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. 1992 Jul;174(13):4197–4204. doi: 10.1128/jb.174.13.4197-4204.1992

An operon of Bacillus subtilis motility genes transcribed by the sigma D form of RNA polymerase.

D B Mirel 1, V M Lustre 1, M J Chamberlin 1
PMCID: PMC206194  PMID: 1624413

Abstract

Two genes controlling motility functions in Bacillus subtilis were identified by DNA sequence analysis of a chromosomal fragment containing a strong promoter for sigma D RNA polymerase. Previous studies had shown that this sigma D-dependent promoter controls synthesis of a 1.6-kb transcript in vivo and in vitro. Sequence analysis revealed that the 1.6-kb transcript contains two open reading frames coding for protein sequences homologous to the Escherichia coli motA and motB gene products, respectively, and ends in a rho-independent termination site. Direct evidence linking these genes to motility functions in B. subtilis was obtained by precise localization by polymerase chain reaction of Tn917 transposon insertion mutations of Mot- strains, isolated by Zuberi et al. (A. R. Zuberi, C. Ying, H. M. Parker, and G. W. Ordal, J. Bacteriol. 172:6841-6848, 1990), to within this mot. operon. Replacement of each wild-type gene by in-frame deletion mutations yielded strains possessing paralyzed flagella and confirmed that both motA and motB are required for the motility of B. subtilis. These current findings support our earlier suggestions that sigma D in B. subtilis plays a central role in the control of gene expression for flagellar assembly, chemotaxis, and motility functions. Sigma F, the enteric homolog of sigma D, controls similar functions in E. coli and Salmonella typhimurium, and these factors appear to be representative of a family of factors implicated in flagellar synthesis in many bacterial species, which we propose to designate the sigma 28 family.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. 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]
  2. 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]
  3. 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]
  4. Bartlett D. H., Frantz B. B., Matsumura P. Flagellar transcriptional activators FlbB and FlaI: gene sequences and 5' consensus sequences of operons under FlbB and FlaI control. J Bacteriol. 1988 Apr;170(4):1575–1581. doi: 10.1128/jb.170.4.1575-1581.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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]
  6. Brendel V., Trifonov E. N. A computer algorithm for testing potential prokaryotic terminators. Nucleic Acids Res. 1984 May 25;12(10):4411–4427. doi: 10.1093/nar/12.10.4411. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. 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]
  8. 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]
  9. Chen E. Y., Seeburg P. H. Supercoil sequencing: a fast and simple method for sequencing plasmid DNA. DNA. 1985 Apr;4(2):165–170. doi: 10.1089/dna.1985.4.165. [DOI] [PubMed] [Google Scholar]
  10. Chun S. Y., Parkinson J. S. Bacterial motility: membrane topology of the Escherichia coli MotB protein. Science. 1988 Jan 15;239(4837):276–278. doi: 10.1126/science.2447650. [DOI] [PubMed] [Google Scholar]
  11. Connell T. D., Black W. J., Kawula T. H., Barritt D. S., Dempsey J. A., Kverneland K., Jr, Stephenson A., Schepart B. S., Murphy G. L., Cannon J. G. Recombination among protein II genes of Neisseria gonorrhoeae generates new coding sequences and increases structural variability in the protein II family. Mol Microbiol. 1988 Mar;2(2):227–236. doi: 10.1111/j.1365-2958.1988.tb00024.x. [DOI] [PubMed] [Google Scholar]
  12. Dean G. E., Macnab R. M., Stader J., Matsumura P., Burks C. Gene sequence and predicted amino acid sequence of the motA protein, a membrane-associated protein required for flagellar rotation in Escherichia coli. J Bacteriol. 1984 Sep;159(3):991–999. doi: 10.1128/jb.159.3.991-999.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Ferrari F. A., Ferrari E., Hoch J. A. Chromosomal location of a Bacillus subtilis DNA fragment uniquely transcribed by sigma-28-containing RNA polymerase. J Bacteriol. 1982 Nov;152(2):780–785. doi: 10.1128/jb.152.2.780-785.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. 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]
  16. 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]
