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Most bacterial proteomic studies done to date utilise bacterial cells harvested from liquid culture media. However, it is widely accepted that many important determinants associated with virulence and host cell adhesion are exclusively expressed during growth on solid media, as a crude mimic of true biofilms. Here, we compare the observed proteome of Escherichia coli K12 from isolated single colonies on solid media with those observed at different growth phases in liquid culture; i.e. early-log, mid-log, early-, mid- and late-stationary growth phases. A total of 2044 protein groups covering approximately 47% of the total proteome were identified across all studied conditions, including 1650 proteins identified from single colonies and 1679 proteins from liquid cultured cells. Label-free quantitative analysis revealed that the E. coli proteome of single colonies on a solid agar differs from that observed in liquid culture. Notably, the presence of proteins in the Suf-operon that are involved in iron mobilisation and swarming motility was associated exclusively with single colony profiles, whereas proteins involved in motility such as motA, motB, fliH, flip, fliD and fliJ were associated exclusively with cells grown in liquid culture. The data presented here provide a valuable resource for understanding the role of key proteins within microenvironments surrounding E. coli single colonies. SIGNIFICANCE: To date, most proteomics studies have used E. coli cells harvested from liquid culture media even though many important determinants associated with virulence and host cell adhesion are exclusively expressed during growth on solid media. In this study, we compare the observed proteome of E. coli K12 from isolated single colonies on solid media with those observed at different growth phases in liquid culture; i.e. early-log, mid-log, early-, mid- and late-stationary growth phases. By using label-free quantitative analysis we demonstrate that the E. coli proteome of single colonies on a solid agar differs from that observed in liquid culture with an overlap of 68% of proteins between the two culture conditions. Our analysis further reveal the presence of proteins in the Suf-operon that are involved in iron mobilisation and swarming motility was associated exclusively with single colony profiles. While those proteins involved in motility such as motA, motB, fliH, flip, fliD and fliJ were associated exclusively with cells grown in liquid culture. By comparison to E. coli proteomic data available on liquid culture and solid media, this research represents a first effort to describe the differential expression of key E. coli proteins within microenvironments surrounding single colonies., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Understanding the cell wall of mycobacteria is crucial for improving drug design or identifying new antigens suitable to vaccination. Yet this remains problematic due to the complexity of the cell wall composition. In this study, we successfully developed gel-free approaches to study cell wall proteins in Mycobacterium smegmatis. The cell wall was subjected to differential centrifugation, differential detergent solubilisation and phase separation to yield the genuine cell wall proteome. Next, protein extracts were digested by filter-assisted sample preparation for LC-MS/MS analysis on a Q Exactive mass spectrometer, and identified proteins filtered through a stringent bioinformatics pipeline. This yielded the unprecedented coverage of 96 lipoproteins, 475 membrane proteins and 73 secreted proteins. Employing this approach, we next quantified changes in the cell wall proteome during exposure of M. smegmatis to sub-lethal concentration of rifampicin. This facilitated detailed characterisation of the dysregulation of ABC transporters, virulence factors such as Mce proteins and PknG, and proteins involved in cell wall and lipid synthesis. Crucially, these cell wall proteins are under-represented in previous proteome analysis of M. smegmatis. This approach enables further quantitative proteomic studies of the role of the cell wall proteome of mycobacteria in virulence or during drug exposure. SIGNIFICANCE: We developed novel gel-free sample preparation workflows for the cell wall fraction of mycobacteria that significantly increase the coverage of the cell wall proteome compared to previous studies. We then provide a data analysis workflow that enables the removal of likely cytosolic contaminants in the cell wall fraction post-measurement. Combined, these approaches increase the coverage of the cell wall proteome while ensuring that the identified proteins are true cell wall proteins and not carry-over of high-abundance contaminants from the cytosol. We have applied these approaches to quantify the dysregulation of cell wall proteins during exposure of M. smegmatis to rifampicin, which has shed new light on the coordinated down-regulation of ABC transporters as well as virulence factors present in the cell wall proteome., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Mycobacterial Ser/Thr kinases play a critical role in bacterial physiology and pathogenesis. Linking kinases to the substrates they phosphorylate in vivo , thereby elucidating their exact functions, is still a challenge. The aim of this work was to associate protein phosphorylation in mycobacteria with important subsequent macro cellular events by identifying the physiological substrates of PknG in Mycobacterium bovis BCG. The study compared the phosphoproteome dynamics during the batch growth of M. bovis BCG versus the respective PknG knock-out mutant (ΔPknG-BCG) strains. We employed TiO 2 phosphopeptide enrichment techniques combined with label-free quantitative phosphoproteomics workflow on LC-MS/MS. The comprehensive analysis of label-free data identified 603 phosphopeptides on 307 phosphoproteins with high confidence. Fifty-five phosphopeptides were differentially phosphorylated, of these, 23 phosphopeptides were phosphorylated in M. bovis BCG wild-type only and not in the mutant. These were further validated through targeted mass spectrometry assays (PRMs). Kinase-peptide docking studies based on a published crystal structure of PknG in complex with GarA revealed that the majority of identified phosphosites presented docking scores close to that seen in previously described PknG substrates, GarA, and ribosomal protein L13. Six out of the 22 phosphoproteins had higher docking scores than GarA, consistent with the proteins identified here being true PknG substrates. Based on protein functional analysis of the PknG substrates identified, this study confirms that PknG plays an important regulatory role in mycobacterial metabolism, through phosphorylation of ATP binding proteins and enzymes in the TCA cycle. This work also reinforces PknG's regulation of protein translation and folding machinery., (© 2018 Nakedi et al.)

