1887

Abstract

Highly branched dendritic swarming of on synthetic B-medium involves a developmental-like process that is absolutely dependent on flagella and surfactin secretion. In order to identify new swarming genes, we targeted the two-component ComPA signalling pathway and associated global regulators. In liquid cultures, the histidine kinase ComP, and the response regulator ComA, respond to secreted pheromones ComX and CSF (encoded by ) in order to control production of surfactin synthases and ComS (competence regulator). In this study, for what is believed to be the first time, we established that distinct early stages of dendritic swarming can be clearly defined, and that they are amenable to genetic analysis. In a mutational analysis producing several mutants with distinctive phenotypes, we were able to assign the genes (activation of surfactin synthases), and (global regulators), (flagellin), and (-like), (motility), to the different swarming stages. Surprisingly, mutations in genes , and , which are normally indispensable for import of CSF, had only modest effects, if any, on swarming and surfactin production. Therefore, during dendritic swarming, surfactin synthesis is apparently subject to novel regulation that is largely independent of the ComXP pathway; we discuss possible alternative mechanisms for driving transcription. We showed that the mutant, largely independent of any effect on surfactin production, was also, nevertheless, blocked early in swarming, forming stunted dendrites, with abnormal dendrite initiation morphology. In a mixed swarm co-inoculated with and (GFP), an apparently normal swarm was produced. In fact, while initiation of all dendrites was of the abnormal type, these were predominantly populated by cells, which migrated faster than the cells. This and other results indicated a specific migration defect in the mutant that could not be -complemented by CSF in a mixed swarm. CSF is the C-terminal pentapeptide of the surface-exposed PhrC pre-peptide and we propose that the residual PhrC 35 aa residue peptide anchored in the exterior of the cytoplasmic membrane has an apparently novel extracellular role in swarming.

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2009-02-01
2019-10-20
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References

  1. Aguilar, C., Vlamakis, H., Losick, R. & Kolter, R. ( 2007; ). Thinking about Bacillus subtilis as a multicellular organism. Curr Opin Microbiol 10, 638–643.[CrossRef]
    [Google Scholar]
  2. Amati, G., Bisicchia, P. & Galizzi, A. ( 2004; ). DegU-P represses expression of the motility fla-che operon in Bacillus subtilis. J Bacteriol 186, 6003–6014.[CrossRef]
    [Google Scholar]
  3. Antelmann, H., Engelmann, S., Schmid, R., Sorokin, A., Lapidus, A. & Hecker, M. ( 1997; ). Expression of a stress- and starvation-induced dps/pexB-homologous gene is controlled by the alternative sigma factor sigmaB in Bacillus subtilis. J Bacteriol 179, 7251–7256.
    [Google Scholar]
  4. Bergara, F., Ibarra, C., Iwamasa, J., Patarroyo, J. C., Aguilera, R. & Marquez-Magana, L. M. ( 2003; ). CodY is a nutritional repressor of flagellar gene expression in Bacillus subtilis. J Bacteriol 185, 3118–3126.[CrossRef]
    [Google Scholar]
  5. Berka, R. M., Hahn, J., Albano, M., Draskovic, I., Persuh, M., Cui, X., Sloma, A., Widner, W. & Dubnau, D. ( 2002; ). Microarray analysis of the Bacillus subtilis K-state: genome-wide expression changes dependent on ComK. Mol Microbiol 43, 1331–1345.[CrossRef]
    [Google Scholar]
  6. Branda, S. S., Vik, S., Friedman, L. & Kolter, R. ( 2005; ). Biofilms: the matrix revisited. Trends Microbiol 13, 20–26.[CrossRef]
    [Google Scholar]
