Boumediene Soufi

Phosphoproteome Analysis of E. coli Reveals Evolutionary Conservation of Bacterial Ser/Thr/Tyr Phosphorylation

Protein phosphorylation on serine, threonine, and tyrosine (Ser/Thr/Tyr) is generally considered the major regulatory posttranslational modification in eukaryotic cells. Increasing evidence at the genome and proteome level shows that this modification is also present and functional in prokaryotes. We have recently reported the first in-depth phosphorylation site-resolved dataset from the model Gram-positive bacterium, Bacillus subtilis, showing that Ser/Thr/Tyr phosphorylation is also present on many essential bacterial proteins. To test whether this modification is common in Eubacteria, here we use a recently developed proteomics approach based on phosphopeptide enrichment and high accuracy MS to analyze the phosphoproteome of the model Gram-negative bacterium Escherichia coli. We report 81 phosphorylation sites on 79 E. coli proteins, with distribution of Ser/Thr/Tyr phosphorylation sites 68%/23%/9%. Despite their phylogenetic distance, phosphoproteomes of E. coli and B. subtilis show striking similarity in size, classes of phosphorylated proteins, and distribution of Ser/Thr/Tyr phosphorylation sites. By combining the two datasets, we created the largest phosphorylation site-resolved database of bacterial phosphoproteins to date (available at www.phosida.com) and used it to study evolutionary conservation of bacterial phosphoproteins and phosphorylation sites across the phylogenetic tree. We demonstrate that bacterial phosphoproteins and phosphorylated residues are significantly more conserved than their nonphosphorylated counterparts, with a number of potential phosphorylation sites conserved from Archaea to humans. Our results establish Ser/Thr/Tyr phosphorylation as a common posttranslational modification in Eubacteria, present since the onset of cellular life.

The Ser/Thr/Tyr phosphoproteome of Lactococcus lactis IL1403 reveals multiply phosphorylated proteins

Recent phosphoproteomics studies of several bacterial species have firmly established protein phosphorylation on Ser/Thr/Tyr residues as a PTM in bacteria. In particular, our recent reports on the Ser/Thr/Tyr phosphoproteomes of bacterial model organisms Bacillus subtilis and Escherichia coli detected over 100 phosphorylation events in each of the bacterial species. Here we extend our analyses to Lactococcus lactis, a lactic acid bacterium widely employed by the food industry, in which protein phosphorylation at Ser/Thr/Tyr residues was barely studied at all. Despite the lack of almost any prior evidence of Ser/Thr/Tyr protein phosphorylation in L. lactis, we identified a phosphoproteome of a size comparable to that of E. coli and B. subtilis, with 73 phosphorylation sites distributed over 63 different proteins. The presence of several multiply phosphorylated proteins, as well as over‐representation of phosphothreonines seems to be the distinguishing features of the L. lactis phosphoproteome. Evolutionary comparison and the conservation of phosphorylation sites in different bacterial organisms indicate that a majority of the detected phosphorylation sites are species–specific, and therefore have probably co‐evolved with the adaptation of the bacterial species to their present‐day ecological niches.

Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) Applied to Quantitative Proteomics of Bacillus subtilis

We applied stable isotope labeling by amino acids in cell culture (SILAC) to large-scale quantitative proteomics analyses of the model bacterium Bacillus subtilis in two physiological conditions: growth on succinate and growth under phosphate starvation. Using a B. subtilis strain auxotrophic for lysine and high accuracy mass spectrometry for downstream analysis, we identified and quantified changes in the levels of more than 1500 proteins in each of the tested conditions with high biological and technical reproducibility. With a total of 1928 identified proteins, this study presents one of the most comprehensive quantitative proteomics studies in bacteria, covering more than 75% of the B. subtilis genes expressed in the log phase of growth. Furthermore, we detect and quantify dynamics of 35 Ser/Thr/Tyr phosphorylation sites under growth on succinate, and 10 phosphorylation sites under phosphate starvation, demonstrating the full compatibility of the method with site-specific detection and quantitation of phosphorylation events in bacteria.

