to 3000-fold), enabling the fast (down to 5 min) and selective fluorogenic detection and labeling of amines, amino acids, and proteins. This approach further benefits from the demonstrated versatility and high reliability of activated ester chem., and background hydrolysis is negligible. The large "turn-on" response is a testament of the extreme sensitivity of meso-carboxyBODIPYs to the minimal changes in electronic properties that distinguish esters from amides. Applications to the detection of food spoilage, staining of proteins on electrophoretic gels or in living cells, and the expedited synthesis of organelle-specific fluorescence microscope imaging agents are further demonstrated.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXnsVyrsrk%253D&md5=6cf7d5edbe94661b749440ef6540defe
  18. 18
    Chen, C. S.; Korobkova, E.; Chen, H.; Zhu, J.; Jian, X.; Tao, S. C.; He, C.; Zhu, H. A proteome chip approach reveals new DNA damage recognition activities in Escherichia coli. Nat. Methods 2008, 5 (1), 69– 74,  DOI: 10.1038/nmeth1148
    Google Scholar
    18
    A proteome chip approach reveals new DNA damage recognition activities in Escherichia coli
    Chen, Chien-Sheng; Korobkova, Ekaterina; Chen, Hao; Zhu, Jian; Jian, Xing; Tao, Sheng-Ce; He, Chuan; Zhu, Heng
    Nature Methods (2008), 5 (1), 69-74CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)
    Despite the fact that many genomes have been decoded, proteome chips comprising individually purified proteins have been reported only for budding yeast, mainly because of the complexity and difficulty of high-throughput protein purifn. To facilitate proteomics studies in prokaryotes, the authors have developed a high-throughput protein purifn. protocol that allowed the authors to purify 4256 proteins encoded by the Escherichia coli K12 strain within 10 h. The purified proteins were then spotted onto glass slides to create E. coli proteome chips. The authors used these chips to develop assays for identifying proteins involved in the recognition of potential base damage in DNA. By using a group of DNA probes, each contg. a mismatched base pair or an abasic site, the authors found a small no. of proteins that could recognize each type of probe with high affinity and specificity. The authors further evaluated two of these proteins, YbaZ and YbcN, by biochem. analyses. The assembly of libraries contg. DNA probes with specific modifications and the availability of E. coli proteome chips have the potential to reveal important interactions between proteins and nucleic acids that are time-consuming and difficult to detect using other techniques.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXht1SksQ%253D%253D&md5=f73a9eb60587b58a6638e236c84e6323
  19. 19
    Kinoshita, E.; Kinoshita-kikuta, E.; Sugiyama, Y.; Fukada, Y.; Ozeki, T.; Koike, T. Highly sensitive detection of protein phosphorylation by using improved Phos-tag Biotin. Proteomics 2012, 12 (7), 932– 937,  DOI: 10.1002/pmic.201100639
    Google Scholar
    There is no corresponding record for this reference.
  20. 20
    Kuo, H. C.; Huang, Y. H.; Chung, F. H.; Chen, P. C.; Sung, T. C.; Chen, Y. W.; Hsieh, K. S.; Chen, C. S.; Syu, G. D. Antibody Profiling of Kawasaki Disease Using Escherichia coli Proteome Microarrays. Mol. Cell. Proteomics 2018, 17 (3), 472– 481,  DOI: 10.1074/mcp.RA117.000198
    Google Scholar
    There is no corresponding record for this reference.
  21. 21
    O’toole, G. A.; Kolter, R. Flagellar and twitching motility are necessary for Pseudomonas aeruginosa biofilm development. Mol. Microbiol. 1998, 30 (2), 295– 304,  DOI: 10.1046/j.1365-2958.1998.01062.x
    Google Scholar
    21
    Flagellar and twitching motility are necessary for Pseudomonas aeruginosa biofilm development
    O'Toole, George A.; Kolter, Roberto
    Molecular Microbiology (1998), 30 (2), 295-304CODEN: MOMIEE; ISSN:0950-382X. (Blackwell Science Ltd.)
    The formation of complex bacterial communities known as biofilms begins with the interaction of planktonic cells with a surface in response to appropriate environmental signals. The authors report the isolation and characterization of mutants of Pseudomonas aeruginosa PA14 defective in the initiation of biofilm formation on an abiotic surface, polyvinylchloride (PVC) plastic. These mutants are designated surface attachment defective (sad). Two classes of sad mutants were analyzed: (i) mutants defective in flagellar-mediated motility; and (ii) mutants defective in biogenesis of the polar-localized type IV pili. The authors followed the development of the bio-film formed by the wild type over 8 h using phase-contrast microscopy. The wild-type strain first formed a monolayer of cells on the abiotic surface, followed by the appearance of microcolonies that were dispersed throughout the monolayer of cells. Using time-lapse microscopy, we present evidence that microcolonies form by aggregation of cells present in the monolayer. As obsd. with the wild-type, strains with mutations in genes required for the synthesis of type IV pili formed a monolayer of cells on the PVC plastic. However, in contrast to the wild-type strain, the type IV pili mutants did not develop microcolonies over the course of the expts., suggesting that these structures play an important role in microcolony formation. Very few cells of a non-motile strain (carrying a mutation in flgK) attached to PVC even after 8 h of incubation, suggesting a role for flagella and/or motility in the initial cell-to-surface interactions. The phenotype of these mutants thus allows us to initiate the dissection of the developmental pathway leading to biofilm formation.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1cXntFOrtr0%253D&md5=90bce56b6075fa11682bb4d9f94655b2
  22. 22
    Di tommaso, P.; Moretti, S.; Xenarios, I.; Orobitg, M.; Montanyola, A.; Chang, J. M.; Taly, J. F.; Notredame, C. T-Coffee: a web server for the multiple sequence alignment of protein and RNA sequences using structural information and homology extension. Nucleic Acids Res. 2011, 39 (Web Server issue), W13– 17,  DOI: 10.1093/nar/gkr245
    Google Scholar
    There is no corresponding record for this reference.
  23. 23
    Trifinopoulos, J.; Nguyen, L.-T.; Von haeseler, A.; Minh, B. Q. W-IQ-TREE: a fast online phylogenetic tool for maximum likelihood analysis. Nucleic Acids Res. 2016, 44 (W1), W232– W235,  DOI: 10.1093/nar/gkw256
    Google Scholar
    23
    W-IQ-TREE: a fast online phylogenetic tool for maximum likelihood analysis
    Trifinopoulos, Jana; Nguyen, Lam-Tung; Von Haeseler, Arndt; Minh, Bui Quang
    Nucleic Acids Research (2016), 44 (W1), W232-W235CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)
    This article presents W-IQ-TREE, an intuitive and user-friendly web interface and server for IQ-TREE, an efficient phylogenetic software for max. likelihood anal. W-IQ-TREE supports multiple sequence types (DNA, protein, codon, binary and morphol.) in common alignment formats and a wide range of evolutionary models including mixt. and partition models. W-IQ-TREE performs fast model selection, partition scheme finding, efficient tree reconstruction, ultrafast bootstrapping, branch tests, and tree topol. tests. All computations are conducted on a dedicated computer cluster and the users receive the results via URL or email. W-IQ-TREE. It is free and open to all users and there is no login requirement.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhtV2isbfN&md5=768b8e5e4d0ff93c7afb09fdcad4f039
  24. 24
    Letunic, I.; Bork, P. Interactive Tree Of Life (iTOL) v5: an online tool for phylogenetic tree display and annotation. Nucleic Acids Res. 2021, 49 (W1), W293– W296,  DOI: 10.1093/nar/gkab301
    Google Scholar
    24
    Interactive tree of life (iTOL) v5: an online tool for phylogenetic tree display and annotation
    Letunic, Ivica; Bork, Peer
    Nucleic Acids Research (2021), 49 (W1), W293-W296CODEN: NARHAD; ISSN:1362-4962. (Oxford University Press)
    A review. The Interactive Tree Of Life is an online tool for the display, manipulation and annotation of phylogenetic and other trees. It is freely available and open to everyone. iTOL version 5 introduces a completely new tree display engine, together with numerous new features. For example, a new dataset type has been added (MEME motifs), while annotation options have been expanded for several existing ones. Node metadata display options have been extended and now also support non-numerical categorical values, as well as multiple values per node. Direct manual annotation is now available, providing a set of basic drawing and labeling tools, allowing users to draw shapes, labels and other features by hand directly onto the trees. Support for tree and dataset scales has been extended, providing fine control over line and label styles. Unrooted tree displays can now use the equal-daylight algorithm, proving a much greater display clarity. The user account system has been streamlined and expanded with new navigation options and currently handles >1 million trees from >70000 individual users.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXhvV2isLvM&md5=87746a1b922fec743a10eadc9be80f72