  7. Calvio, C., Celandroni, F., Ghelardi, E., Amati, G., Salvetti, S., Cecilliani, F., Galizzi, A. & Sensi, S. ( 2005; ). Swarming differentiation and swimming motility in Bacillus subtilis are controlled by swrA, a newly identified dicistronic operon. J Bacteriol 187, 5356–5366.[CrossRef]
    [Google Scholar]
  8. Comella, N. & Grossman, A. D. ( 2005; ). Conservation of genes and processes controlled by the quorum response in bacteria: characterization of genes controlled by the quorum-sensing transcription factor ComA in Bacillus subtilis. Mol Microbiol 57, 1159–1174.[CrossRef]
    [Google Scholar]
  9. Connelly, M. B., Young, G. M. & Sloma, A. ( 2004; ). Extracellular proteolytic activity plays a central role in swarming motility in Bacillus subtilis. J Bacteriol 186, 4159–4167.[CrossRef]
    [Google Scholar]
  10. Core, L. & Perego, M. ( 2003; ). TPR-mediated interaction of RapC with ComA inhibits response regulator–DNA binding for competence development in Bacillus subtilis. Mol Microbiol 49, 1509–1522.[CrossRef]
    [Google Scholar]
  11. Cosby, W. M. & Zuber, P. ( 1997; ). Regulation of Bacillus subtilis sigmaH (spo0H) and AbrB in response to changes in external pH. J Bacteriol 179, 6778–6787.
    [Google Scholar]
  12. Cosby, W. M., Vollenbroich, D., Lee, O. H. & Zuber, P. ( 1998; ). Altered srf expression in Bacillus subtilis resulting from changes in culture pH is dependent on the Spo0K oligopeptide permease and the ComQX system of extracellular control. J Bacteriol 180, 1438–1445.
    [Google Scholar]
  13. Debois, D., Hamze, K., Guérineau, V., Le Caër, J. P., Holland, I. B., Lopes, P., Ouazzani, J., Séror, S. J., Brunelle, A. & Laprévote, O. ( 2008; ). In situ localization and quantification of surfactins in a Bacillus subtilis swarming community by imaging mass spectrometry. Proteomics 8, 3682–3691.[CrossRef]
    [Google Scholar]
  14. Dixit, M., Murudkar, C. S. & Rao, K. K. ( 2002; ). epr is transcribed from a final sigma(D) promoter and is involved in swarming of Bacillus subtilis. J Bacteriol 184, 596–599.[CrossRef]
    [Google Scholar]
  15. D'Souza, C., Nakano, M. M., Frisby, D. L. & Zuber, P. ( 1995; ). Translation of the open reading frame encoded by comS, a gene of the srf operon, is necessary for the development of genetic competence, but not surfactin biosynthesis, in Bacillus subtilis. J Bacteriol 177, 4144–4148.
    [Google Scholar]
  16. Hahn, J. & Dubnau, D. ( 1991; ). Growth stage signal transduction and the requirements for srfA induction in development of competence. J Bacteriol 173, 7275–7282.
    [Google Scholar]
  17. Hahn, J., Bylund, J., Haines, M., Higgins, M. & Dubnau, D. ( 1995; ). Inactivation of mecA prevents recovery from the competent state and interferes with cell division and the partitioning of nucleoids in Bacillus subtilis. Mol Microbiol 18, 755–767.[CrossRef]
    [Google Scholar]
  18. Hamoen, L. W., Eshuis, H., Jongbloed, J., Venema, G. & Van Sinderen, D. ( 1995; ). A small gene, designated comS, located within the coding region of the fourth amino acid-activation domain of srfA, is required for competence development in Bacillus subtilis. Mol Microbiol 15, 55–63.[CrossRef]
    [Google Scholar]
  19. Hamoen, L. W., Venema, G. & Kuipers, O. P. ( 2003; ). Controlling competence in Bacillus subtilis: shared use of regulators. Microbiology 149, 9–17.[CrossRef]
    [Google Scholar]
  20. Hamon, M. A., Stanley, N. R., Britton, R. A., Grossman, A. D. & Lazazzera, B. A. ( 2004; ). Identification of AbrB-regulated genes involved in biofilm formation by Bacillus subtilis. Mol Microbiol 52, 847–860.[CrossRef]
    [Google Scholar]
  21. Hanlon, D. W. & Ordal, G. W. ( 1994; ). Cloning and characterization of genes encoding methyl-accepting chemotaxis proteins in Bacillus subtilis. J Biol Chem 269, 14038–14046.
