Bacillus subtilis Two-Component System Sensory Kinase DegS Is Regulated by Serine Phosphorylation in Its Input Domain

Jers, Carsten; Kobir, Ahasanul; Søndergaard, Elsebeth Oline; Jensen, Peter Ruhdal; Mijaković, Ivan

doi:10.1371/journal.pone.0014653

Cited by 47 publications

(48 citation statements)

References 45 publications

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“…The DegS domain (residues 12 to 170), with an unknown function, is another candidate site regulating the interaction. Although the physiological significance is obscure, Ser76 in this domain is phosphorylated in vivo, and YbdM was proposed previously to be a kinase responsible for Ser76 phosphorylation (11). The expression of the pgs operon is regulated not only by cell density but also by nutritional signals from the environment (13).…”

Section: Discussionmentioning

confidence: 99%

Mutations Suppressing the Loss of DegQ Function in Bacillus subtilis (natto) Poly-γ-Glutamate Synthesis

Huyen

Suzuki

Abe

et al. 2011

Appl Environ Microbiol

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The degQ gene of Bacillus subtilis (natto), encoding a small peptide of 46 amino acids, is essential for the synthesis of extracellular poly-gamma-glutamate (␥PGA). To elucidate the role of DegQ in ␥PGA synthesis, we knocked out the degQ gene in Bacillus subtilis (natto) and screened for suppressor mutations that restored ␥PGA synthesis in the absence of DegQ. Suppressor mutations were found in degS, the receptor kinase gene of the DegS-DegU two-component system. Recombinant DegS-His 6 mutant proteins were expressed in Escherichia coli cells and subjected to an in vitro phosphorylation assay. Compared with the wild type, mutant DegS-His 6 proteins showed higher levels of autophosphorylation (R208Q, M195I, L248F, and D250N), reduced autodephosphorylation (D250N), reduced phosphatase activity toward DegU, or a reduced ability to stimulate the autodephosphorylation activity of DegU (R208Q, D249G, M195I, L248F, and D250N) and stabilized DegU in the phosphorylated form. These mutant DegS proteins mimic the effect of DegQ on wild-type DegSU in vitro. Interestingly, DegQ stabilizes phosphorylated DegS only in the presence of DegU, indicating a complex interaction of these three proteins.Extracellular poly-gamma-glutamate (␥PGA) is a glutamate polymer linked through a gamma-peptide bond produced outside the cells by bacilli, including Bacillus subtilis, B. licheniformis, and B. anthracis, and Staphylococcus epidermidis (4). It acts as a nutrient reservoir to prevent starvation in the stationary phase and as a barrier against bacteriophage or phagocytotic attack by the host immune system (4, 12, 14, 16). Microbial functions that are not essential for the life cycle under laboratory conditions but that are required for survival in the natural environment are sometimes lost in domesticated laboratory strains. The loss of the ability to synthesize ␥PGA in a laboratory strain of B. subtilis is an example of such an event, and a mutation in degQ is involved in it (30).degQ function was characterized mainly through studies of the ␥PGA-negative laboratory lineage B. subtilis 168. degQ encodes a small peptide of 46 amino acids, and it is a pleiotropic regulator of degradation enzymes, including alkaline protease, ␣-amylase, and levansucrase (1). The expression of degQ is dependent on comA, the global transcriptional regulator in the stationary phase, and is strongly induced under dense-cell conditions (18). The regulation of these exoenzymes by degQ requires the presence of the degS-degU operon that comprises a two-component system (17, 18). The sensor histidine kinase DegS has four activities: it acts as a self-kinase (autophosphorylation of His189), a self-phosphatase (autodephosphorylation), a phosphotransferase (phosphorylation of Asp56 of DegU), and a phosphatase (dephosphorylation of DegU) (5,6,15,20,31). Exoenzyme expression is controlled directly by DegU, a DNA-binding protein, and DegU phosphorylation by DegS (DegU-P i ) enhances it (25,29,34). A recent study reported that DegQ stimulates phosphate transfer from phospho...