  25. 25
    Vincent, C.; Duclos, B.; Grangeasse, C.; Vaganay, E.; Riberty, M.; Cozzone, A. J.; Doublet, P. Relationship between exopolysaccharide production and protein-tyrosine phosphorylation in gram-negative bacteria. J. Mol. Biol. 2000, 304 (3), 311– 321,  DOI: 10.1006/jmbi.2000.4217
    Google Scholar
    25
    Relationship Between Exopolysaccharide Production and Protein-tyrosine Phosphorylation in Gram-negative Bacteria
    Vincent, Carole; Duclos, Bertrand; Grangeasse, Christophe; Vaganay, Elisabeth; Riberty, Mylene; Cozzone, Alain J.; Doublet, Patricia
    Journal of Molecular Biology (2000), 304 (3), 311-321CODEN: JMOBAK; ISSN:0022-2836. (Academic Press)
    The phosphorylation of proteins at tyrosine residues is known to play a key role in the control of numerous fundamental processes in animal systems. In contrast, the biol. significance of protein-tyrosine phosphorylation in bacteria, which has only been recognized recently, is still unclear. Here, we have analyzed the role in Escherichia coli cells of an autophosphorylating protein-tyrosine kinase, Wzc, and a phosphotyrosine-protein phosphatase, Wzb, by performing knock-out expts. on the corresponding genes, wzc and wzb, and looking at the metabolic consequences induced. The results demonstrate that the phosphorylation of Wzc, as regulated by Wzb, is directly connected with the prodn. of a particular capsular polysaccharide, colanic acid. Thus, when Wzc is phosphorylated on tyrosine, no colanic acid is synthesized by bacteria, but when dephosphorylated by Wzb, colanic acid is produced. This process is rather specific to the pair of proteins Wzc/Wzb. Indeed, a much lesser effect, if any, on colanic acid synthesis is obsd. when knock-out expts. are performed on another pair of genes, etk and etp, which also encode resp. a protein-tyrosine kinase, Etk, and a phosphotyrosine-protein phosphatase, Etp, in E. coli. In addn., the anal. of the phosphorylation reaction at the mol. level reveals differences between Gram-neg. and Gram-pos. bacteria, namely in the no. of protein components required for this reaction to occur. (c) 2000 Academic Press.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3cXot1KltL0%253D&md5=02b7cd80d990305355d7229fbcf016be
  26. 26
    Gratani, F. L.; Englert, T.; Nashier, P.; Sass, P.; Czech, L.; Neumann, N.; Doello, S.; Mann, P.; Blobelt, R.; Alberti, S. E. coli Toxin YjjJ (HipH) Is a Ser/Thr Protein Kinase That Impacts Cell Division, Carbon Metabolism, and Ribosome Assembly. mSystems 2023, 8 (1), e0104322  DOI: 10.1128/msystems.01043-22
    Google Scholar
    There is no corresponding record for this reference.
  27. 27
    Ptacek, J.; Devgan, G.; Michaud, G.; Zhu, H.; Zhu, X.; Fasolo, J.; Guo, H.; Jona, G.; Breitkreutz, A.; Sopko, R. Global analysis of protein phosphorylation in yeast. Nature 2005, 438 (7068), 679– 684,  DOI: 10.1038/nature04187
    Google Scholar
    There is no corresponding record for this reference.
  28. 28
    Cendra mdel, M.; Juárez, A.; Torrents, E. Biofilm modifies expression of ribonucleotide reductase genes in Escherichia coli. PLoS One 2012, 7 (9), e46350  DOI: 10.1371/journal.pone.0046350
    Google Scholar
    There is no corresponding record for this reference.
  29. 29
    Scott, P. M.; Erickson, K. M.; Troutman, J. M. Identification of the Functional Roles of Six Key Proteins in the Biosynthesis of Enterobacteriaceae Colanic Acid. Biochemistry 2019, 58 (13), 1818– 1830,  DOI: 10.1021/acs.biochem.9b00040
    Google Scholar
    29
    Identification of the functional roles of six key proteins in the biosynthesis of Enterobacteriaceae colanic acid
    Scott, Phillip M.; Erickson, Katelyn M.; Troutman, Jerry M.
    Biochemistry (2019), 58 (13), 1818-1830CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
    When subjected to harsh conditions such as low pH, pathogenic Escherichia coli can secrete colanic acid to establish a protective barrier between the organism and the acidic environment. The colanic acid consists of a six-sugar repeating unit polymer comprised of glucose, fucose, galactose, and glucuronic acid. The region of the E. coli genome that encodes colanic acid biosynthesis has been reported, and the first enzyme in the biosynthesis pathway has been biochem. characterized. However, the specific roles of the remaining genes required for colanic acid biosynthesis have not been identified. Here we report the in vitro reconstitution of the next six steps in the assembly of the colanic acid repeating unit. To do this, we have cloned and overexpressed each gene within the colanic acid biosynthesis operon. We then tested the activity of the protein product of these genes using high-performance liq. chromatog. anal. and a fluorescent analog of the isoprenoid anchor bactoprenyl diphospho-glucose as a starting substrate. To ensure that retention time changes were assocd. with varying sugar addns. or modifications, we developed a liq. chromatog.-mass spectrometry method for anal. of the products produced by each enzyme. We have identified the function of all but one encoded glycosyltransferase and have identified the function of two acetyltransferases. This work demonstrates the centrality of acetylation in the biosynthesis of colanic acid and provides insight into the activity of key proteins involved in the prodn. of an important and highly conserved bacterial glycopolymer.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXjvVylt7c%253D&md5=f8cab122e4790136da4063aec178997b
  30. 30
    Andrianopoulos, K.; Wang, L.; Reeves, P. R. Identification of the Fucose Synthetase Gene in the Colanic Acid Gene Cluster of Escherichia coli K-12. J. Bacteriol. 1998, 180 (4), 998– 1001,  DOI: 10.1128/JB.180.4.998-1001.1998
    Google Scholar
    30
    Identification of the fucose synthetase gene in the colanic acid gene cluster of Escherichia coli K-12
    Andrianopoulos, Kanella; Wang, Lei; Reeves, Peter R.
    Journal of Bacteriology (1998), 180 (4), 998-1001CODEN: JOBAAY; ISSN:0021-9193. (American Society for Microbiology)
    GDP-L-fucose, the substrate for fucosyltransferases for addn. of fucose to polysaccharides or glycoproteins in both prokaryotes and eukaryotes, is made from GDP-D-mannose. L-Fucose is a component of bacterial surface antigens, including the extracellular polysaccharide colanic acid produced by most Escherichia coli strains. We previously sequenced the E. coli colanic acid gene cluster and identified one of the GDP-L-fucose biosynthetic pathway genes, gmd. We report here the identification of the gene (fcl), located downstream of gmd, encoding the fucose synthetase.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1cXhtVGltr0%253D&md5=a2abd4d302d77aa7d49ec799aa5833de
  31. 31
    Mcnulty, C.; Thompson, J.; Barrett, B.; Lord, L.; Andersen, C.; Roberts, I. S. The cell surface expression of group 2 capsular polysaccharides in Escherichia coli: the role of KpsD, RhsA and a multi-protein complex at the pole of the cell. Mol. Microbiol. 2006, 59 (3), 907– 922,  DOI: 10.1111/j.1365-2958.2005.05010.x
    Google Scholar
    There is no corresponding record for this reference.
  32. 32
    Eliasson, R.; Pontis, E.; Fontecave, M.; Gerez, C.; Harder, J.; Jörnvall, H.; Krook, M.; Reichard, P. Characterization of components of the anaerobic ribonucleotide reductase system from Escherichia coli. J. Biol. Chem. 1992, 267 (35), 25541– 25547,  DOI: 10.1016/S0021-9258(19)74074-5
    Google Scholar
    There is no corresponding record for this reference.
  33. 33
    Grangeasse, C.; Nessler, S.; Mijakovic, I. Bacterial tyrosine kinases: evolution, biological function and structural insights. Philos. Trans R Soc. Lond B Biol. Sci. 2012, 367 (1602), 2640– 2655,  DOI: 10.1098/rstb.2011.0424
    Google Scholar
    There is no corresponding record for this reference.
  34. 34
    Aravind, L.; Wolf, Y. I.; Koonin, E. V. The ATP-cone: an evolutionarily mobile, ATP-binding regulatory domain. J. Mol. Microbiol. Biotechnol. 2000, 2 (2), 191– 194
    Google Scholar
    There is no corresponding record for this reference.

