Bacterial single-stranded DNA-binding proteins are phosphorylated on tyrosine

Mijaković, Ivan; Petranović, Dina; Maček, Boris; Čepo, Tina; Mann, Matthias; Davies, Julian; Jensen, Peter Ruhdal; Vujaklija, Dušica

doi:10.1093/nar/gkj514

Cited by 128 publications

(136 citation statements)

References 55 publications

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“…The N terminus of the purified proteins was sequenced by automatic Edman degradation. The corresponding molar extinction coefficient for RecA, SsbA, Ssb SPP1 , RecO, and RecR was calculated as 15,200,11,460,15,340,19,620, and 10,680 M Ϫ1 cm Ϫ1 , respectively, at 280 nm, as previously described (43). The protein concentrations were determined using the above molar extinction coefficients, and RecA and RecR are expressed as mole of protein monomers, Ssb SPP1 and RecO as dimers, and SsbA as tetramers.…”

Section: Methodsmentioning

confidence: 99%

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Bacillus subtilis RecO Nucleates RecA onto SsbA-coated Single-stranded DNA

Manfredi¹,

Carrasco

Ayora

et al. 2008

Journal of Biological Chemistry

113

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Subsaturating amounts of Bacillus subtilis SsbA, independently of the order of addition, partially inhibit the singlestranded DNA-dependent dATPase activity of RecA. This negative effect is fully overcome when a substoichiometric amount of RecO is added. SsbA added prior to RecA does not stimulate the dATP-dependent DNA strand exchange activity; however, added after RecA it enhances the extent of strand exchange. The addition of RecO stimulates RecA-mediated joint molecule formation, although it limits the accumulation of final recombination products. Thus we suggest that RecO has a dual activity: RecO acts as a RecA mediator enabling RecA to utilize SsbAcoated single-stranded DNA as a polymerization substrate and controls RecA-mediated DNA strand exchange by limiting its extent. We herein discuss the possible mechanisms of RecO involvement in the regulation of double strand break repair and genetic transformation.

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Section: Methodsmentioning

confidence: 99%

“…The SsbA protein becomes phosphorylated (19). The biochemical activities associated with SsbA in DNA repair are poorly understood, as well as the role of its phosphorylation.…”

mentioning

confidence: 99%

Bacillus subtilis RecO Nucleates RecA onto SsbA-coated Single-stranded DNA

Manfredi¹,

Carrasco

Ayora

et al. 2008

Journal of Biological Chemistry

113

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“…Interestingly, kinases with dual specificity have already been identified in plants (25), and multiple lines of evidence point to the presence of kinases with dual specificity in bacteria. Streptomyces griseus contains a single-stranded DNA binding protein that is tyrosine phosphorylated (21), but current methods of genome sequence annotation have not identified tyrosine kinases in the streptomycetes, suggesting the presence of unconventional kinase activity, perhaps catalyzed by STPKs, in these bacteria. Additionally, the DivL protein from Caulobacter crescentus is a predicted histidine kinase that displays unprecedented kinase activity by autophosphorylating on tyrosine (34).…”

Section: Vol 189 2007 Topology and Dual Specificity Of C Pneumoniamentioning

confidence: 99%

Chlamydophila pneumoniae PknD Exhibits Dual Amino Acid Specificity and Phosphorylates Cpn0712, a Putative Type III Secretion YscD Homolog

Johnson

Mahony

2007

J Bacteriol

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Chlamydophila pneumoniae is an obligate intracellular bacterium that causes bronchitis, pharyngitis, and pneumonia and may be involved in atherogenesis and Alzheimer's disease. Genome sequencing has identified three eukaryote-type serine/threonine protein kinases, Pkn1, Pkn5, and PknD, that may be important signaling molecules in Chlamydia. Full-length PknD was cloned and expressed as a histidine-tagged protein in Escherichia coli. Differential centrifugation followed by sodium carbonate treatment of E. coli membranes demonstrated that His-PknD is an integral membrane protein. Fusions of overlapping PknD fragments to alkaline phosphatase revealed that PknD contains a single transmembrane domain and that the kinase domain is in the cytoplasm. To facilitate solubility, the kinase domain was cloned and expressed as a glutathione Stransferase (GST) fusion protein in E. coli. Purified GST-PknD kinase domain autophosphorylated, and catalytic mutants (K33G, D156G, and K33G-D156G mutants) and activation loop mutants (T185A and T193A) were inactive. PknD phosphorylated recombinant Cpn0712, a type III secretion YscD homolog that has two forkhead-associated domains. Thin-layer chromatography revealed that the PknD kinase domain autophosphorylated on threonine and tyrosine and phosphorylated the FHA-2 domain of Cpn0712 on serine and tyrosine. To our knowledge, this is the first demonstration of a bacterial protein kinase with amino acid specificity for both serine/threonine and tyrosine residues and this is the first study to show phosphorylation of a predicted type III secretion structural protein.

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“…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), and it plays a role in transcriptional regulation via phosphorylation of the fatty aciddisplaced repressor FatR (14).…”

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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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Bacterial single-stranded DNA-binding proteins are phosphorylated on tyrosine

Cited by 128 publications

References 55 publications

Bacillus subtilis RecO Nucleates RecA onto SsbA-coated Single-stranded DNA

Bacillus subtilis RecO Nucleates RecA onto SsbA-coated Single-stranded DNA

Chlamydophila pneumoniae PknD Exhibits Dual Amino Acid Specificity and Phosphorylates Cpn0712, a Putative Type III Secretion YscD Homolog

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

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