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

Abstract

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...

Self-regulation of exopolysaccharide production in Bacillus subtilis by a tyrosine kinase

“…S2), a region known to exhibit phosphorylation in other bacterial tyrosine kinases ). Phosphorylation of Y227 was also reported in a recent phosphoproteomic study (Ravikumar et al 2014). As a further test of whether these tyrosines are the only or principal sites of phosphorylation in vivo, we purified a His 6 -EpsB variant in which the two Table 1.…”

Global analysis of bacterial membrane proteins and their modifications

“…This resulted in 503 hits out of a potential 898 total hits to be predicted as membrane proteins in the E. coli proteome, corresponding to nearly a 60% coverage of membrane proteins without any specialized enrichment. It is noteworthy to mention that other similar proteomics approaches previously performed could potentially show this same trend of membrane protein coverage without specialized membrane protein extraction protocols (Macek et al, 2007Ravikumar et al, 2014;Soares et al, 2013;Soufi et al, 2008Soufi et al, , 2010Wolff et al, 2007).…”

Bacterial Electron Transfer Chains Primed by Proteomics

“…However, it is largely unknown how those kinases and phosphatases are activated. The elucidation of these regulatory mechanisms and the identification of protein substrates of the kinases and phosphatases are topics of extensive research (Macek & Mijakovic, 2011;Mijakovic & Deutscher, 2015;Ravikumar et al, 2014). One of the outcomes of these research efforts is that different phosphorylation systems are able of crosstalk (Jers, Kobir, Søndergaard, Jensen, & Mijakovic, 2011;Shi et al, 2014;van Noort et al, 2012).…”