Cited By

Click to copy section linkSection link copied!

This article has not yet been cited by other publications.

Download PDF
Go to Analytical Chemistry
Get e-Alerts
Get e-Alerts

Analytical Chemistry

Cite this: Anal. Chem. 2024, 96, 21, 8721–8729
Click to copy citationCitation copied!
https://doi.org/10.1021/acs.analchem.4c00965
Published April 29, 2024

Copyright © 2024 The Authors. Published by American Chemical Society. This publication is licensed under

CC-BY 4.0 .

Article Views

1139

Altmetric

-

Citations

-
Learn about these metrics

Article Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.

Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.

The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated.

  • Abstract

    High Resolution Image
    Download MS PowerPoint Slide

    Figure 1

    Figure 1. Workflow of BY-Kinase interrogation by the ECLA. The overall workflow of tyrosine phosphorylation profiling of ECLA starts with (A) ECLA fabrication by high-throughput culturing, induction, lysis, clarify, and printing of the 4126 overexpressed ASKA clones onto aldehyde-coated slides. (B) NHS succinyl ester Dylight 650 was used to visualize total protein on ECLA slides (left). Overexpression of 6xHis-tagged protein in the lysate spots was monitored by anti-His fluorescence staining (right). (C) Phosphorylation levels in each lysate clone were monitored by anti-pTyr antibody staining on ECLA. The BY-kinase candidates were selected by overlapping two different anti-pTyr antibodies. (D) BY-kinase candidates were verified by in vivo overexpressing (left) and in vitro addition (right). The level of phosphorylation in the lysates was monitored by Western blot.