    [Google Scholar]
  22. Harshey, R. M. ( 2003; ). Bacterial motility on a surface: many ways to a common goal. Annu Rev Microbiol 57, 249–273.[CrossRef]
    [Google Scholar]
  23. Jiang, M., Grau, R. & Perego, M. ( 2000; ). Differential processing of propeptide inhibitors of Rap phosphatases in Bacillus subtilis. J Bacteriol 182, 303–310.[CrossRef]
    [Google Scholar]
  24. Joseph, P., Ratnayake-Lecamwasam, M. & Sonenshein, A. L. ( 2005; ). A region of Bacillus subtilis CodY protein required for interaction with DNA. J Bacteriol 187, 4127–4139.[CrossRef]
    [Google Scholar]
  25. Julkowska, D., Obuchowski, M., Holland, I. B. & Séror, S. J. ( 2004; ). Branched swarming patterns on a synthetic medium formed by wild-type Bacillus subtilis strain 3610: detection of different cellular morphologies and constellations of cells as the complex architecture develops. Microbiology 150, 1839–1849.[CrossRef]
    [Google Scholar]
  26. Julkowska, D., Obuchowski, M., Holland, I. B. & Séror, S. J. ( 2005; ). Comparative analysis of the development of swarming communities of Bacillus subtilis 168 and a natural wild type: critical effects of surfactin and the composition of the medium. J Bacteriol 187, 65–76.[CrossRef]
    [Google Scholar]
  27. Kearns, D. B. & Losick, R. ( 2003; ). Swarming motility in undomesticated Bacillus subtilis. Mol Microbiol 49, 581–590.
    [Google Scholar]
  28. Kearns, D. B. & Losick, R. ( 2005; ). Cell population heterogeneity during growth of Bacillus subtilis. Genes Dev 19, 3083–3094.[CrossRef]
    [Google Scholar]
  29. Kearns, D. B., Chu, F., Rudner, R. & Losick, R. ( 2004; ). Genes governing swarming in Bacillus subtilis and evidence for a phase variation mechanism controlling surface motility. Mol Microbiol 52, 357–369.[CrossRef]
    [Google Scholar]
  30. Kim, S. B., Shin, B. S., Choi, S. K., Kim, C. K. & Park, S. H. ( 2001; ). Involvement of acetyl phosphate in the in vivo activation of the response regulator ComA in Bacillus subtilis. FEMS Microbiol Lett 195, 179–183.[CrossRef]
    [Google Scholar]
  31. Kinsinger, R. F., Shirk, M. C. & Fall, R. ( 2003; ). Rapid surface motility in Bacillus subtilis is dependent on extracellular surfactin and potassium ion. J Bacteriol 185, 5627–5631.[CrossRef]
    [Google Scholar]
  32. Kinsinger, R. F., Kearns, D. B., Hale, M. & Fall, R. ( 2005; ). Genetic requirements for potassium ion-dependent colony spreading in Bacillus subtilis. J Bacteriol 187, 8462–8469.[CrossRef]
    [Google Scholar]
  33. Kobayashi, K. ( 2007; ). Bacillus subtilis pellicle formation proceeds through genetically defined morphological changes. J Bacteriol 189, 4920–4931.[CrossRef]
    [Google Scholar]
  34. Koide, A. & Hoch, J. A. ( 1994; ). Identification of a second oligopeptide transport system in Bacillus subtilis and determination of its role in sporulation. Mol Microbiol 13, 417–426.[CrossRef]
    [Google Scholar]
