Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1994 Dec;176(24):7630–7637. doi: 10.1128/jb.176.24.7630-7637.1994

Molecular characterization of the Salmonella typhimurium flhB operon and its protein products.

T Minamino 1, T Iino 1, K Kutuskake 1
PMCID: PMC197220  PMID: 8002587

Abstract

The flhB and flhA genes constitute an operon called flhB operon on the Salmonella typhimurium chromosome. Their gene products are required for formation of the rod structure of flagellar apparatus. Furthermore, several lines of evidence suggest that they, together with FliI and FliH, may constitute the export apparatus of flagellin, the component protein of flagellar filament. In this study, we determined the nucleotide sequence of the entire flhB operon from S. typhimurium. It was shown that the flhB and flhA genes encode highly hydrophobic polypeptides with calculated molecular masses of 42,322 and 74,848 Da, respectively. Both proteins have several potential membrane-spanning segments, suggesting that they may be integral membrane proteins. The flhB operon was found to contain an additional open reading frame capable of encoding a polypeptide with a calculated molecular mass of 14,073 Da. We designated this open reading frame flhE. The N-terminal 16 amino acids of FlhE displays a feature of a typical signal sequence. A maxicell labeling experiment enabled us to identify the precursor and mature forms of the flhE gene products. Insertion of a kanamycin-resistant gene cartridge into the chromosomal flhE gene did not affect the motility of the cells, indicating that the flhE gene is not essential for flagellar formation and function. We have overproduced and purified N-terminally truncated FlhB and FlhA proteins and raised antibodies against them. By use of these antibodies, localization of the FlhB and FlhA proteins was analyzed by Western blotting (immunoblotting) with the fractionated cell extracts. The results obtained indicated that both proteins are localized in the cytoplasmic membrane.