    High Resolution Image
    Download MS PowerPoint Slide

    Figure 2

    Figure 2. Quality control of ECLAs. Quality control of ECLAs was assessed by Dylight 650 NHS Ester for total protein (A,C,E) and anti-His for overexpressed protein (B,D,F). (A) Distribution of total protein from ECLA spots was obtained from two independent assays. Fluorescence intensity from the buffer was indicated with the dotted line. (B) Distribution of 6xHis-tagged protein from ECLA spots was obtained from two independent assays. Fluorescence intensities from WT lysate signals were indicated with the dotted line. (C) Representative image of an ECLA stained by Dylight 650 NHS Ester. (D) Representative image of an ECLA block stained by anti-His. Control spots were indicated in the bottom row. (E) Reproducibility of total protein staining obtained from two independent ECLA assays. (F) Reproducibility of overexpressed protein in the lysates obtained from two independent ECLA assays.

    High Resolution Image
    Download MS PowerPoint Slide

    Figure 3

    Figure 3. Lysate profiles of overexpressed candidates. There were 6 overlapped hits from the two anti-pTyr ECLA assays. (A) Images from the three assays were done with P-Tyr-100 antibody followed by fluorescent-labeled antimouse, and three assays were done with fluorescent-labeled antimouse as blanks. The WT lysate spots were listed as baseline phosphorylation. (B) Each ASKA clone was listed on top, including etk, wzc, argG, dogD, yjjJ, and nrdD. Immunoblots were performed with P-Tyr-100 antibody to show the phosphorylation profiles in the lysates with or without IPTG inductions. (C) Each purified candidate was incubated with or without WT lysates in the kinase buffer supplemented with ATP before analysis by SDS-PAGE. Pro-Q Diamond staining was used to image the phosphorylated levels.

    High Resolution Image
    Download MS PowerPoint Slide

    Figure 4

    Figure 4. Contribution of nrdD to autophosphorylation and biofilm formation. (A) Purified nrdD was treated with a kinase buffer supplemented with ATP for various time points. The level of phosphorylation was quantified by Phos-tag and P-Tyr-100 antibody with the SDS–PAGE. The same membrane was stained for Ponceau S for loading controls. (B) Biofilms were quantified based on the crystal violet staining in nrdD clones with or without IPTG induction. Data were analyzed from five replicates using an unpaired two-tailed t test, ****p < 0.0001.

    High Resolution Image
    Download MS PowerPoint Slide

  • References

    This article references 34 other publications.

