An alternate route to phosphorylating DegU of Bacillus subtilis using acetyl phosphate

BMC Microbiology

Nicola R Stanley-Wall³  Keith Bromley⁴  Jessica E Martyn²  Lynne S Cairns¹ 

[1] Current address: Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston 02111, MA, USA;Current address: Sir William Dunn School of Pathology, South Parks Road Oxford, Oxford University, Oxford OX1 3RE, UK;Division of Molecular Microbiology, College of Life Sciences, University of Dundee, Dundee DD1 5EH, UK;James Clerk Maxwell Building, School of Physics, University of Edinburgh, Edinburgh EH9 3JZ, UK

关键词: Swarming;    Biofilm;    Acetyl phosphate;    DegU;    Bacillus subtilis;   

Others  :  1206340
DOI  :  10.1186/s12866-015-0410-z

 received in 2015-01-23, accepted in 2015-03-12,  发布年份 2015

【 摘 要 】

Background

Two-component signal transduction pathways allow bacteria to sense and respond to the environment. Typically such pathways comprise a sensor histidine kinase and a response regulator. Phosphorylation of the response regulator commonly results in its activation, allowing the protein to bind to target promoter elements to regulate transcription. Several mechanisms are used to prevent inappropriate phosphorylation of the response regulator, thereby ensuring a specific response. In Bacillus subtilis, the DegS-DegU two-component system controls transcription of target genes in a manner dependent on the level of the phosphorylated response regulator, DegU. Previous work has tentatively indicated that DegU, and DegU H¹²L, a DegU variant which displays enhanced stability of the phosphoryl moiety, can be phosphorylated in the absence of the kinase, DegS.

Results

The data presented here reveal that DegU H¹²L requires aspartic acid 56 (D⁵⁶), the identified DegU phosphorylation site, for its activity. By indirectly measuring the level of DegU ~ P in the cell by assessment of several well recognised DegU regulated processes it was shown that DegU H¹²L retains its activity in the absence of DegS, and that mutation of D⁵⁶ produced an inactive protein. Further experiments designed to raise the level of acetyl phosphate within the cell suggest that DegU can be phosphorylated by acetyl phosphate in the absence of degS. Additionally, the phenotypic and biochemical experiments presented indicate that DegU H¹²L can reliably mimic high levels of phosphorylated DegU.

Conclusions

The ability of acetyl phosphate to modify DegU, and indeed DegU H¹²L, reveal an additional layer of regulation for DegU phosphorylation that will be relevant when the level of DegS is low or in the absence of degS. Given the number of processes that DegU can activate or inhibit, extensive regulation at a number of levels is required to ensure that the system is not inappropriately stimulated. DegS has both kinase and phosphatase activity and our findings demonstrate that the phosphatase activity of DegS is essential to control the level of DegU phosphate. Overall we contribute to our understanding of how the intricate signalling pathway DegS-DegU is regulated in B. subtilis.

