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. 1994 Aug;176(15):4492–4500. doi: 10.1128/jb.176.15.4492-4500.1994

Identification of flagellar synthesis regulatory and structural genes in a sigma D-dependent operon of Bacillus subtilis.

D B Mirel 1, P Lauer 1, M J Chamberlin 1
PMCID: PMC196267  PMID: 8045879

Abstract

The sigma D form of RNA polymerase from Bacillus subtilis has been shown previously to direct the synthesis of several transcription units bearing genes for flagellin, motility proteins, and autolysins. In this report, we describe an operon of genes transcribed from the sigma D-dependent promoter PD-1. We have identified three complete open reading frames and one partial one downstream of this promoter; immediately upstream is the previously identified comF locus. The PD-1 operon encodes the presumptive B. subtilis homologs of two Salmonella typhimurium late flagellar genes, flgM and flgK. Also present in this operon are two genes of unknown function, orf139 and orf160, whose products show similarities to the eukaryotic cytoskeletal proteins myosin and vimentin, respectively. orf139 and orf160 may encode proteins that form extended alpha-helical secondary structures and coiled-coil quaternary structures which may be filamentous components of the gram-positive bacterial flagellum. We have characterized the B. subtilis flgM gene further by constructing an in-frame deletion mutation, flgM delta 80, and creating strains of B. subtilis in which this allele has replaced the wild-type copy. By primer extension analysis of cellular RNA, we have shown that the flgM delta 80 mutation relieves the block to transcription of two other sigma D-dependent operons imposed by an unlinked mutation in a gene directing early flagellar synthesis. We conclude that, as in the case of S. typhimurium, early flagellar synthesis in B. subtilis is coupled to late flagellar synthesis through repression of sigma D-dependent transcription by the flgM gene product.