  35. Kong, L. & Dubnau, D. ( 1994; ). Regulation of competence-specific gene expression by Mec-mediated protein–protein interaction in Bacillus subtilis. Proc Natl Acad Sci U S A 91, 5793–5797.[CrossRef]
    [Google Scholar]
  36. Kunst, F., Ogasawara, N., Moszer, I., Albertini, A. M., Alloni, G., Azevedo, V., Bertero, M. G., Bessieres, P., Bolotin, A. & other authors ( 1997; ). The complete genome sequence of the Gram-positive bacterium Bacillus subtilis. Nature 390, 249–256.[CrossRef]
    [Google Scholar]
  37. Lambalot, R. H., Gehring, A. M., Flugel, R. S., Zuber, P., LaCelle, M., Marahiel, M. A., Reid, R., Khosla, C. & Walsh, C. T. ( 1996; ). A new enzyme superfamily – the phosphopantetheinyl transferases. Chem Biol 3, 923–936.[CrossRef]
    [Google Scholar]
  38. Lanigan-Gerdes, S., Dooley, A. N., Faull, K. F. & Lazazzera, B. A. ( 2007; ). Identification of subtilisin, Epr and Vpr as enzymes that produce CSF, an extracellular signalling peptide of Bacillus subtilis. Mol Microbiol 65, 1321–1333.[CrossRef]
    [Google Scholar]
  39. Lazazzera, B. A., Solomon, J. M. & Grossman, A. D. ( 1997; ). An exported peptide functions intracellularly to contribute to cell density signaling in B. subtilis. Cell 89, 917–925.[CrossRef]
    [Google Scholar]
  40. Lazazzera, B. A., Kurtser, I. G., McQuade, R. S. & Grossman, A. D. ( 1999; ). An autoregulatory circuit affecting peptide signaling in Bacillus subtilis. J Bacteriol 181, 5193–5200.
    [Google Scholar]
  41. Liu, J. & Zuber, P. ( 1998; ). A molecular switch controlling competence and motility: competence regulatory factors ComS, MecA, and ComK control sigmaD-dependent gene expression in Bacillus subtilis. J Bacteriol 180, 4243–4251.
    [Google Scholar]
  42. Macek, B., Mijakovic, I., Olsen, J. V., Gnad, F., Kumar, C., Jensen, P. R. & Mann, M. ( 2007; ). The serine/threonine/tyrosine phosphoproteome of the model bacterium Bacillus subtilis. Mol Cell Proteomics 6, 697–707.[CrossRef]
    [Google Scholar]
  43. Mäder, U., Antelmann, H., Buder, T., Dahl, M. K., Hecker, M. & Homuth, G. ( 2002; ). Bacillus subtilis functional genomics: genome-wide analysis of the DegS–DegU regulon by transcriptomics and proteomics. Mol Genet Genomics 268, 455–467.[CrossRef]
    [Google Scholar]
  44. Marahiel, M. A., Nakano, M. M. & Zuber, P. ( 1993; ). Regulation of peptide antibiotic production in Bacillus. Mol Microbiol 7, 631–636.[CrossRef]
    [Google Scholar]
  45. Mirel, D. B., Estacio, W. F., Mathieu, M., Olmsted, E., Ramirez, J. & Marquez-Magana, L. M. ( 2000; ). Environmental regulation of Bacillus subtilis sigma(D)-dependent gene expression. J Bacteriol 182, 3055–3062.[CrossRef]
    [Google Scholar]
  46. Msadek, T., Kunst, F., Klier, A. & Rapoport, G. ( 1991; ). DegS–DegU and ComP–ComA modulator-effector pairs control expression of the Bacillus subtilis pleiotropic regulatory gene degQ. J Bacteriol 173, 2366–2377.