【 授权许可】

   
2015 Cairns et al.; licensee BioMed Central.

【 图 表 】

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【 参考文献 】

• [1]Callewaert L, Vanoirbeek KGA, Lurquin I, Michiels CW, Aertsen A. The Rcs Two-Component System Regulates Expression of Lysozyme Inhibitors and Is Induced by Exposure to Lysozyme. J Bacteriol. 2009; 191(6):1979-81.
• [2]Aguilar PS, Hernandez-Arriaga AM, Cybulski LE, Erazo AC, de Mendoza D. Molecular basis of thermosensing: a two-component signal transduction thermometer in Bacillus subtilis. EMBO J. 2001; 20(7):1681-91.
• [3]Igo MM, Ninfa AJ, Silhavy TJ. A bacterial environmental sensor that functions as a protein-kinase and stimulates transcriptional activation. Genes Dev. 1989; 3(5):598-605.
• [4]Georgellis D, Kwon O, Lin EC. Quinones as the redox signal for the arc two-component system of bacteria. Science. 2001; 292(5525):2314-6.
• [5]Neiditch MB, Federle MJ, Pompeani AJ, Kelly RC, Swem DL, Jeffrey PD et al.. Ligand-induced asymmetry in histidine sensor kinase complex regulates quorum sensing. Cell. 2006; 126(6):1095-108.
• [6]Galperin MY. Diversity of structure and function of response regulator output domains. Curr Opin Microbiol. 2010; 13(2):150-9.
• [7]Laub MT, Goulian M. Specificity in two-component signal transduction pathways. Annu Rev Genet. 2007; 41:121-45.
• [8]Bourret RB. Receiver domain structure and function in response regulator proteins. Curr Opin Microbiol. 2010; 13(2):142-9.
• [9]Gao R, Mack TR, Stock AM. Bacterial response regulators: versatile regulatory strategies from common domains. Trends Biochem Sci. 2007; 32(5):225-34.
• [10]Fabret C, Feher VA, Hoch JA. Two-component signal transduction in Bacillus subtilis: how one organism sees its world. J Bacteriol. 1999; 181(7):1975-83.
• [11]Msadek T, Kunst F, Henner D, Klier A, Rapoport G, Dedonder R. Signal transduction pathway controlling synthesis of a class of degradative enzymes in Bacillus subtilis: expression of the regulatory genes and analysis of mutations in degS and degU. J Bacteriol. 1990; 172(2):824-34.
• [12]Dahl MK, Msadek T, Kunst F, Rapoport G. Mutational analysis of the Bacillus subtilis DegU regulator and its phosphorylation by the DegS protein kinase. J Bacteriol. 1991; 173(8):2539-47.
• [13]Kunst F, Debarbouille M, Msadek T, Young M, Mauel C, Karamata D et al.. Deduced polypeptides encoded by the Bacillus subtilis sacU locus share homology with two-component sensor-regulator systems. J Bacteriol. 1988; 170(11):5093-101.
• [14]Do TH, Suzuki Y, Abe N, Kaneko J, Itoh Y, Kimura K. Mutations suppressing the loss of DegQ function in Bacillus subtilis (natto) poly-gamma-glutamate synthesis. Appl Environ Microbiol. 2011; 77(23):8249-58.
• [15]Tanaka T, Kawata M, Mukai K. Altered phosphorylation of Bacillus subtilis DegU caused by single amino acid changes in DegS. J Bacteriol. 1991; 173(17):5507-15.
• [16]Murray EJ, Kiley TB, Stanley-Wall NR. A pivotal role for the response regulator DegU in controlling multicellular behaviour. Microbiology. 2009; 155(Pt 1):1-8.
• [17]Cairns LS, Marlow VL, Bissett E, Ostrowski A, Stanley-Wall NR. A mechanical signal transmitted by the flagellum controls signalling in Bacillus subtilis. Mol Microbiol. 2013; 90(1):6-21.
• [18]Verhamme DT, Kiley TB, Stanley-Wall NR. DegU co-ordinates multicellular behaviour exhibited by Bacillus subtilis. Mol Microbiol. 2007; 65(2):554-68.
• [19]Kobayashi K. Gradual activation of the response regulator DegU controls serial expression of genes for flagellum formation and biofilm formation in Bacillus subtilis. Mol Microbiol. 2007; 66(2):395-409.
• [20]Hamoen LW, Van Werkhoven AF, Venema G, Dubnau D. The pleiotropic response regulator DegU functions as a priming protein in competence development in Bacillus subtilis. Proc Natl Acad Sci U S A. 2000; 97(16):9246-51.
• [21]Stanley NR, Lazazzera BA. Defining the genetic differences between wild and domestic strains of Bacillus subtilis that affect poly-gamma-dl-glutamic acid production and biofilm formation. Mol Microbiol. 2005; 57(4):1143-58.
• [22]Amati G, Bisicchia P, Galizzi A. DegU-P represses expression of the motility fla-che operon in Bacillus subtilis. J Bacteriol. 2004; 186(18):6003-14.
• [23]Marlow VL, Porter M, Hobley L, Kiley TB, Swedlow JR, Davidson FA et al.. Phosphorylated DegU manipulates cell fate differentiation in the Bacillus subtilis biofilm. J Bacteriol. 2014; 196(1):16-27.
• [24]Mordini S, Osera C, Marini S, Scavone F, Bellazzi R, Galizzi A et al.. The role of SwrA, DegU and P(D3) in fla/che expression in B. subtilis. PLoS One. 2013; 8(12):e85065.
• [25]Koshland DE. Effect of Catalysts on the Hydrolysis of Acetyl Phosphate - Nucleophilic Displacement Mechanisms in Enzymatic Reactions. J Am Chem Soc. 1952; 74(9):2286-92.