  17. Guerry P., Logan S. M., Thornton S., Trust T. J. Genomic organization and expression of Campylobacter flagellin genes. J Bacteriol. 1990 Apr;172(4):1853–1860. doi: 10.1128/jb.172.4.1853-1860.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hanafusa T., Sakai A., Tominaga A., Enomoto M. Isolation and characterization of Escherichia coli hag operator mutants whose hag48 expression has become repressible by a Salmonella H1 repressor. Mol Gen Genet. 1989 Mar;216(1):44–50. doi: 10.1007/BF00332229. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. 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]
  21. Helmann J. D., Chamberlin M. J. DNA sequence analysis suggests that expression of flagellar and chemotaxis genes in Escherichia coli and Salmonella typhimurium is controlled by an alternative sigma factor. Proc Natl Acad Sci U S A. 1987 Sep;84(18):6422–6424. doi: 10.1073/pnas.84.18.6422. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. 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]
  24. Komeda Y. Transcriptional control of flagellar genes in Escherichia coli K-12. J Bacteriol. 1986 Dec;168(3):1315–1318. doi: 10.1128/jb.168.3.1315-1318.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kustu S., Santero E., Keener J., Popham D., Weiss D. Expression of sigma 54 (ntrA)-dependent genes is probably united by a common mechanism. Microbiol Rev. 1989 Sep;53(3):367–376. doi: 10.1128/mr.53.3.367-376.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. 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]
  27. Kyte J., Doolittle R. F. A simple method for displaying the hydropathic character of a protein. J Mol Biol. 1982 May 5;157(1):105–132. doi: 10.1016/0022-2836(82)90515-0. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. 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]
  30. 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]
  31. Nuijten P. J., van Asten F. J., Gaastra W., van der Zeijst B. A. Structural and functional analysis of two Campylobacter jejuni flagellin genes. J Biol Chem. 1990 Oct 15;265(29):17798–17804. [PubMed] [Google Scholar]
  32. 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]
  33. Perkins J. B., Youngman P. J. Construction and properties of Tn917-lac, a transposon derivative that mediates transcriptional gene fusions in Bacillus subtilis. Proc Natl Acad Sci U S A. 1986 Jan;83(1):140–144. doi: 10.1073/pnas.83.1.140. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Pleier E., Schmitt R. Identification and sequence analysis of two related flagellin genes in Rhizobium meliloti. J Bacteriol. 1989 Mar;171(3):1467–1475. doi: 10.1128/jb.171.3.1467-1475.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. 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]
  36. Silverman M., Simon M. Operon controlling motility and chemotoxis in E. coli. Nature. 1976 Dec 9;264(5586):577–580. doi: 10.1038/264577a0. [DOI] [PubMed] [Google Scholar]
  37. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  38. Stader J., Matsumura P., Vacante D., Dean G. E., Macnab R. M. Nucleotide sequence of the Escherichia coli motB gene and site-limited incorporation of its product into the cytoplasmic membrane. J Bacteriol. 1986 Apr;166(1):244–252. doi: 10.1128/jb.166.1.244-252.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. 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]
  40. Totten P. A., Lory S. Characterization of the type a flagellin gene from Pseudomonas aeruginosa PAK. J Bacteriol. 1990 Dec;172(12):7188–7199. doi: 10.1128/jb.172.12.7188-7199.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. 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]
  42. Wilson M. L., Macnab R. M. Co-overproduction and localization of the Escherichia coli motility proteins motA and motB. J Bacteriol. 1990 Jul;172(7):3932–3939. doi: 10.1128/jb.172.7.3932-3939.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Wilson M. L., Macnab R. M. Overproduction of the MotA protein of Escherichia coli and estimation of its wild-type level. J Bacteriol. 1988 Feb;170(2):588–597. doi: 10.1128/jb.170.2.588-597.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. 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]
  45. 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]
  46. 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]
  47. 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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