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Selected References

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  1. Albers K., Fuchs E. The molecular biology of intermediate filament proteins. Int Rev Cytol. 1992;134:243–279. doi: 10.1016/s0074-7696(08)62030-6. [DOI] [PubMed] [Google Scholar]
  2. Albertini A. M., Caramori T., Crabb W. D., Scoffone F., Galizzi A. The flaA locus of Bacillus subtilis is part of a large operon coding for flagellar structures, motility functions, and an ATPase-like polypeptide. J Bacteriol. 1991 Jun;173(11):3573–3579. doi: 10.1128/jb.173.11.3573-3579.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Altschul S. F., Gish W., Miller W., Myers E. W., Lipman D. J. Basic local alignment search tool. J Mol Biol. 1990 Oct 5;215(3):403–410. doi: 10.1016/S0022-2836(05)80360-2. [DOI] [PubMed] [Google Scholar]
  4. Anagnostopoulos C., Spizizen J. REQUIREMENTS FOR TRANSFORMATION IN BACILLUS SUBTILIS. J Bacteriol. 1961 May;81(5):741–746. doi: 10.1128/jb.81.5.741-746.1961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Arnosti D. N., Chamberlin M. J. Secondary sigma factor controls transcription of flagellar and chemotaxis genes in Escherichia coli. Proc Natl Acad Sci U S A. 1989 Feb;86(3):830–834. doi: 10.1073/pnas.86.3.830. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bischoff D. S., Ordal G. W. Sequence and characterization of Bacillus subtilis CheB, a homolog of Escherichia coli CheY, and its role in a different mechanism of chemotaxis. J Biol Chem. 1991 Jul 5;266(19):12301–12305. [PubMed] [Google Scholar]
  8. Brutlag D. L., Clayton J., Friedland P., Kedes L. H. SEQ: a nucleotide sequence analysis and recombination system. Nucleic Acids Res. 1982 Jan 11;10(1):279–294. doi: 10.1093/nar/10.1.279. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Brutlag D. L., Dautricourt J. P., Maulik S., Relph J. Improved sensitivity of biological sequence database searches. Comput Appl Biosci. 1990 Jul;6(3):237–245. doi: 10.1093/bioinformatics/6.3.237. [DOI] [PubMed] [Google Scholar]
  10. Carpenter P. B., Hanlon D. W., Ordal G. W. flhF, a Bacillus subtilis flagellar gene that encodes a putative GTP-binding protein. Mol Microbiol. 1992 Sep;6(18):2705–2713. doi: 10.1111/j.1365-2958.1992.tb01447.x. [DOI] [PubMed] [Google Scholar]
  11. Carpenter P. B., Ordal G. W. Bacillus subtilis FlhA: a flagellar protein related to a new family of signal-transducing receptors. Mol Microbiol. 1993 Mar;7(5):735–743. doi: 10.1111/j.1365-2958.1993.tb01164.x. [DOI] [PubMed] [Google Scholar]
  12. Chou P. Y., Fasman G. D. Prediction of the secondary structure of proteins from their amino acid sequence. Adv Enzymol Relat Areas Mol Biol. 1978;47:45–148. doi: 10.1002/9780470122921.ch2. [DOI] [PubMed] [Google Scholar]
  13. Coleman J. E. Zinc proteins: enzymes, storage proteins, transcription factors, and replication proteins. Annu Rev Biochem. 1992;61:897–946. doi: 10.1146/annurev.bi.61.070192.004341. [DOI] [PubMed] [Google Scholar]
  14. Fuhrer D. K., Ordal G. W. Bacillus subtilis CheN, a homolog of CheA, the central regulator of chemotaxis in Escherichia coli. J Bacteriol. 1991 Dec;173(23):7443–7448. doi: 10.1128/jb.173.23.7443-7448.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Gillen K. L., Hughes K. T. Molecular characterization of flgM, a gene encoding a negative regulator of flagellin synthesis in Salmonella typhimurium. J Bacteriol. 1991 Oct;173(20):6453–6459. doi: 10.1128/jb.173.20.6453-6459.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Gillen K. L., Hughes K. T. Negative regulatory loci coupling flagellin synthesis to flagellar assembly in Salmonella typhimurium. J Bacteriol. 1991 Apr;173(7):2301–2310. doi: 10.1128/jb.173.7.2301-2310.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Gillen K. L., Hughes K. T. Transcription from two promoters and autoregulation contribute to the control of expression of the Salmonella typhimurium flagellar regulatory gene flgM. J Bacteriol. 1993 Nov;175(21):7006–7015. doi: 10.1128/jb.175.21.7006-7015.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Gilman M. Z., Chamberlin M. J. Developmental and genetic regulation of Bacillus subtilis genes transcribed by sigma 28-RNA polymerase. Cell. 1983 Nov;35(1):285–293. doi: 10.1016/0092-8674(83)90231-3. [DOI] [PubMed] [Google Scholar]