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Section: Discussionmentioning

confidence: 99%

Mutations Suppressing the Loss of DegQ Function in Bacillus subtilis (natto) Poly-γ-Glutamate Synthesis

Huyen

Suzuki

Abe

et al. 2011

Appl Environ Microbiol

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“…Interestingly, DegS was found to be phosphorylated on residue Ser76, situated in its signal-sensing domain (Macek et al, 2007). Jers et al (2011) demonstrated that DegS Ser76 can be phosphorylated by two B. subtilis STKs: PrkD (YbdM) and YabT (Fig. 3a).…”

Section: Phosphorylation Of Tcs Hks and Rrs By Stksmentioning

confidence: 94%

Serine/threonine/tyrosine phosphorylation regulates DNA binding of bacterial transcriptional regulators

et al. 2015

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Reversible phosphorylation of bacterial transcriptional regulators (TRs) belonging to the family of two-component systems (TCSs) is a well-established mechanism for regulating gene expression. Recent evidence points to the fact that reversible phosphorylation of bacterial TRs on other types of residue, i.e. serine, threonine, tyrosine and cysteine, is also quite common. The phosphorylation of the ester type (phospho-serine/threonine/tyrosine) is more stable than the aspartate phosphorylation of TCSs. The kinases which catalyse these phosphorylation events (Hanks-type serine/threonine protein kinases and bacterial protein tyrosine kinases) are also much more promiscuous than the TCS kinases, i.e. each of them can phosphorylate several substrate proteins. As a consequence, the dynamics and topology of the signal transduction networks depending on these kinases differ significantly from the TCSs. Here, we present an overview of different classes of bacterial TR phosphorylated and regulated by serine/threonine and tyrosine kinases. Particular attention is given to examples when serine/threonine and tyrosine kinases interact with TCSs, phosphorylating either the histidine kinases or the response regulators. We argue that these promiscuous kinases connect several signal transduction pathways and serve the role of signal integration. Bacterial two-component systems (TCSs)TCSs are signal transduction devices that were initially discovered in bacteria (Ninfa & Magasani, 1986;Nixon et al., 1986). They play an important role in signal sensing and response to various stimuli, enabling the organisms to adapt to environmental changes. A typical TCS consists of a histidine kinase (HK) and a corresponding response regulator (RR) (Stock et al., 2000;Gao & Stock, 2009). HK usually possesses a highly variable sensor domain and a conserved kinase core. Following environmental stimulus, a signal ligand binds to the sensor domain and results in the autophosphorylation of the kinase core at a conserved histidine residue, at the expense of ATP. Next, the phosphoryl group is transferred from HK to a conserved aspartate in the regulatory domain of the RR. RRs usually contain two domains: a regulatory domain with the conserved phosphorylatable aspartate and a variable effector domain. Phosphorylation activates the effector domain of RRs, triggering the physiological response. As phosphohistidine has a very short half-life in aqueous solutions at neutral pH, in the absence of the environmental signal the system switches itself off very rapidly.Many effector domains of bacterial RRs have DNA-binding capacity. This allows RRs to function as transcriptional regulators (TRs) and consequently change gene transcription when they become phosphorylated. In Escherichia coli, osmoregulation of porin proteins OmpF and OmpC is under transcriptional control of the TCS EnvZ/OmpR. The phosphorylation of OmpR by EnvZ changes its affinity for the promoter region of ompF and ompC, resulting in different transcriptional levels of these genes (Forst et al., 1...

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“…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 Rsb-proteins (9). 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).…”

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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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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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Bacillus subtilis Two-Component System Sensory Kinase DegS Is Regulated by Serine Phosphorylation in Its Input Domain

Cited by 47 publications

References 45 publications

Mutations Suppressing the Loss of DegQ Function in Bacillus subtilis (natto) Poly-γ-Glutamate Synthesis

Mutations Suppressing the Loss of DegQ Function in Bacillus subtilis (natto) Poly-γ-Glutamate Synthesis

Serine/threonine/tyrosine phosphorylation regulates DNA binding of bacterial transcriptional regulators

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

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