Analysis of the dynamic <b><i>Bacillus subtilis</i></b> Ser/Thr/Tyr phosphoproteome implicated in a wide variety of cellular processes

Levine, Alain; Vannier, Françoise; Absalon, Cédric; Kuhn, Lauriane; Jackson, Peter; Scrivener, Elaine; Labas, Valérie; Vinh, Joëlle; Courtney, Patrick; Garin, Jérôme; Séror, Simone J.

doi:10.1002/pmic.200500352

Cited by 85 publications

(92 citation statements)

References 52 publications

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“…Of particular interest, proteins involved in phosphate metabolism exhibited strong differential phosphorylation. Phosphate binding ABC transporter protein PstS has previously been identified in the phosphoproteome of B. subtilis (22), and is known to be strongly induced by phosphate starvation (45). Our data set indicates that PstS is very strongly dephosphorylated in early and late stationary phase (where phosphate depletion is expected), suggesting that the dephosphorylated state may be more active in phosphate import.…”

Section: Discussionmentioning

confidence: 99%

“…Comprehensive phosphoproteomics analysis has been conducted in various bacterial systems such as E. coli (18), Latococcus lactis (19), Pseudomonas species (20), Mycobacterium tuberculosis (21), to name a few. Dynamics of Ser/Thr/Tyr cellular phosphorylation events have been further investigated in the Gram-positive model organism Bacillus subtilis by employing 2D-gel electrophoresis mass spectrometry (22,23) or a global, gel-free, site-specific quantitative analysis (24,25). Recently a large transcriptomic, proteomic and metabolomic study was conducted in B. subtilis to gain an understanding of the molecular changes occurring on glucose starvation (26).…”

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confidence: 99%

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Quantitative Phosphoproteome Analysis of Bacillus subtilis Reveals Novel Substrates of the Kinase PrkC and Phosphatase PrpC

Ravikumar¹,

Shi²,

Krug³

et al. 2014

Molecular & Cellular Proteomics

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Reversible protein phosphorylation on serine, threonine, and tyrosine (Ser/Thr/Tyr) residues plays a critical role in regulation of vital processes in the cell. Despite of considerable progress in our understanding of the role of this modification in bacterial physiology, the dynamics of protein phosphorylation during bacterial growth has rarely been systematically addressed. In addition, little is known about in vivo substrates of bacterial Ser/Thr/Tyr kinases and phosphatases. An excellent candidate to study these questions is the Gram-positive bacterium Bacillus subtilis, one of the most intensively investigated bacterial model organism with both research and industrial applications. Here we employed gel-free phosphoproteomics combined with SILAC labeling and high resolution mass spectrometry to study the proteome and phosphoproteome dynamics during the batch growth of B. subtilis. We measured the dynamics of 1666 proteins and 64 phosphorylation sites in five distinct phases of growth. Enzymes of the central carbon metabolism and components of the translation machinery appear to be highly phosphorylated in the stationary phase, coinciding with stronger expression of Ser/Thr kinases. We further used the SILAC workflow to identify novel putative substrates of the Ser/Thr kinase PrkC and the phosphatase PrpC during stationary phase. The overall number of putative substrates was low, pointing to a high kinase and phosphatase specificity. One of the phosphorylation sites affected by both, PrkC and PrpC, was the Ser281 on the oxidoreductase YkwC. We showed that PrkC phosphorylates and PrpC dephosphorylates YkwC in vitro and that phosphorylation at Protein phosphorylation on serine, threonine and tyrosine (Ser/Thr/Tyr) is rapidly becoming a prominent avenue of research in microbiology. Hanks-type Ser/Thr kinases and BYkinases (bacterial Tyr kinases) were shown to have implications in vital processes such as pathogenicity (1, 2), DNA repair, heat shock response (3), cell morphology, and separation (4). In certain pathogenic species like Salmonella, Listeria and Shigella they play a vital role in virulence (5). Functions of Ser/Thr/Tyr phosphorylation have been extensively studied in Bacillus subtilis, a Gram-positive model bacterium widely used in basic research and industrial applications. It was shown that B. subtilis Ser/Thr kinases are involved in regulation of catabolic repression via phosphorylation of the CcpA co-repressor HPr (6). They are also involved in spore development via phosphorylation of a recombinase RecA (7), in spore germination (8), and in regulation of the general stress sigma factor SigB via phosphorylation of . Importantly, Ser/Thr kinases can also regulate complementary signal transduction systems as shown by phosphorylation of the two-component kinase DegS (10). In addition, B. subtilis tyrosine kinase PtkA plays an important role in DNA replication by phosphorylating SSB proteins (11,12). It is involved in exopolysaccharide synthesis via phosphorylation of UDP-glucose dehydrogenases (13), a...