Vitamin C has been found to affect mycobacteria in multiple ways, including increasing susceptibility to antimicrobial drugs, inducing dormancy, and having a bactericidal effect. However, the regulatory events mediating vitamin C related adaptations remain largely elusive. Ser/Thr/Tyr protein phosphorylation plays an important regulatory role in mycobacteria, contributing to environmental adaptation, including dormancy and drug resistance. This study utilised the model organism, Mycobacterium smegmatis, and TiO 2 phosphopeptide enrichment combined with mass spectrometry-based proteomics methods to elucidate the mycobacterial signalling and regulatory response to sub-lethal concentrations of vitamin C. After initial validation of peptide spectra, 224 non-redundant phosphosites in 154 proteins were retained with high confidence. Data analysis revealed that 30 peptides were differentially phosphorylated with Vitamin C treatment, including novel phosphosites found on both PknG and GarA. Of these significant proteins, we validated 11 by parallel reaction monitoring of high-confidence phosphopeptides. Interestingly, 17/30 phosphopeptides were annotated as part of transmembrane proteins, suggesting that it is likely vitamin C triggers typical signal transduction events in which the protein periplasmic domain perceives environmental signals and the cytoplasmic domain is then phosphorylated. Finally, the diverse nature of phosphorylated proteins involved in signalling, transport, and carbohydrate biosynthesis indicates the extent of such regulatory phosphorylation events., Biological Significance: Our findings provide new mechanistic insight into a coordinated network of signalling and regulatory responses to sub-lethal vitamin C in Mycobacterium smegmatis and provide evidence that vitamin C is able to act as a novel extracellular signalling molecule. Vitamin C treatment caused changes in both the proteome and phosphoproteome associated with response to oxidative stress, a shift in metabolic regulation and progression toward dormancy, as well as phospho-dependent activation of specific secretory pathways and activation of specific two component and Ser/Thr/Tyr protein kinase activities. This study confirms the potential of vitamin C as convenient means to study aspects of mycobacterial dormancy, including those regulated at post-translational level., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Shiga-toxin-producing Escherichia coli (STEC) and enteropathogenic Escherichia coli (EPEC) are key diarrhoea-causing foodborne pathogens. We used proteomics to characterize the virulence and antimicrobial resistance protein profiles of three clinical pathogenic E. coli isolates (two EPEC [one resistant to ciprofloxacin] and one STEC) cultured on CHROMagar™STEC solid media after minimal laboratory passage. We identified 4767 unique peptides from 1630 protein group across all three clinical E. coli strains. Label-free proteomic analysis allowed the identification of virulence and drug resistance proteins that were unique to each of the clinical isolates compared in this study. The B subunit of Shiga toxin, ToxB, was uniquely detected in the STEC strain while several other virulence factors including SheA, OmpF, OmpC and OmpX were significantly more abundant in the STEC strain. The ciprofloxacin resistant EPEC isolate possessed reduced levels of key virulence proteins compared to the ciprofloxacin susceptible EPEC and STEC strains. Parallel reaction monitoring assays validated the presence of biologically relevant proteins across biologically-replicated cultures. Propagation of clinical isolates on a relevant solid medium followed by mass spectrometry analysis represents a convenient means to quantify virulence factors and drug resistance determinants that might otherwise be lost through extensive in vitro passage in enteropathogenic bacteria., Significance: Through the use of quantitative proteomics, we have characterized the virulence and antimicrobial resistance attributes of three clinically isolated, pathogenic E. coli strains cultured on solid media. Our results provide new, quantitative data on the expressed proteomes of these tellurite-resistant, diarrhoeagenic E. coli strains and reveal a subset of antimicrobial resistance and virulence proteins that are differentially abundant between these clinical strains. Our quantitative proteomics-based approach should thus have applicability in microbiological diagnostic labs for the identification of pathogenic/drug resistant E. coli in the future., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The comprehensive characterisation of the cell wall proteome of mycobacteria is of considerable relevance to both the discovery of new drug targets as well as to the design of new vaccines against Mycobacterium tuberculosis . However, due to its extremely hydrophobic nature, the coverage of proteomic studies of this subcellular compartment is still far from complete. Here, we report novel gel-free cell wall sample preparation procedures and quantitative LC-MS/MS measurements on a Q Exactive mass spectrometer. We combine these with a novel post-measurement bioinformatic analysis to filter out likely cytosolic contaminants. This reveals a subset of proteins that are highly enriched for cell wall proteins. The success of this approach is verified by peptide-centric measurement of the abundance of known subcellular markers, as well as analysis of the percentage of predicted membrane proteins within the purified fraction. While M. smegmatis was used during this study to establish and optimise the sample preparation procedures, these can easily be applied to other mycobacterial species, such as M. bovis BCG or M. tuberculosis . •Improved gel-free cell wall sample preparation gives higher yields of tryptic peptides for LC-MS/MS measurement.•Higher yields of tryptic peptides provide better quantitation and coverage of cell wall proteome.•Post-measurement enrichment analysis filters out high abundance cytosolic contaminants that have carried through the experimental analysis.