    1. 1
      Hajredini, F.; Alphonse, S.; Ghose, R. BY-kinases: Protein tyrosine kinases like no other. J. Biol. Chem. 2023, 299 (1), 102737  DOI: 10.1016/j.jbc.2022.102737
      There is no corresponding record for this reference.
    2. 2
      Ilan, O.; Bloch, Y.; Frankel, G.; Ullrich, H.; Geider, K.; Rosenshine, I. Protein tyrosine kinases in bacterial pathogens are associated with virulence and production of exopolysaccharide. EMBO J. 1999, 18 (12), 3241– 3248,  DOI: 10.1093/emboj/18.12.3241
      There is no corresponding record for this reference.
    3. 3
      Vincent, C.; Doublet, P.; Grangeasse, C.; Vaganay, E.; Cozzone, A. J.; Duclos, B. Cells of Escherichia coli Contain a Protein-Tyrosine Kinase, Wzc, and a Phosphotyrosine-Protein Phosphatase. Wzb. J. Bacteriol. 1999, 181 (11), 3472– 3477,  DOI: 10.1128/JB.181.11.3472-3477.1999
      There is no corresponding record for this reference.
    4. 4
      Hansen, A.-M.; Chaerkady, R.; Sharma, J.; Díaz-mejía, J. J.; Tyagi, N.; Renuse, S.; Jacob, H. K. C.; Pinto, S. M.; Sahasrabuddhe, N. A.; Kim, M.-S. The Escherichia coli Phosphotyrosine Proteome Relates to Core Pathways and Virulence. PLoS Pathol. 2013, 9 (6), e1003403  DOI: 10.1371/journal.ppat.1003403
      There is no corresponding record for this reference.
    5. 5
      Schastnaya, E.; Raguz nakic, Z.; Gruber, C. H.; Doubleday, P. F.; Krishnan, A.; Johns, N. I.; Park, J.; Wang, H. H.; Sauer, U. Extensive regulation of enzyme activity by phosphorylation in Escherichia coli. Nat. Commun. 2021, 12 (1), 5650,  DOI: 10.1038/s41467-021-25988-4
      There is no corresponding record for this reference.
    6. 6
      Rajagopalan, K.; Dworkin, J. Escherichia coli YegI is a novel Ser/Thr kinase lacking conserved motifs that localizes to the inner membrane. FEBS Lett. 2020, 594 (21), 3530– 3541,  DOI: 10.1002/1873-3468.13920
      There is no corresponding record for this reference.
    7. 7
      Dueñas, M. E.; Peltier-heap, R. E.; Leveridge, M.; Annan, R. S.; Büttner, F. H.; Trost, M. Advances in high-throughput mass spectrometry in drug discovery. EMBO Mol. Med. 2023, 15 (1), e14850  DOI: 10.15252/emmm.202114850
      7
      Advances in high-throughput mass spectrometry in drug discovery
      Duenas, Maria Emilia; Peltier-Heap, Rachel E.; Leveridge, Melanie; Annan, Roland S.; Buettner, Frank H.; Trost, Matthias
      EMBO Molecular Medicine (2023), 15 (1), e14850CODEN: EMMMAM; ISSN:1757-4684. (Wiley-Blackwell)
      A review. High-throughput (HT) screening drug discovery, during which thousands or millions of compds. are screened, remains the key methodol. for identifying active chem. matter in early drug discovery pipelines. Recent technol. developments in mass spectrometry (MS) and automation have revolutionized the application of MS for use in HT screens. These methods allow the targeting of unlabeled biomols. in HT assays, thereby expanding the breadth of targets for which HT assays can be developed compared to traditional approaches. Moreover, these label-free MS assays are often cheaper, faster, and more physiol. relevant than competing assay technologies. In this review, we will describe current MS techniques used in drug discovery and explain their advantages and disadvantages. We will highlight the power of mass spectrometry in label-free in vitro assays, and its application for setting up multiplexed cellular phenotypic assays, providing an exciting new tool for screening compds. in cell lines, and even primary cells. Finally, we will give an outlook on how technol. advances will increase the future use and the capabilities of mass spectrometry in drug discovery.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XjtFWqtL7I&md5=068329c09fb22018c83791dd0d9b29d6
    8. 8
      Gavriilidou, A. F. M.; Sokratous, K.; Yen, H.-Y.; De Colibus, L. High-Throughput Native Mass Spectrometry Screening in Drug Discovery. Front. Mol. Biosci. 2022, 9, 837901  DOI: 10.3389/fmolb.2022.837901
      8
      High-Throughput Native Mass Spectrometry Screening in Drug Discovery
      Gavriilidou Agni F M; Sokratous Kleitos; Yen Hsin-Yung; De Colibus Luigi
      Frontiers in molecular biosciences (2022), 9 (), 837901 ISSN:2296-889X.
      The design of new therapeutic molecules can be significantly informed by studying protein-ligand interactions using biophysical approaches directly after purification of the protein-ligand complex. Well-established techniques utilized in drug discovery include isothermal titration calorimetry, surface plasmon resonance, nuclear magnetic resonance spectroscopy, and structure-based drug discovery which mainly rely on protein crystallography and, more recently, cryo-electron microscopy. Protein-ligand complexes are dynamic, heterogeneous, and challenging systems that are best studied with several complementary techniques. Native mass spectrometry (MS) is a versatile method used to study proteins and their non-covalently driven assemblies in a native-like folded state, providing information on binding thermodynamics and stoichiometry as well as insights on ternary and quaternary protein structure. Here, we discuss the basic principles of native mass spectrometry, the field's recent progress, how native MS is integrated into a drug discovery pipeline, and its future developments in drug discovery.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB2Mrks1Ohuw%253D%253D&md5=80ce8f1d387bace38193aa9c29ce383e
    9. 9
      Kitagawa, M.; Ara, T.; Arifuzzaman, M.; Ioka-nakamichi, T.; Inamoto, E.; Toyonaga, H.; Mori, H. Complete set of ORF clones of Escherichia coli ASKA library (a complete set of E. coli K-12 ORF archive): unique resources for biological research. DNA Res. 2006, 12 (5), 291– 299,  DOI: 10.1093/dnares/dsi012
      There is no corresponding record for this reference.
    10. 10
      Jehle, S.; Kunowska, N.; Benlasfer, N.; Woodsmith, J.; Weber, G.; Wahl, M. C.; Stelzl, U. A human kinase yeast array for the identification of kinases modulating phosphorylation-dependent protein–protein interactions. Mol. Syst. Biol. 2022, 18 (3), e10820  DOI: 10.15252/msb.202110820
      There is no corresponding record for this reference.
    11. 11
      Chen, J.; Bell, J.; Lau, B. T.; Whittaker, T.; Stapleton, D.; Ji, H. P. A functional CRISPR/Cas9 screen identifies kinases that modulate FGFR inhibitor response in gastric cancer. Oncogenesis 2019, 8 (5), 33,  DOI: 10.1038/s41389-019-0145-z
      There is no corresponding record for this reference.
    12. 12
      Coarfa, C.; Grimm, S. L.; Rajapakshe, K.; Perera, D.; Lu, H. Y.; Wang, X.; Christensen, K. R.; Mo, Q.; Edwards, D. P.; Huang, S. Reverse-Phase Protein Array: Technology, Application, Data Processing, and Integration. J. Biomol Tech 2021, 32 (1), 15– 29,  DOI: 10.7171/jbt.21-3202-001
      12
      Reverse-Phase Protein Array: Technology, Application, Data Processing, and Integration
      Coarfa Cristian; Grimm Sandra L; Rajapakshe Kimal; Edwards Dean P; Huang Shixia; Coarfa Cristian; Mo Qianxing; Edwards Dean P; Huang Shixia; Coarfa Cristian; Perera Dimuthu; Lu Hsin-Yi; Wang Xuan; Christensen Kurt R; Edwards Dean P; Huang Shixia
      Journal of biomolecular techniques : JBT (2021), 32 (1), 15-29 ISSN:.