    [Google Scholar]
  47. Nakano, M. M., Marahiel, M. A. & Zuber, P. ( 1988; ). Identification of a genetic locus required for biosynthesis of the lipopeptide antibiotic surfactin in Bacillus subtilis. J Bacteriol 170, 5662–5668.
    [Google Scholar]
  48. Nakano, M. M., Xia, L. A. & Zuber, P. ( 1991; ). Transcription initiation region of the srfA operon, which is controlled by the comP–comA signal transduction system in Bacillus subtilis. J Bacteriol 173, 5487–5493.
    [Google Scholar]
  49. Nakano, M. M., Corbell, N., Besson, J. & Zuber, P. ( 1992; ). Isolation and characterization of sfp: a gene that functions in the production of the lipopeptide biosurfactant, surfactin, in Bacillus subtilis. Mol Gen Genet 232, 313–321.
    [Google Scholar]
  50. Ogura, M., Yamaguchi, H., Yoshida, K. I., Fujita, Y. & Tanaka, T. ( 2001; ). DNA microarray analysis of Bacillus subtilis DegU, ComA and PhoP regulons: an approach to comprehensive analysis of B. subtilis two-component regulatory systems. Nucleic Acids Res 29, 3804–3813.[CrossRef]
    [Google Scholar]
  51. Perego, M. ( 1997; ). A peptide export–import control circuit modulating bacterial development regulates protein phosphatases of the phosphorelay. Proc Natl Acad Sci U S A 94, 8612–8617.[CrossRef]
    [Google Scholar]
  52. Peypoux, F., Bonmatin, J. M. & Wallach, J. ( 1999; ). Recent trends in the biochemistry of surfactin. Appl Microbiol Biotechnol 51, 553–563.[CrossRef]
    [Google Scholar]
  53. Piazza, F., Tortosa, P. & Dubnau, D. ( 1999; ). Mutational analysis and membrane topology of ComP, a quorum-sensing histidine kinase of Bacillus subtilis controlling competence development. J Bacteriol 181, 4540–4548.
    [Google Scholar]
  54. Qazi, S. N., Rees, C. E., Mellits, K. H. & Hill, P. J. ( 2001; ). Development of gfp vectors for expression in Listeria monocytogenes and other low G+C Gram-positive bacteria. Microb Ecol 41, 301–309.
    [Google Scholar]
  55. Rashid, M. H., Tamakoshi, A. & Sekiguchi, J. ( 1996; ). Effects of mecA and mecB (clpC) mutations on expression of sigD, which encodes an alternative sigma factor, and autolysin operons and on flagellin synthesis in Bacillus subtilis. J Bacteriol 178, 4861–4869.
    [Google Scholar]
  56. Schneider, K. B., Palmer, T. M. & Grossman, A. D. ( 2002; ). Characterization of comQ and comX, two genes required for production of ComX pheromone in Bacillus subtilis. J Bacteriol 184, 410–419.[CrossRef]
    [Google Scholar]
  57. Serror, P. & Sonenshein, A. L. ( 1996; ). CodY is required for nutritional repression of Bacillus subtilis genetic competence. J Bacteriol 178, 5910–5915.
