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 Ser281 abolishes the oxidoreductase activity of YkwC in vitro and in vivo. Our results present the most detailed phosphoproteomic analysis of B. subtilis growth to date and provide the first global in vivo screen of PrkC and PrpC substrates.
Highlights
From the ‡Proteome Center Tuebingen, Interfaculty Institute for Cell Biology, University of Tuebingen, Germany; §Micalis UMR 1319, AgroParisTech/Institut National de la Recherche Agronomique, Jouy en Josas, France; ¶Systems and Synthetic Biology, Department of Chemical and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden
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
The main objective of this study was to analyze the global dynamics of proteins and phosphorylation events as the B. subtilis culture undergoes different stages of growth
Summary
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 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 Rsb-proteins [9]. 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], and it plays a role in transcriptional regulation via phosphorylation of the fatty aciddisplaced repressor FatR [14]. An in vivo study of PrkC and PrpC substrates has not been reported so far
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