  19. Gilman M. Z., Glenn J. S., Singer V. L., Chamberlin M. J. Isolation of sigma-28-specific promoters from Bacillus subtilis DNA. Gene. 1984 Dec;32(1-2):11–20. doi: 10.1016/0378-1119(84)90027-1. [DOI] [PubMed] [Google Scholar]
  20. Gilman M. Z., Wiggs J. L., Chamberlin M. J. Nucleotide sequences of two Bacillus subtilis promoters used by Bacillus subtilis sigma-28 RNA polymerase. Nucleic Acids Res. 1981 Nov 25;9(22):5991–6000. doi: 10.1093/nar/9.22.5991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hanlon D. W., Márquez-Magaña L. M., Carpenter P. B., Chamberlin M. J., Ordal G. W. Sequence and characterization of Bacillus subtilis CheW. J Biol Chem. 1992 Jun 15;267(17):12055–12060. [PubMed] [Google Scholar]
  22. Heidecker G., Messing J., Gronenborn B. A versatile primer for DNA sequencing in the M13mp2 cloning system. Gene. 1980 Jun;10(1):69–73. doi: 10.1016/0378-1119(80)90145-6. [DOI] [PubMed] [Google Scholar]
  23. Helmann J. D. Alternative sigma factors and the regulation of flagellar gene expression. Mol Microbiol. 1991 Dec;5(12):2875–2882. doi: 10.1111/j.1365-2958.1991.tb01847.x. [DOI] [PubMed] [Google Scholar]
  24. Helmann J. D., Masiarz F. R., Chamberlin M. J. Isolation and characterization of the Bacillus subtilis sigma 28 factor. J Bacteriol. 1988 Apr;170(4):1560–1567. doi: 10.1128/jb.170.4.1560-1567.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Helmann J. D., Márquez L. M., Chamberlin M. J. Cloning, sequencing, and disruption of the Bacillus subtilis sigma 28 gene. J Bacteriol. 1988 Apr;170(4):1568–1574. doi: 10.1128/jb.170.4.1568-1574.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Henikoff S. Unidirectional digestion with exonuclease III in DNA sequence analysis. Methods Enzymol. 1987;155:156–165. doi: 10.1016/0076-6879(87)55014-5. [DOI] [PubMed] [Google Scholar]
  27. Henner D. J., 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 Nov;170(11):5102–5109. doi: 10.1128/jb.170.11.5102-5109.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Homma M., DeRosier D. J., Macnab R. M. Flagellar hook and hook-associated proteins of Salmonella typhimurium and their relationship to other axial components of the flagellum. J Mol Biol. 1990 Jun 20;213(4):819–832. doi: 10.1016/S0022-2836(05)80266-9. [DOI] [PubMed] [Google Scholar]
  29. Homma M., Kutsukake K., Hasebe M., Iino T., Macnab R. M. FlgB, FlgC, FlgF and FlgG. A family of structurally related proteins in the flagellar basal body of Salmonella typhimurium. J Mol Biol. 1990 Jan 20;211(2):465–477. doi: 10.1016/0022-2836(90)90365-S. [DOI] [PubMed] [Google Scholar]
  30. Hughes K. T., Gillen K. L., Semon M. J., Karlinsey J. E. Sensing structural intermediates in bacterial flagellar assembly by export of a negative regulator. Science. 1993 Nov 19;262(5137):1277–1280. doi: 10.1126/science.8235660. [DOI] [PubMed] [Google Scholar]
  31. Jones C. J., Macnab R. M. Flagellar assembly in Salmonella typhimurium: analysis with temperature-sensitive mutants. J Bacteriol. 1990 Mar;172(3):1327–1339. doi: 10.1128/jb.172.3.1327-1339.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Kunkel T. A., Roberts J. D., Zakour R. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. doi: 10.1016/0076-6879(87)54085-x. [DOI] [PubMed] [Google Scholar]
  33. Kutsukake K., Ohya Y., Iino T. Transcriptional analysis of the flagellar regulon of Salmonella typhimurium. J Bacteriol. 1990 Feb;172(2):741–747. doi: 10.1128/jb.172.2.741-747.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Lazarevic V., Margot P., Soldo B., Karamata D. Sequencing and analysis of the Bacillus subtilis lytRABC divergon: a regulatory unit encompassing the structural genes of the N-acetylmuramoyl-L-alanine amidase and its modifier. J Gen Microbiol. 1992 Sep;138(9):1949–1961. doi: 10.1099/00221287-138-9-1949. [DOI] [PubMed] [Google Scholar]
  35. Londoño-Vallejo J. A., Dubnau D. comF, a Bacillus subtilis late competence locus, encodes a protein similar to ATP-dependent RNA/DNA helicases. Mol Microbiol. 1993 Jul;9(1):119–131. doi: 10.1111/j.1365-2958.1993.tb01674.x. [DOI] [PubMed] [Google Scholar]
  36. Macnab R. M. Genetics and biogenesis of bacterial flagella. Annu Rev Genet. 1992;26:131–158. doi: 10.1146/annurev.ge.26.120192.001023. [DOI] [PubMed] [Google Scholar]