• [26]Dahl MK, Msadek T, Kunst F, Rapoport G. The phosphorylation state of the DegU response regulator acts as a molecular switch allowing either degradative enzyme synthesis or expression of genetic competence in Bacillus subtilis. J Biol Chem. 1992; 267(20):14509-14.
• [27]Henner DJ, Yang M, Ferrari E. Localization of Bacillus subtilis sacU(Hy) mutations to two linked genes with similarities to the conserved procaryotic family of two-component signalling systems. J Bacteriol. 1988; 170(11):5102-9.
• [28]Branda SS, Gonzalez-Pastor JE, Ben-Yehuda S, Losick R, Kolter R. Fruiting body formation by Bacillus subtilis. Proc Natl Acad Sci U S A. 2001; 98(20):11621-6.
• [29]Quisel JD, Burkholder WF, Grossman AD. In vivo effects of sporulation kinases on mutant Spo0A proteins in Bacillus subtilis. J Bacteriol. 2001; 183(22):6573-8.
• [30]Kunst F, Pascal M, Lepesant-Kejzlarova J, Lepesant JA, Billault A, Dedonder R. Pleiotropic mutations affecting sporulation conditions and the syntheses of extracellular enzymes in Bacillus subtilis 168. Biochimie. 1974; 56(11–12):1481-9.
• [31]Ostrowski A, Merhert A, Prescott A, Kiley TB, Stanley-Wall NR. YuaB functions synergistically with the exopolysaccharide and TasA amyloid fibers to allow biofilm formation by Bacillus subtilis. J Bacteriol. 2011; 193(18):4821-31.
• [32]Branda SS, Chu F, Kearns DB, Losick R, Kolter R. A major protein component of the Bacillus subtilis biofilm matrix. Mol Microbiol. 2006; 59(4):1229-38.
• [33]Srivatsan A, Han Y, Peng J, Tehranchi AK, Gibbs R, Wang JD et al.. High-precision, whole-genome sequencing of laboratory strains facilitates genetic studies. PLoS Genet. 2008; 4(8):e1000139.
• [34]Earl AM, Eppinger M, Fricke WF, Rosovitz MJ, Rasko DA, Daugherty S et al.. Whole-genome sequences of Bacillus subtilis and close relatives. J Bacteriol. 2012; 194(9):2378-9.
• [35]Steinmetz M, Kunst F, Dedonder R. Mapping of mutations affecting synthesis of exocellular enzymes in Bacillus subtilis. Identity of the sacUh, amyB and pap mutations. Mol Gen Genet. 1976; 148(3):281-5.
• [36]Mukai K, Kawata-Mukai M, Tanaka T. Stabilization of phosphorylated Bacillus subtilis DegU by DegR. J Bacteriol. 1992; 174(24):7954-62.
• [37]Kobayashi K, Iwano M. BslA (YuaB) forms a hydrophobic layer on the surface of Bacillus subtilis biofilms. Mol Microbiol. 2012; 85(1):51-66.
• [38]Hobley L, Ostrowski A, Rao FV, Bromley KM, Porter M, Prescott AR et al.. BslA is a self-assembling bacterial hydrophobin that coats the Bacillus subtilis biofilm. Proc Natl Acad Sci U S A. 2013; 110(33):13600-5.
• [39]Cairns LS, Hobley L, Stanley-Wall NR. Biofilm formation by Bacillus subtilis: new insights into regulatory strategies and assembly mechanisms. Mol Microbiol. 2014; 93(4):587-98.
• [40]Verhamme DT, Murray EJ, Stanley-Wall NR. DegU and Spo0A jointly control transcription of two loci required for complex colony development by Bacillus subtilis. J Bacteriol. 2009; 191(1):100-8.
• [41]Wolfe AJ. Physiologically relevant small phosphodonors link metabolism to signal transduction. Curr Opin Microbiol. 2010; 13(2):204-9.
• [42]Feng J, Atkinson MR, McCleary W, Stock JB, Wanner BL, Ninfa AJ. Role of phosphorylated metabolic intermediates in the regulation of glutamine synthetase synthesis in Escherichia coli. J Bacteriol. 1992; 174(19):6061-70.
• [43]Xu H, Caimano MJ, Lin T, He M, Radolf JD, Norris SJ et al.. Role of acetyl-phosphate in activation of the Rrp2-RpoN-RpoS pathway in Borrelia burgdorferi. PLoS Pathog. 2010; 6(9):e1001104.
• [44]Pruss BM, Wolfe AJ. Regulation of acetyl phosphate synthesis and degradation, and the control of flagellar expression in Escherichia coli. Mol Microbiol. 1994; 12(6):973-84.
• [45]Gueriri I, Bay S, Dubrac S, Cyncynatus C, Msadek T. The Pta-AckA pathway controlling acetyl phosphate levels and the phosphorylation state of the DegU orphan response regulator both play a role in regulating Listeria monocytogenes motility and chemotaxis. Mol Microbiol. 2008; 70(6):1342-57.
• [46]Gueriri I, Cyncynatus C, Dubrac S, Arana AT, Dussurget O, Msadek T. The DegU orphan response regulator of Listeria monocytogenes autorepresses its own synthesis and is required for bacterial motility, virulence and biofilm formation. Microbiology. 2008; 154(Pt 8):2251-64.
• [47]Wolfe AJ. The acetate switch. Microbiol Mol Biol Rev. 2005; 69(1):12-50.
• [48]Schrecke K, Jordan S, Mascher T. Stoichiometry and perturbation studies of the LiaFSR system of Bacillus subtilis. Mol Microbiol. 2013; 87(4):769-88.
• [49]Lukat GS, McCleary WR, Stock AM, Stock JB. Phosphorylation of bacterial response regulator proteins by low molecular weight phospho-donors. Proc Natl Acad Sci U S A. 1992; 89(2):718-22.
• [50]Perego M, Hoch JA. Sequence analysis and regulation of the hpr locus, a regulatory gene for protease production and sporulation in Bacillus subtilis. J Bacteriol. 1988; 170(6):2560-7.