Interplay of the Serine/Threonine-Kinase StkP and the Paralogs DivIVA and GpsB in Pneumococcal Cell Elongation and Division

Despite years of intensive research, much remains to be discovered to understand the regulatory networks coordinating bacterial cell growth and division. The mechanisms by which Streptococcus pneumoniae achieves its characteristic ellipsoid-cell shape remain largely unknown. In this study, we analyzed the interplay of the cell division paralogs DivIVA and GpsB with the ser/thr kinase StkP. We observed that the deletion of divIVA hindered cell elongation and resulted in cell shortening and rounding. By contrast, the absence of GpsB resulted in hampered cell division and triggered cell elongation. Remarkably, ΔgpsB elongated cells exhibited a helical FtsZ pattern instead of a Z-ring, accompanied by helical patterns for DivIVA and peptidoglycan synthesis. Strikingly, divIVA deletion suppressed the elongated phenotype of ΔgpsB cells. These data suggest that DivIVA promotes cell elongation and that GpsB counteracts it. Analysis of protein-protein interactions revealed that GpsB and DivIVA do not interact with FtsZ but with the cell division protein EzrA, which itself interacts with FtsZ. In addition, GpsB interacts directly with DivIVA. These results are consistent with DivIVA and GpsB acting as a molecular switch to orchestrate peripheral and septal PG synthesis and connecting them with the Z-ring via EzrA. The cellular co-localization of the transpeptidases PBP2x and PBP2b as well as the lipid-flippases FtsW and RodA in ΔgpsB cells further suggest the existence of a single large PG assembly complex. Finally, we show that GpsB is required for septal localization and kinase activity of StkP, and therefore for StkP-dependent phosphorylation of DivIVA. Altogether, we propose that the StkP/DivIVA/GpsB triad finely tunes the two modes of peptidoglycan (peripheral and septal) synthesis responsible for the pneumococcal ellipsoid cell shape.

Phosphoproteomics in bacteria: towards a systemic understanding of bacterial phosphorylation networks

Bacteria use protein phosphorylation to regulate all kinds of physiological processes. Protein phosphorylation plays a role in several key steps of the infection process of bacterial pathogens, such as adhesion to the host, triggering and regulation of pathogenic functions as well as biochemical warfare; scrambling the host signaling cascades and impairing its defense mechanisms. Recent phosphoproteomic studies indicate that the bacterial protein phosphorylation networks could be more complex than initially expected, comprising promiscuous kinases that regulate several distinct cellular functions by phosphorylating different protein substrates. Recent advances in protein labeling with stable isotopes in the field of quantitative mass spectrometry phosphoproteomics will enable us to chart the global phosphorylation networks and to understand the implication of protein phosphorylation in cellular regulation on the systems scale. For the study of bacterial pathogens, in particular, this research avenue will enable us to dissect phosphorylation-related events during different stages of infection and stimulate our efforts to find inhibitors for key kinases and phosphatases implicated therein.

Proteomics reveals evidence of cross-talk between protein modifications in bacteria: focus on acetylation and phosphorylation

Recent advances in gel-free, mass spectrometry-based proteomics have firmly established existence of serine phosphorylation, threonine phosphorylation, tyrosine phosphorylation and lysine acetylation on many bacterial proteins. Intriguingly, numerous proteins have been shown to be modified by both modifications, leading to the emerging concept of cross-talk between posttranslational modifications in bacteria. This concept is further supported by biological follow-up studies that are starting to reveal bacterial proteins and processes regulated by multiple modifications. In this review, we provide an overview of the large-scale studies involving protein phosphorylation and acetylation in bacteria and discuss some of the current examples of cross-talk between these and other bacterial modifications.