      Reverse-phase protein array (RPPA) is a high-throughput antibody-based targeted proteomics platform that can quantify hundreds of proteins in thousands of samples derived from tissue or cell lysates, serum, plasma, or other body fluids. Protein samples are robotically arrayed as microspots on nitrocellulose-coated glass slides. Each slide is probed with a specific antibody that can detect levels of total protein expression or post-translational modifications, such as phosphorylation as a measure of protein activity. Here we describe workflow protocols and software tools that we have developed and optimized for RPPA in a core facility setting that includes sample preparation, microarray mapping and printing of protein samples, antibody labeling, slide scanning, image analysis, data normalization and quality control, data reporting, statistical analysis, and management of data. Our RPPA platform currently analyzes ∼240 validated antibodies that primarily detect proteins in signaling pathways and cellular processes that are important in cancer biology. This is a robust technology that has proven to be of value for both validation and discovery proteomic research and integration with other omics data sets.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB2c%252FotVSrtw%253D%253D&md5=9f70d7e3e3a1dd18b42442072e1447da
    13. 13
      Wang, X.; Shi, Z.; Lu, H.-Y.; Kim, J. J.; Bu, W.; Villalobos, J. A.; Perera, D. N.; Jung, S. Y.; Wang, T.; Grimm, S. L. High-throughput profiling of histone post-translational modifications and chromatin modifying proteins by reverse phase protein array. Journal of Proteomics 2022, 262, 104596  DOI: 10.1016/j.jprot.2022.104596
      There is no corresponding record for this reference.
    14. 14
      Cheng, L.; Liu, C.-X.; Jiang, S.; Hou, S.; Huang, J.-G.; Chen, Z.-Q.; Sun, Y.-Y.; Qi, H.; Jiang, H.-W.; Wang, J.-F. Cell Lysate Microarray for Mapping the Network of Genetic Regulators for Histone Marks. Molecular & cellular proteomics: MCP 2018, 17 (9), 1720– 1736,  DOI: 10.1074/mcp.RA117.000550
      There is no corresponding record for this reference.
    15. 15
      Baba, T.; Ara, T.; Hasegawa, M.; Takai, Y.; Okumura, Y.; Baba, M.; Datsenko, K. A.; Tomita, M.; Wanner, B. L.; Mori, H. Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol. Syst. Biol. 2006, 2, 2006.0008  DOI: 10.1038/msb4100050
      There is no corresponding record for this reference.
    16. 16
      Du, P.-X.; Chou, Y.-Y.; Santos, H. M.; Keskin, B. B.; Hsieh, M.-H.; Ho, T.-S.; Wang, J.-Y.; Lin, Y.-L.; Syu, G.-D. Development and Application of Human Coronavirus Protein Microarray for Specificity Analysis. Anal. Chem. 2021, 93 (21), 7690– 7698,  DOI: 10.1021/acs.analchem.1c00614
      There is no corresponding record for this reference.
    17. 17
      Jeon, S.; Kim, T.-I.; Jin, H.; Lee, U.; Bae, J.; Bouffard, J.; Kim, Y. Amine-Reactive Activated Esters of meso-CarboxyBODIPY: Fluorogenic Assays and Labeling of Amines, Amino Acids, and Proteins. J. Am. Chem. Soc. 2020, 142 (20), 9231– 9239,  DOI: 10.1021/jacs.9b13982
      17
      Amine-Reactive Activated Esters of meso-CarboxyBODIPY: Fluorogenic Assays and Labeling of Amines, Amino Acids, and Proteins
      Jeon, Sungjin; Kim, Tae-Il; Jin, Hanyong; Lee, Uisung; Bae, Jeehyeon; Bouffard, Jean; Kim, Youngmi
      Journal of the American Chemical Society (2020), 142 (20), 9231-9239CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Fluorescence-based amine-reactive dyes are highly valuable for the sensing of amines and the labeling of biomols. Although it would be highly desirable, large changes in emission spectra and intensity seldom accompany the conjugation of known amine-reactive dyes to their target mols. On the contrary, amide bond formation between amines and the pentafluorophenyl (2-PFP) and succinimidyl (2-NHS) esters of meso-carboxyBODIPY results in significant changes in emission maxima (Δλ: 70-100 nm) and intensity (up to 3000-fold), enabling the fast (down to 5 min) and selective fluorogenic detection and labeling of amines, amino acids, and proteins. This approach further benefits from the demonstrated versatility and high reliability of activated ester chem., and background hydrolysis is negligible. The large "turn-on" response is a testament of the extreme sensitivity of meso-carboxyBODIPYs to the minimal changes in electronic properties that distinguish esters from amides. Applications to the detection of food spoilage, staining of proteins on electrophoretic gels or in living cells, and the expedited synthesis of organelle-specific fluorescence microscope imaging agents are further demonstrated.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXnsVyrsrk%253D&md5=6cf7d5edbe94661b749440ef6540defe
    18. 18
      Chen, C. S.; Korobkova, E.; Chen, H.; Zhu, J.; Jian, X.; Tao, S. C.; He, C.; Zhu, H. A proteome chip approach reveals new DNA damage recognition activities in Escherichia coli. Nat. Methods 2008, 5 (1), 69– 74,  DOI: 10.1038/nmeth1148
      18
      A proteome chip approach reveals new DNA damage recognition activities in Escherichia coli
      Chen, Chien-Sheng; Korobkova, Ekaterina; Chen, Hao; Zhu, Jian; Jian, Xing; Tao, Sheng-Ce; He, Chuan; Zhu, Heng
      Nature Methods (2008), 5 (1), 69-74CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)
      Despite the fact that many genomes have been decoded, proteome chips comprising individually purified proteins have been reported only for budding yeast, mainly because of the complexity and difficulty of high-throughput protein purifn. To facilitate proteomics studies in prokaryotes, the authors have developed a high-throughput protein purifn. protocol that allowed the authors to purify 4256 proteins encoded by the Escherichia coli K12 strain within 10 h. The purified proteins were then spotted onto glass slides to create E. coli proteome chips. The authors used these chips to develop assays for identifying proteins involved in the recognition of potential base damage in DNA. By using a group of DNA probes, each contg. a mismatched base pair or an abasic site, the authors found a small no. of proteins that could recognize each type of probe with high affinity and specificity. The authors further evaluated two of these proteins, YbaZ and YbcN, by biochem. analyses. The assembly of libraries contg. DNA probes with specific modifications and the availability of E. coli proteome chips have the potential to reveal important interactions between proteins and nucleic acids that are time-consuming and difficult to detect using other techniques.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXht1SksQ%253D%253D&md5=f73a9eb60587b58a6638e236c84e6323
    19. 19
      Kinoshita, E.; Kinoshita-kikuta, E.; Sugiyama, Y.; Fukada, Y.; Ozeki, T.; Koike, T. Highly sensitive detection of protein phosphorylation by using improved Phos-tag Biotin. Proteomics 2012, 12 (7), 932– 937,  DOI: 10.1002/pmic.201100639
      There is no corresponding record for this reference.
    20. 20
      Kuo, H. C.; Huang, Y. H.; Chung, F. H.; Chen, P. C.; Sung, T. C.; Chen, Y. W.; Hsieh, K. S.; Chen, C. S.; Syu, G. D. Antibody Profiling of Kawasaki Disease Using Escherichia coli Proteome Microarrays. Mol. Cell. Proteomics 2018, 17 (3), 472– 481,  DOI: 10.1074/mcp.RA117.000198