  37. McLachlan A. D. Repeating sequences and gene duplication in proteins. J Mol Biol. 1972 Mar 14;64(2):417–437. doi: 10.1016/0022-2836(72)90508-6. [DOI] [PubMed] [Google Scholar]
  38. Mirel D. B., Chamberlin M. J. The Bacillus subtilis flagellin gene (hag) is transcribed by the sigma 28 form of RNA polymerase. J Bacteriol. 1989 Jun;171(6):3095–3101. doi: 10.1128/jb.171.6.3095-3101.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Mirel D. B., Lustre V. M., Chamberlin M. J. An operon of Bacillus subtilis motility genes transcribed by the sigma D form of RNA polymerase. J Bacteriol. 1992 Jul;174(13):4197–4204. doi: 10.1128/jb.174.13.4197-4204.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Márquez-Magaña L. M., Chamberlin M. J. Characterization of the sigD transcription unit of Bacillus subtilis. J Bacteriol. 1994 Apr;176(8):2427–2434. doi: 10.1128/jb.176.8.2427-2434.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Márquez L. M., Helmann J. D., Ferrari E., Parker H. M., Ordal G. W., Chamberlin M. J. Studies of sigma D-dependent functions in Bacillus subtilis. J Bacteriol. 1990 Jun;172(6):3435–3443. doi: 10.1128/jb.172.6.3435-3443.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Ohnishi K., Kutsukake K., Suzuki H., Iino T. Gene fliA encodes an alternative sigma factor specific for flagellar operons in Salmonella typhimurium. Mol Gen Genet. 1990 Apr;221(2):139–147. doi: 10.1007/BF00261713. [DOI] [PubMed] [Google Scholar]
  43. Pooley H. M., Karamata D. Genetic analysis of autolysin-deficient and flagellaless mutants of Bacillus subtilis. J Bacteriol. 1984 Dec;160(3):1123–1129. doi: 10.1128/jb.160.3.1123-1129.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Russel M., Kidd S., Kelley M. R. An improved filamentous helper phage for generating single-stranded plasmid DNA. Gene. 1986;45(3):333–338. doi: 10.1016/0378-1119(86)90032-6. [DOI] [PubMed] [Google Scholar]
  45. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Stahl M. L., Ferrari E. Replacement of the Bacillus subtilis subtilisin structural gene with an In vitro-derived deletion mutation. J Bacteriol. 1984 May;158(2):411–418. doi: 10.1128/jb.158.2.411-418.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Warrick H. M., Spudich J. A. Myosin structure and function in cell motility. Annu Rev Cell Biol. 1987;3:379–421. doi: 10.1146/annurev.cb.03.110187.002115. [DOI] [PubMed] [Google Scholar]
  48. Wiggs J. L., Gilman M. Z., Chamberlin M. J. Heterogeneity of RNA polymerase in Bacillus subtilis: evidence for an additional sigma factor in vegetative cells. Proc Natl Acad Sci U S A. 1981 May;78(5):2762–2766. doi: 10.1073/pnas.78.5.2762. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Yang M. Y., Ferrari E., Henner D. J. Cloning of the neutral protease gene of Bacillus subtilis and the use of the cloned gene to create an in vitro-derived deletion mutation. J Bacteriol. 1984 Oct;160(1):15–21. doi: 10.1128/jb.160.1.15-21.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]
  51. Zuberi A. R., Bischoff D. S., Ordal G. W. Nucleotide sequence and characterization of a Bacillus subtilis gene encoding a flagellar switch protein. J Bacteriol. 1991 Jan;173(2):710–719. doi: 10.1128/jb.173.2.710-719.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Zuberi A. R., Ying C. W., Parker H. M., Ordal G. W. Transposon Tn917lacZ mutagenesis of Bacillus subtilis: identification of two new loci required for motility and chemotaxis. J Bacteriol. 1990 Dec;172(12):6841–6848. doi: 10.1128/jb.172.12.6841-6848.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Zuberi A. R., Ying C. W., Weinreich M. R., Ordal G. W. Transcriptional organization of a cloned chemotaxis locus of Bacillus subtilis. J Bacteriol. 1990 Apr;172(4):1870–1876. doi: 10.1128/jb.172.4.1870-1876.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Zuberi A. R., Ying C., Bischoff D. S., Ordal G. W. Gene-protein relationships in the flagellar hook-basal body complex of Bacillus subtilis: sequences of the flgB, flgC, flgG, fliE and fliF genes. Gene. 1991 May 15;101(1):23–31. doi: 10.1016/0378-1119(91)90220-6. [DOI] [PubMed] [Google Scholar]

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