Bacillus subtilis BY‐kinase PtkA controls enzyme activity and localization of its protein substrates

Bacillus subtilis BY‐kinase PtkA was previously shown to phosphorylate, and thereby regulate the activity of two classes of protein substrates: UDP‐glucose dehydrogenases and single‐stranded DNA‐binding proteins. Our recent phosphoproteome study identified nine new tyrosine‐phosphorylated proteins in B. subtilis. We found that the majority of these proteins could be phosphorylated by PtkA in vitro. Among these new substrates, single‐stranded DNA exonuclease YorK, and aspartate semialdehyde dehydrogenase Asd were activated by PtkA‐dependent phosphorylation. Because enzyme activity was not affected in other cases, we used fluorescent protein tags to study the impact of PtkA on localization of these proteins in vivo. For several substrates colocalization with PtkA was observed, and more importantly, the localization pattern of the proteins enolase, YjoA, YnfE, YvyG, Ugd and SsbA was dramatically altered in ΔptkA background. Our results confirm that PtkA can control enzyme activity of its substrates in some cases, but also reveal a new mode of action for PtkA, namely ensuring correct cellular localization of its targets.

NetPhosBac – A predictor for Ser/Thr phosphorylation sites in bacterial proteins†

There is ample evidence for the involvement of protein phosphorylation on serine/threonine/tyrosine in bacterial signaling and regulation, but very few exact phosphorylation sites have been experimentally determined. Recently, gel‐free high accuracy MS studies reported over 150 phosphorylation sites in two bacterial model organisms Bacillus subtilis and Escherichia coli. Interestingly, the analysis of these phosphorylation sites revealed that most of them are not characteristic for eukaryotic‐type protein kinases, which explains the poor performance of eukaryotic data‐trained phosphorylation predictors on bacterial systems. We used these large bacterial datasets and neural network algorithms to create the first bacteria‐specific protein phosphorylation predictor: NetPhosBac. With respect to predicting bacterial phosphorylation sites, NetPhosBac significantly outperformed all benchmark predictors. Moreover, NetPhosBac predictions of phosphorylation sites in E. coli proteins were experimentally verified on protein and site‐specific levels. In conclusion, NetPhosBac clearly illustrates the advantage of taxa‐specific predictors and we hope it will provide a useful asset to the microbiological community.

Characterization of the E. coli proteome and its modifications during growth and ethanol stress

We set out to provide a resource to the microbiology community especially with respect to systems biology based endeavors. To this end, we generated a comprehensive dataset monitoring the changes in protein expression, copy number, and post translational modifications in a systematic fashion during growth and ethanol stress in E. coli. We utilized high-resolution mass spectrometry (MS) combined with the Super-SILAC approach. In a single experiment, we have identified over 2300 proteins, which represent approximately 88% of the estimated expressed proteome of E. coli and estimated protein copy numbers using the Intensity Based Absolute Quantitation (iBAQ). The dynamic range of protein expression spanned up to six orders of magnitude, with the highest protein copy per cell estimated at approximately 300,000. We focused on the proteome dynamics involved during stationary phase growth. A global up-regulation of proteins related to stress response was detected in later stages of growth. We observed the down-regulation of the methyl directed mismatch repair system containing MutS and MutL of E. coli growing in long term growth cultures, confirming that higher incidence of mutations presents an important mechanism in the increase in genetic diversity and stationary phase survival in E. coli. During ethanol stress, known markers such as alcohol dehydrogenase and aldehyde dehydrogenase were induced, further validating the dataset. Finally, we performed unbiased protein modification detection and revealed changes of many known and unknown protein modifications in both experimental conditions. Data are available via ProteomeXchange with identifier PXD001648.

Cell-selective labeling using amino acid precursors for proteomic studies of multicellular environments

We report a technique to selectively and continuously label the proteomes of individual cell types in coculture, named cell type–specific labeling using amino acid precursors (CTAP). Through transgenic expression of exogenous amino acid biosynthesis enzymes, vertebrate cells overcome their dependence on supplemented essential amino acids and can be selectively labeled through metabolic incorporation of amino acids produced from heavy isotope–labeled precursors. When testing CTAP in several human and mouse cell lines, we could differentially label the proteomes of distinct cell populations in coculture and determine the relative expression of proteins by quantitative mass spectrometry. In addition, using CTAP we identified the cell of origin of extracellular proteins secreted from cells in coculture. We believe that this method, which allows linking of proteins to their cell source, will be useful in studies of cell-cell communication and potentially for discovery of biomarkers.