    [Google Scholar]
  58. Shapiro, J. A. ( 1998; ). Thinking about bacterial populations as multicellular organisms. Annu Rev Microbiol 52, 81–104.[CrossRef]
    [Google Scholar]
  59. Solomon, J. M., Lazazzera, B. A. & Grossman, A. D. ( 1996; ). Purification and characterization of an extracellular peptide factor that affects two different developmental pathways in Bacillus subtilis. Genes Dev 10, 2014–2024.[CrossRef]
    [Google Scholar]
  60. Solomon, J., Su, L., Shyn, S. & Grossman, A. D. ( 2003; ). Isolation and characterization of mutants of the Bacillus subtilis oligopeptide permease with altered specificity of oligopeptide transport. J Bacteriol 185, 6425–6433.[CrossRef]
    [Google Scholar]
  61. Steller, S., Sokoll, A., Wilde, C., Bernhard, F., Franke, P. & Vater, J. ( 2004; ). Initiation of surfactin biosynthesis and the role of the SrfD-thioesterase protein. Biochemistry 43, 11331–11343.[CrossRef]
    [Google Scholar]
  62. Stephenson, S., Mueller, C., Jiang, M. & Perego, M. ( 2003; ). Molecular analysis of Phr peptide processing in Bacillus subtilis. J Bacteriol 185, 4861–4871.[CrossRef]
    [Google Scholar]
  63. Süel, G. M., Garcia-Ojalvo, J., Liberman, L. M. & Elowitz, M. B. ( 2006; ). An excitable gene regulatory circuit induces transient cellular differentiation. Nature 440, 545–550.[CrossRef]
    [Google Scholar]
  64. Turgay, K., Hahn, J., Burghoorn, J. & Dubnau, D. ( 1998; ). Competence in Bacillus subtilis is controlled by regulated proteolysis of a transcription factor. EMBO J 17, 6730–6738.[CrossRef]
    [Google Scholar]
  65. van Sinderen, D., Withoff, S., Boels, H. & Venema, G. ( 1990; ). Isolation and characterization of comL, a transcription unit involved in competence development of Bacillus subtilis. Mol Gen Genet 224, 396–404.[CrossRef]
    [Google Scholar]
  66. van Sinderen, D., Luttinger, A., Kong, L., Dubnau, D., Venema, G. & Hamoen, L. ( 1995; ). comK encodes the competence transcription factor, the key regulatory protein for competence development in Bacillus subtilis. Mol Microbiol 15, 455–462.[CrossRef]
    [Google Scholar]
  67. Verhamme, D. T., Kiley, T. B. & Stanley-Wall, N. R. ( 2007; ). DegU co-ordinates multicellular behaviour exhibited by Bacillus subtilis. Mol Microbiol 65, 554–568.[CrossRef]
    [Google Scholar]
  68. Werhane, H., Lopez, P., Mendel, M., Zimmer, M., Ordal, G. W. & Marquez-Magana, L. M. ( 2004; ). The last gene of the fla/che operon in Bacillus subtilis, ylxL, is required for maximal sigmaD function. J Bacteriol 186, 4025–4029.[CrossRef]
    [Google Scholar]
  69. Wolfe, A. J., Chang, D., Walker, J. D., Seitz-Partridge, J. E., Vidaurri, M. D., Lange, C. F., Prüß, B. M., Henk, M. C., Larkin, J. C. & Conway, T. ( 2003; ). Evidence that acetyl phosphate functions as a global signal during biofilm development. Mol Microbiol 48, 977–988.[CrossRef]
    [Google Scholar]
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Scheme of regulatory circuits controlling surfactin production and competence expression in liquid cultures. ComPA constitutes a two-component signal transduction system controlled by the interaction of the secreted peptide pheromone, ComX, with the histidine kinase ComP. In addition, the secreted pentapeptide CSF, ultimately re-enters the cell, via the Opp permease, in order to modulate the inhibition of the response regulator, ComAp by the RapC phosphatase. ComA, also possibly phosphorylated by acetyl phosphate, regulates a large number of genes in addition to A. The promoter is subject to multiple regulation and, in addition to the expression of the surfactin synthases (a, b, c) Directs the expression of (competence activator) embedded in an alternative reading frame in . Following production, surfactin is secreted to the medium probably involving an ABC transporter. ComS negatively regulates the ability of MecA to target ComK (competence regulon transcription factor) for proteolysis. Other relevant players indicated in these networks include the global regulators AbrB, CodY (GTP sensor) and DegU (Mäder , 2002), the oxidative stress transcription factor Spx (Nakano , 2002), Hag (flagellin) and ComQ (modification and export of ComX). Black boxes represent genes. Black arrows and black bars represent positive and negative regulators, respectively.

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