      There is no corresponding record for this reference.
    21. 21
      O’toole, G. A.; Kolter, R. Flagellar and twitching motility are necessary for Pseudomonas aeruginosa biofilm development. Mol. Microbiol. 1998, 30 (2), 295– 304,  DOI: 10.1046/j.1365-2958.1998.01062.x
      21
      Flagellar and twitching motility are necessary for Pseudomonas aeruginosa biofilm development
      O'Toole, George A.; Kolter, Roberto
      Molecular Microbiology (1998), 30 (2), 295-304CODEN: MOMIEE; ISSN:0950-382X. (Blackwell Science Ltd.)
      The formation of complex bacterial communities known as biofilms begins with the interaction of planktonic cells with a surface in response to appropriate environmental signals. The authors report the isolation and characterization of mutants of Pseudomonas aeruginosa PA14 defective in the initiation of biofilm formation on an abiotic surface, polyvinylchloride (PVC) plastic. These mutants are designated surface attachment defective (sad). Two classes of sad mutants were analyzed: (i) mutants defective in flagellar-mediated motility; and (ii) mutants defective in biogenesis of the polar-localized type IV pili. The authors followed the development of the bio-film formed by the wild type over 8 h using phase-contrast microscopy. The wild-type strain first formed a monolayer of cells on the abiotic surface, followed by the appearance of microcolonies that were dispersed throughout the monolayer of cells. Using time-lapse microscopy, we present evidence that microcolonies form by aggregation of cells present in the monolayer. As obsd. with the wild-type, strains with mutations in genes required for the synthesis of type IV pili formed a monolayer of cells on the PVC plastic. However, in contrast to the wild-type strain, the type IV pili mutants did not develop microcolonies over the course of the expts., suggesting that these structures play an important role in microcolony formation. Very few cells of a non-motile strain (carrying a mutation in flgK) attached to PVC even after 8 h of incubation, suggesting a role for flagella and/or motility in the initial cell-to-surface interactions. The phenotype of these mutants thus allows us to initiate the dissection of the developmental pathway leading to biofilm formation.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1cXntFOrtr0%253D&md5=90bce56b6075fa11682bb4d9f94655b2
    22. 22
      Di tommaso, P.; Moretti, S.; Xenarios, I.; Orobitg, M.; Montanyola, A.; Chang, J. M.; Taly, J. F.; Notredame, C. T-Coffee: a web server for the multiple sequence alignment of protein and RNA sequences using structural information and homology extension. Nucleic Acids Res. 2011, 39 (Web Server issue), W13– 17,  DOI: 10.1093/nar/gkr245
      There is no corresponding record for this reference.
    23. 23
      Trifinopoulos, J.; Nguyen, L.-T.; Von haeseler, A.; Minh, B. Q. W-IQ-TREE: a fast online phylogenetic tool for maximum likelihood analysis. Nucleic Acids Res. 2016, 44 (W1), W232– W235,  DOI: 10.1093/nar/gkw256
      23
      W-IQ-TREE: a fast online phylogenetic tool for maximum likelihood analysis
      Trifinopoulos, Jana; Nguyen, Lam-Tung; Von Haeseler, Arndt; Minh, Bui Quang
      Nucleic Acids Research (2016), 44 (W1), W232-W235CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)
      This article presents W-IQ-TREE, an intuitive and user-friendly web interface and server for IQ-TREE, an efficient phylogenetic software for max. likelihood anal. W-IQ-TREE supports multiple sequence types (DNA, protein, codon, binary and morphol.) in common alignment formats and a wide range of evolutionary models including mixt. and partition models. W-IQ-TREE performs fast model selection, partition scheme finding, efficient tree reconstruction, ultrafast bootstrapping, branch tests, and tree topol. tests. All computations are conducted on a dedicated computer cluster and the users receive the results via URL or email. W-IQ-TREE. It is free and open to all users and there is no login requirement.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhtV2isbfN&md5=768b8e5e4d0ff93c7afb09fdcad4f039
    24. 24
      Letunic, I.; Bork, P. Interactive Tree Of Life (iTOL) v5: an online tool for phylogenetic tree display and annotation. Nucleic Acids Res. 2021, 49 (W1), W293– W296,  DOI: 10.1093/nar/gkab301
      24
      Interactive tree of life (iTOL) v5: an online tool for phylogenetic tree display and annotation
      Letunic, Ivica; Bork, Peer
      Nucleic Acids Research (2021), 49 (W1), W293-W296CODEN: NARHAD; ISSN:1362-4962. (Oxford University Press)
      A review. The Interactive Tree Of Life is an online tool for the display, manipulation and annotation of phylogenetic and other trees. It is freely available and open to everyone. iTOL version 5 introduces a completely new tree display engine, together with numerous new features. For example, a new dataset type has been added (MEME motifs), while annotation options have been expanded for several existing ones. Node metadata display options have been extended and now also support non-numerical categorical values, as well as multiple values per node. Direct manual annotation is now available, providing a set of basic drawing and labeling tools, allowing users to draw shapes, labels and other features by hand directly onto the trees. Support for tree and dataset scales has been extended, providing fine control over line and label styles. Unrooted tree displays can now use the equal-daylight algorithm, proving a much greater display clarity. The user account system has been streamlined and expanded with new navigation options and currently handles >1 million trees from >70000 individual users.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXhvV2isLvM&md5=87746a1b922fec743a10eadc9be80f72
    25. 25
      Vincent, C.; Duclos, B.; Grangeasse, C.; Vaganay, E.; Riberty, M.; Cozzone, A. J.; Doublet, P. Relationship between exopolysaccharide production and protein-tyrosine phosphorylation in gram-negative bacteria. J. Mol. Biol. 2000, 304 (3), 311– 321,  DOI: 10.1006/jmbi.2000.4217
      25
      Relationship Between Exopolysaccharide Production and Protein-tyrosine Phosphorylation in Gram-negative Bacteria
      Vincent, Carole; Duclos, Bertrand; Grangeasse, Christophe; Vaganay, Elisabeth; Riberty, Mylene; Cozzone, Alain J.; Doublet, Patricia
      Journal of Molecular Biology (2000), 304 (3), 311-321CODEN: JMOBAK; ISSN:0022-2836. (Academic Press)