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Quantitative Phosphoproteome Analysis of Bacillus subtilis Reveals Novel Substrates of the Kinase PrkC and Phosphatase PrpC

Ravikumar¹,

Shi²,

Krug³

et al. 2014

Molecular & Cellular Proteomics

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“…This exceeds the percentage of phosphorylated proteins in other bacteria by far. For example, only 2.5% of all proteins of B. subtilis are known to be phosphorylated on a serine, threonine, or tyrosine residue (9,10,12). This raises the question whether protein phosphorylation is more common in M. pneumoniae than in other bacteria.…”

Section: Discussionmentioning

confidence: 99%

The Phosphoproteome of the Minimal Bacterium Mycoplasma pneumoniae

Schmidl

Gronau

Pietack

et al. 2010

Molecular & Cellular Proteomics

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Mycoplasma pneumoniae belongs to the Mollicutes, the group of organisms with the smallest genomes that are capable of host-independent life. These bacteria show little regulation in gene expression, suggesting an important role for the control of protein activities. We have studied protein phosphorylation in M. pneumoniae to identify phosphorylated proteins. Two-dimensional gel electrophoresis and mass spectrometry allowed the detection of 63 phosphorylated proteins, many of them enzymes of central carbon metabolism and proteins related to host cell adhesion. We identified 16 phosphorylation sites, among them 8 serine and 8 threonine residues, respectively. A phosphoproteome analysis with mutants affected in the two annotated protein kinase genes or in the single known protein phosphatase gene suggested that only one protein (HPr) is phosphorylated by the HPr kinase, HPrK, whereas four adhesion-related or surface proteins were targets of the protein kinase C, PrkC. A comparison with the phosphoproteomes of other bacteria revealed that protein phosphorylation is evolutionarily only poorly conserved. Only one single protein with an identified phosphorylation site, a phosphosugar mutase (ManB in M. pneumoniae), is phosphorylated on a conserved serine residue in all studied organisms from archaea and bacteria to man. We demonstrate that this protein undergoes autophosphorylation. This explains the strong conservation of this phosphorylation event. For most other proteins, even if they are phosphorylated in different species, the actual phosphorylation sites are different. This suggests that protein phosphorylation is a form of adaptation of the bacteria to the specific needs of their particular ecological niche. Molecular & Cellular Proteomics 9:1228 -1242, 2010.Bacteria of the group called Mollicutes are unique among all living organisms because of their small genome that nevertheless allows them to grow independently from any host cell.The two most intensively studied representatives of the Mollicutes are Mycoplasma genitalium and Mycoplasma pneumoniae with genome sizes of 580 and 816 kb, respectively. The approximately 475 protein-coding and 43 RNA-coding genes of M. genitalium define the lower limit of the genetic equipment that permits independent life. The small genomes of the Mollicutes reflect their adaptation to rarely changing ecosystems, such as lung epithelia for M. pneumoniae (1). Furthermore, M. pneumoniae has only limited metabolic capabilities: this bacterium can utilize only a few carbon sources (glucose, fructose, and glycerol) (2), and its only way to produce ATP is by substrate level phosphorylation in glycolysis. The citric acid cycle, respiration, and most anabolic reactions are not carried out by M. pneumoniae (3).M. pneumoniae is a human pathogen that causes usually mild infections such as atypical pneumonia; however, the infections may be severe in children and elderly people. In addition, M. pneumoniae is involved in extrapulmonary complications, such as erythema multiforme and pediatric en...

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“…The essential translational GTPase elongation factor EF-G is phosphorylated in this PrkC-dependent germination pathway (160). PrkC-dependent phosphorylation of EF-G on a Thr residue(s) in vivo has also been described for vegetative B. subtilis cells (45,50,91,98). In addition to the phylogenetically conserved phosphorylation of EF-G identified in the phosphoproteomes of Corynebacterium glutamicum (17), E. coli (97), and Mycoplasma pneumoniae (156), other proteins involved in translation have been implicated as targets of eSTKs.…”

Section: Estks In Bacteriamentioning

confidence: 99%

Eukaryote-Like Serine/Threonine Kinases and Phosphatases in Bacteria

Pereira

Goss

Dworkin

2011

Microbiol Mol Biol Rev

327

348

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SUMMARY Genomic studies have revealed the presence of Ser/Thr kinases and phosphatases in many bacterial species, although their physiological roles have largely been unclear. Here we review bacterial Ser/Thr kinases (eSTKs) that show homology in their catalytic domains to eukaryotic Ser/Thr kinases and their partner phosphatases (eSTPs) that are homologous to eukaryotic phosphatases. We first discuss insights into the enzymatic mechanism of eSTK activation derived from structural studies on both the ligand-binding and catalytic domains. We then turn our attention to the identified substrates of eSTKs and eSTPs for a number of species and to the implications of these findings for understanding their physiological roles in these organisms.

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Analysis of the dynamic Bacillus subtilis Ser/Thr/Tyr phosphoproteome implicated in a wide variety of cellular processes

Cited by 85 publications

References 52 publications

Quantitative Phosphoproteome Analysis of Bacillus subtilis Reveals Novel Substrates of the Kinase PrkC and Phosphatase PrpC

Quantitative Phosphoproteome Analysis of Bacillus subtilis Reveals Novel Substrates of the Kinase PrkC and Phosphatase PrpC

The Phosphoproteome of the Minimal Bacterium Mycoplasma pneumoniae

Eukaryote-Like Serine/Threonine Kinases and Phosphatases in Bacteria

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