Full text

PDF
7634

Images in this article

7635Image
on p.7635
7635Image
on p.7635

Selected References

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

  1. Amann E., Ochs B., Abel K. J. Tightly regulated tac promoter vectors useful for the expression of unfused and fused proteins in Escherichia coli. Gene. 1988 Sep 30;69(2):301–315. doi: 10.1016/0378-1119(88)90440-4. [DOI] [PubMed] [Google Scholar]
  2. Andrews G. P., Maurelli A. T. mxiA of Shigella flexneri 2a, which facilitates export of invasion plasmid antigens, encodes a homolog of the low-calcium-response protein, LcrD, of Yersinia pestis. Infect Immun. 1992 Aug;60(8):3287–3295. doi: 10.1128/iai.60.8.3287-3295.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bergman T., Erickson K., Galyov E., Persson C., Wolf-Watz H. The lcrB (yscN/U) gene cluster of Yersinia pseudotuberculosis is involved in Yop secretion and shows high homology to the spa gene clusters of Shigella flexneri and Salmonella typhimurium. J Bacteriol. 1994 May;176(9):2619–2626. doi: 10.1128/jb.176.9.2619-2626.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. Carpenter P. B., Zuberi A. R., Ordal G. W. Bacillus subtilis flagellar proteins FliP, FliQ, FliR and FlhB are related to Shigella flexneri virulence factors. Gene. 1993 Dec 31;137(2):243–245. doi: 10.1016/0378-1119(93)90014-t. [DOI] [PubMed] [Google Scholar]
  6. Daniels C. J., Bole D. G., Quay S. C., Oxender D. L. Role for membrane potential in the secretion of protein into the periplasm of Escherichia coli. Proc Natl Acad Sci U S A. 1981 Sep;78(9):5396–5400. doi: 10.1073/pnas.78.9.5396. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dreyfus G., Williams A. W., Kawagishi I., Macnab R. M. Genetic and biochemical analysis of Salmonella typhimurium FliI, a flagellar protein related to the catalytic subunit of the F0F1 ATPase and to virulence proteins of mammalian and plant pathogens. J Bacteriol. 1993 May;175(10):3131–3138. doi: 10.1128/jb.175.10.3131-3138.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Filip C., Fletcher G., Wulff J. L., Earhart C. F. Solubilization of the cytoplasmic membrane of Escherichia coli by the ionic detergent sodium-lauryl sarcosinate. J Bacteriol. 1973 Sep;115(3):717–722. doi: 10.1128/jb.115.3.717-722.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Galán J. E., Ginocchio C., Costeas P. Molecular and functional characterization of the Salmonella invasion gene invA: homology of InvA to members of a new protein family. J Bacteriol. 1992 Jul;174(13):4338–4349. doi: 10.1128/jb.174.13.4338-4349.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Groisman E. A., Ochman H. Cognate gene clusters govern invasion of host epithelial cells by Salmonella typhimurium and Shigella flexneri. EMBO J. 1993 Oct;12(10):3779–3787. doi: 10.1002/j.1460-2075.1993.tb06056.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Henikoff S. Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene. 1984 Jun;28(3):351–359. doi: 10.1016/0378-1119(84)90153-7. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Homma M., Komeda Y., Iino T., Macnab R. M. The flaFIX gene product of Salmonella typhimurium is a flagellar basal body component with a signal peptide for export. J Bacteriol. 1987 Apr;169(4):1493–1498. doi: 10.1128/jb.169.4.1493-1498.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. Iino T. Polarity of flagellar growth in salmonella. J Gen Microbiol. 1969 May;56(2):227–239. doi: 10.1099/00221287-56-2-227. [DOI] [PubMed] [Google Scholar]
  16. Jones C. J., Homma M., Macnab R. M. L-, P-, and M-ring proteins of the flagellar basal body of Salmonella typhimurium: gene sequences and deduced protein sequences. J Bacteriol. 1989 Jul;171(7):3890–3900. doi: 10.1128/jb.171.7.3890-3900.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kubori T., Shimamoto N., Yamaguchi S., Namba K., Aizawa S. Morphological pathway of flagellar assembly in Salmonella typhimurium. J Mol Biol. 1992 Jul 20;226(2):433–446. doi: 10.1016/0022-2836(92)90958-m. [DOI] [PubMed] [Google Scholar]
  18. Kutsukake K., Iino T., Komeda Y., Yamaguchi S. Functional homology of fla genes between Salmonella typhimurium and Escherichia coli. Mol Gen Genet. 1980 Apr;178(1):59–67. doi: 10.1007/BF00267213. [DOI] [PubMed] [Google Scholar]
  19. Kutsukake K., Iino T. Refined genetic analysis of the region II che mutants in Salmonella typhimurium. Mol Gen Genet. 1985;199(3):406–409. doi: 10.1007/BF00330750. [DOI] [PubMed] [Google Scholar]
  20. Kutsukake K., Iino T. Role of the FliA-FlgM regulatory system on the transcriptional control of the flagellar regulon and flagellar formation in Salmonella typhimurium. J Bacteriol. 1994 Jun;176(12):3598–3605. doi: 10.1128/jb.176.12.3598-3605.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. 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]