      The phosphorylation of proteins at tyrosine residues is known to play a key role in the control of numerous fundamental processes in animal systems. In contrast, the biol. significance of protein-tyrosine phosphorylation in bacteria, which has only been recognized recently, is still unclear. Here, we have analyzed the role in Escherichia coli cells of an autophosphorylating protein-tyrosine kinase, Wzc, and a phosphotyrosine-protein phosphatase, Wzb, by performing knock-out expts. on the corresponding genes, wzc and wzb, and looking at the metabolic consequences induced. The results demonstrate that the phosphorylation of Wzc, as regulated by Wzb, is directly connected with the prodn. of a particular capsular polysaccharide, colanic acid. Thus, when Wzc is phosphorylated on tyrosine, no colanic acid is synthesized by bacteria, but when dephosphorylated by Wzb, colanic acid is produced. This process is rather specific to the pair of proteins Wzc/Wzb. Indeed, a much lesser effect, if any, on colanic acid synthesis is obsd. when knock-out expts. are performed on another pair of genes, etk and etp, which also encode resp. a protein-tyrosine kinase, Etk, and a phosphotyrosine-protein phosphatase, Etp, in E. coli. In addn., the anal. of the phosphorylation reaction at the mol. level reveals differences between Gram-neg. and Gram-pos. bacteria, namely in the no. of protein components required for this reaction to occur. (c) 2000 Academic Press.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3cXot1KltL0%253D&md5=02b7cd80d990305355d7229fbcf016be
    26. 26
      Gratani, F. L.; Englert, T.; Nashier, P.; Sass, P.; Czech, L.; Neumann, N.; Doello, S.; Mann, P.; Blobelt, R.; Alberti, S. E. coli Toxin YjjJ (HipH) Is a Ser/Thr Protein Kinase That Impacts Cell Division, Carbon Metabolism, and Ribosome Assembly. mSystems 2023, 8 (1), e0104322  DOI: 10.1128/msystems.01043-22
      There is no corresponding record for this reference.
    27. 27
      Ptacek, J.; Devgan, G.; Michaud, G.; Zhu, H.; Zhu, X.; Fasolo, J.; Guo, H.; Jona, G.; Breitkreutz, A.; Sopko, R. Global analysis of protein phosphorylation in yeast. Nature 2005, 438 (7068), 679– 684,  DOI: 10.1038/nature04187
      There is no corresponding record for this reference.
    28. 28
      Cendra mdel, M.; Juárez, A.; Torrents, E. Biofilm modifies expression of ribonucleotide reductase genes in Escherichia coli. PLoS One 2012, 7 (9), e46350  DOI: 10.1371/journal.pone.0046350
      There is no corresponding record for this reference.
    29. 29
      Scott, P. M.; Erickson, K. M.; Troutman, J. M. Identification of the Functional Roles of Six Key Proteins in the Biosynthesis of Enterobacteriaceae Colanic Acid. Biochemistry 2019, 58 (13), 1818– 1830,  DOI: 10.1021/acs.biochem.9b00040
      29
      Identification of the functional roles of six key proteins in the biosynthesis of Enterobacteriaceae colanic acid
      Scott, Phillip M.; Erickson, Katelyn M.; Troutman, Jerry M.
      Biochemistry (2019), 58 (13), 1818-1830CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
      When subjected to harsh conditions such as low pH, pathogenic Escherichia coli can secrete colanic acid to establish a protective barrier between the organism and the acidic environment. The colanic acid consists of a six-sugar repeating unit polymer comprised of glucose, fucose, galactose, and glucuronic acid. The region of the E. coli genome that encodes colanic acid biosynthesis has been reported, and the first enzyme in the biosynthesis pathway has been biochem. characterized. However, the specific roles of the remaining genes required for colanic acid biosynthesis have not been identified. Here we report the in vitro reconstitution of the next six steps in the assembly of the colanic acid repeating unit. To do this, we have cloned and overexpressed each gene within the colanic acid biosynthesis operon. We then tested the activity of the protein product of these genes using high-performance liq. chromatog. anal. and a fluorescent analog of the isoprenoid anchor bactoprenyl diphospho-glucose as a starting substrate. To ensure that retention time changes were assocd. with varying sugar addns. or modifications, we developed a liq. chromatog.-mass spectrometry method for anal. of the products produced by each enzyme. We have identified the function of all but one encoded glycosyltransferase and have identified the function of two acetyltransferases. This work demonstrates the centrality of acetylation in the biosynthesis of colanic acid and provides insight into the activity of key proteins involved in the prodn. of an important and highly conserved bacterial glycopolymer.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXjvVylt7c%253D&md5=f8cab122e4790136da4063aec178997b
    30. 30
      Andrianopoulos, K.; Wang, L.; Reeves, P. R. Identification of the Fucose Synthetase Gene in the Colanic Acid Gene Cluster of Escherichia coli K-12. J. Bacteriol. 1998, 180 (4), 998– 1001,  DOI: 10.1128/JB.180.4.998-1001.1998
      30
      Identification of the fucose synthetase gene in the colanic acid gene cluster of Escherichia coli K-12
      Andrianopoulos, Kanella; Wang, Lei; Reeves, Peter R.
      Journal of Bacteriology (1998), 180 (4), 998-1001CODEN: JOBAAY; ISSN:0021-9193. (American Society for Microbiology)
      GDP-L-fucose, the substrate for fucosyltransferases for addn. of fucose to polysaccharides or glycoproteins in both prokaryotes and eukaryotes, is made from GDP-D-mannose. L-Fucose is a component of bacterial surface antigens, including the extracellular polysaccharide colanic acid produced by most Escherichia coli strains. We previously sequenced the E. coli colanic acid gene cluster and identified one of the GDP-L-fucose biosynthetic pathway genes, gmd. We report here the identification of the gene (fcl), located downstream of gmd, encoding the fucose synthetase.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1cXhtVGltr0%253D&md5=a2abd4d302d77aa7d49ec799aa5833de
    31. 31
      Mcnulty, C.; Thompson, J.; Barrett, B.; Lord, L.; Andersen, C.; Roberts, I. S. The cell surface expression of group 2 capsular polysaccharides in Escherichia coli: the role of KpsD, RhsA and a multi-protein complex at the pole of the cell. Mol. Microbiol. 2006, 59 (3), 907– 922,  DOI: 10.1111/j.1365-2958.2005.05010.x
      There is no corresponding record for this reference.
    32. 32
      Eliasson, R.; Pontis, E.; Fontecave, M.; Gerez, C.; Harder, J.; Jörnvall, H.; Krook, M.; Reichard, P. Characterization of components of the anaerobic ribonucleotide reductase system from Escherichia coli. J. Biol. Chem. 1992, 267 (35), 25541– 25547,  DOI: 10.1016/S0021-9258(19)74074-5
      There is no corresponding record for this reference.
    33. 33
      Grangeasse, C.; Nessler, S.; Mijakovic, I. Bacterial tyrosine kinases: evolution, biological function and structural insights. Philos. Trans R Soc. Lond B Biol. Sci. 2012, 367 (1602), 2640– 2655,  DOI: 10.1098/rstb.2011.0424
      There is no corresponding record for this reference.
    34. 34
      Aravind, L.; Wolf, Y. I.; Koonin, E. V. The ATP-cone: an evolutionarily mobile, ATP-binding regulatory domain. J. Mol. Microbiol. Biotechnol. 2000, 2 (2), 191– 194
      There is no corresponding record for this reference.

  • Supporting Information

    Supporting Information

    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.analchem.4c00965.

    • List of ECLA spots with elevated tyrosine phosphorylation ranking, phylogenetic tree, list and figures of nrdD protein from other bacteria, list of nrdD substrates obtained from in vitro kinase assay on E. coli proteome microarray, in vitro kinase assay results on E. coli proteome array to verify ssuD as a substrate, and additional supporting Western blot images (PDF)


    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.