  22. Kutsukake K., Ohya Y., Yamaguchi S., Iino T. Operon structure of flagellar genes in Salmonella typhimurium. Mol Gen Genet. 1988 Sep;214(1):11–15. doi: 10.1007/BF00340172. [DOI] [PubMed] [Google Scholar]
  23. Kutsukake K., Okada T., Yokoseki T., Iino T. Sequence analysis of the flgA gene and its adjacent region in Salmonella typhimurium, and identification of another flagellar gene, flgN. Gene. 1994 May 27;143(1):49–54. doi: 10.1016/0378-1119(94)90603-3. [DOI] [PubMed] [Google Scholar]
  24. Kuwajima G., Kawagishi I., Homma M., Asaka J., Kondo E., Macnab R. M. Export of an N-terminal fragment of Escherichia coli flagellin by a flagellum-specific pathway. Proc Natl Acad Sci U S A. 1989 Jul;86(13):4953–4957. doi: 10.1073/pnas.86.13.4953. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]
  26. 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]
  27. Malakooti J., Ely B., Matsumura P. Molecular characterization, nucleotide sequence, and expression of the fliO, fliP, fliQ, and fliR genes of Escherichia coli. J Bacteriol. 1994 Jan;176(1):189–197. doi: 10.1128/jb.176.1.189-197.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Namba K., Yamashita I., Vonderviszt F. Structure of the core and central channel of bacterial flagella. Nature. 1989 Dec 7;342(6250):648–654. doi: 10.1038/342648a0. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. Plano G. V., Barve S. S., Straley S. C. LcrD, a membrane-bound regulator of the Yersinia pestis low-calcium response. J Bacteriol. 1991 Nov;173(22):7293–7303. doi: 10.1128/jb.173.22.7293-7303.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Ramakrishnan G., Zhao J. L., Newton A. The cell cycle-regulated flagellar gene flbF of Caulobacter crescentus is homologous to a virulence locus (lcrD) of Yersinia pestis. J Bacteriol. 1991 Nov;173(22):7283–7292. doi: 10.1128/jb.173.22.7283-7292.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Sanders L. A., Van Way S., Mullin D. A. Characterization of the Caulobacter crescentus flbF promoter and identification of the inferred FlbF product as a homolog of the LcrD protein from a Yersinia enterocolitica virulence plasmid. J Bacteriol. 1992 Feb;174(3):857–866. doi: 10.1128/jb.174.3.857-866.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Sasakawa C., Komatsu K., Tobe T., Suzuki T., Yoshikawa M. Eight genes in region 5 that form an operon are essential for invasion of epithelial cells by Shigella flexneri 2a. J Bacteriol. 1993 Apr;175(8):2334–2346. doi: 10.1128/jb.175.8.2334-2346.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Stock A. M., Stock J. B. Purification and characterization of the CheZ protein of bacterial chemotaxis. J Bacteriol. 1987 Jul;169(7):3301–3311. doi: 10.1128/jb.169.7.3301-3311.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Suzuki T., Iino T., Horiguchi T., Yamaguchi S. Incomplete flagellar structures in nonflagellate mutants of Salmonella typhimurium. J Bacteriol. 1978 Feb;133(2):904–915. doi: 10.1128/jb.133.2.904-915.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Tomoyasu T., Yamanaka K., Murata K., Suzaki T., Bouloc P., Kato A., Niki H., Hiraga S., Ogura T. Topology and subcellular localization of FtsH protein in Escherichia coli. J Bacteriol. 1993 Mar;175(5):1352–1357. doi: 10.1128/jb.175.5.1352-1357.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Vieira J., Messing J. Production of single-stranded plasmid DNA. Methods Enzymol. 1987;153:3–11. doi: 10.1016/0076-6879(87)53044-0. [DOI] [PubMed] [Google Scholar]
  38. Viitanen A. M., Toivanen P., Skurnik M. The lcrE gene is part of an operon in the lcr region of Yersinia enterocolitica O:3. J Bacteriol. 1990 Jun;172(6):3152–3162. doi: 10.1128/jb.172.6.3152-3162.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Vogler A. P., Homma M., Irikura V. M., Macnab R. M. Salmonella typhimurium mutants defective in flagellar filament regrowth and sequence similarity of FliI to F0F1, vacuolar, and archaebacterial ATPase subunits. J Bacteriol. 1991 Jun;173(11):3564–3572. doi: 10.1128/jb.173.11.3564-3572.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Yamada M., Hakura A., Sofuni T., Nohmi T. New method for gene disruption in Salmonella typhimurium: construction and characterization of an ada-deletion derivative of Salmonella typhimurium TA1535. J Bacteriol. 1993 Sep;175(17):5539–5547. doi: 10.1128/jb.175.17.5539-5547.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. 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]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES