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. 1997 Apr;179(7):2389–2400. doi: 10.1128/jb.179.7.2389-2400.1997

The flk gene of Salmonella typhimurium couples flagellar P- and L-ring assembly to flagellar morphogenesis.

J E Karlinsey 1, A J Pease 1, M E Winkler 1, J L Bailey 1, K T Hughes 1
PMCID: PMC178978  PMID: 9079927

Abstract

The flagellum of Salmonella typhimurium is assembled in stages, and the negative regulatory protein, FlgM, is able to sense the completion of an intermediate stage of assembly, the basal body-hook (BBH) structure. Mutations in steps leading to the formation of the BBH structure do not express the flagellar filament structural genes, fliC and fljB, due to negative regulation by FlgM (K. L. Gillen and K. T. Hughes, J. Bacteriol. 173:6453-6459, 1991). We have discovered another novel regulatory gene, flk, which appears to sense the completion of another assembly stage in the flagellar morphogenic pathway just prior to BBH formation: the completion of the P- and L-rings. Cells that are unable to assemble the L- or P-rings do not express the flagellin structural genes. Mutations by insertional inactivation in either the flk or flgM locus allow expression of the fljB flagellin structural gene in strains defective in flagellar P- and L-ring assembly. Mutations in the flgM gene, but not mutations in the flk gene, allow expression of the fljB gene in strains defective in all of the steps leading to BBH formation. The flk gene was mapped to min 52 of the S. typhimurium linkage map between the pdxB and fabB loci. A null allele of flk was complemented in trans by a flk+ allele present in a multicopy pBR-based plasmid. DNA sequence analysis of the flk gene has revealed it to be identical to a gene of Escherichia coli of unknown function which has an overlapping, divergent promoter with the pdxB gene promoter (P. A. Schoenlein, B. B. Roa, and M. E. Winkler, J. Bacteriol. 174:6256-6263, 1992). An open reading frame of 333 amino acids corresponding to the flk gene product of S. typhimurium and 331 amino acids from the E. coli sequence was identified. The transcriptional start site of the S. typhimurium flk gene was determined and transcription of the flk gene was independent of the FlhDC and sigma28 flagellar transcription factors. The Flk protein observed in a T7 RNA polymerase-mediated expression system showed an apparent molecular mass of 35 kDa, slightly smaller than the predicted size of 37 kDa. The predicted structure of Flk is a mostly hydrophilic protein with a very C-terminal membrane-spanning segment preceded by positively charged amino acids. This finding predicts Flk to be inserted into the cytoplasmic membrane facing inside the cytoplasm.

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Selected References

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  1. Aizawa S. I. Flagellar assembly in Salmonella typhimurium. Mol Microbiol. 1996 Jan;19(1):1–5. doi: 10.1046/j.1365-2958.1996.344874.x. [DOI] [PubMed] [Google Scholar]
  2. Beck C. F., Warren R. A. Divergent promoters, a common form of gene organization. Microbiol Rev. 1988 Sep;52(3):318–326. doi: 10.1128/mr.52.3.318-326.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bertin P., Terao E., Lee E. H., Lejeune P., Colson C., Danchin A., Collatz E. The H-NS protein is involved in the biogenesis of flagella in Escherichia coli. J Bacteriol. 1994 Sep;176(17):5537–5540. doi: 10.1128/jb.176.17.5537-5540.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Blair D. F. How bacteria sense and swim. Annu Rev Microbiol. 1995;49:489–522. doi: 10.1146/annurev.mi.49.100195.002421. [DOI] [PubMed] [Google Scholar]
  5. Bolivar F., Rodriguez R. L., Greene P. J., Betlach M. C., Heyneker H. L., Boyer H. W., Crosa J. H., Falkow S. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene. 1977;2(2):95–113. [PubMed] [Google Scholar]
  6. Chumley F. G., Menzel R., Roth J. R. Hfr formation directed by tn10. Genetics. 1979 Apr;91(4):639–655. doi: 10.1093/genetics/91.4.639. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Emerson S. U., Tokuyasu K., Simon M. I. Bacterial flagella: polarity of elongation. Science. 1970 Jul 10;169(3941):190–192. doi: 10.1126/science.169.3941.190. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. 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]
  11. 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]
  12. Groisman E. A. In vivo genetic engineering with bacteriophage Mu. Methods Enzymol. 1991;204:180–212. doi: 10.1016/0076-6879(91)04010-l. [DOI] [PubMed] [Google Scholar]
  13. Heijne G. The distribution of positively charged residues in bacterial inner membrane proteins correlates with the trans-membrane topology. EMBO J. 1986 Nov;5(11):3021–3027. doi: 10.1002/j.1460-2075.1986.tb04601.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hirano T., Yamaguchi S., Oosawa K., Aizawa S. Roles of FliK and FlhB in determination of flagellar hook length in Salmonella typhimurium. J Bacteriol. 1994 Sep;176(17):5439–5449. doi: 10.1128/jb.176.17.5439-5449.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. Homma M., Ohnishi K., Iino T., Macnab R. M. Identification of flagellar hook and basal body gene products (FlaFV, FlaFVI, FlaFVII and FlaFVIII) in Salmonella typhimurium. J Bacteriol. 1987 Aug;169(8):3617–3624. doi: 10.1128/jb.169.8.3617-3624.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hong J. S., Ames B. N. Localized mutagenesis of any specific small region of the bacterial chromosome. Proc Natl Acad Sci U S A. 1971 Dec;68(12):3158–3162. doi: 10.1073/pnas.68.12.3158. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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]
  19. Hughes K. T., Roth J. R. Transitory cis complementation: a method for providing transposition functions to defective transposons. Genetics. 1988 May;119(1):9–12. doi: 10.1093/genetics/119.1.9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Ikeda T., Homma M., Iino T., Asakura S., Kamiya R. Localization and stoichiometry of hook-associated proteins within Salmonella typhimurium flagella. J Bacteriol. 1987 Mar;169(3):1168–1173. doi: 10.1128/jb.169.3.1168-1173.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kitamura E., Nakayama Y., Matsuzaki H., Matsumoto K., Shibuya I. Acidic-phospholipid deficiency represses the flagellar master operon through a novel regulatory region in Escherichia coli. Biosci Biotechnol Biochem. 1994 Dec;58(12):2305–2307. doi: 10.1271/bbb.58.2305. [DOI] [PubMed] [Google Scholar]
  22. Komeda Y., Suzuki H., Ishidsu J. I., Iino T. The role of cAMP in flagellation of Salmonella typhimurium. Mol Gen Genet. 1976 Dec 31;142(4):289–298. doi: 10.1007/BF00271253. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Kutsukake K. Excretion of the anti-sigma factor through a flagellar substructure couples flagellar gene expression with flagellar assembly in Salmonella typhimurium. Mol Gen Genet. 1994 Jun 15;243(6):605–612. doi: 10.1007/BF00279569. [DOI] [PubMed] [Google Scholar]
  25. Kutsukake K., Ide N. Transcriptional analysis of the flgK and fliD operons of Salmonella typhimurium which encode flagellar hook-associated proteins. Mol Gen Genet. 1995 May 10;247(3):275–281. doi: 10.1007/BF00293195. [DOI] [PubMed] [Google Scholar]
  26. Kutsukake K., Minamino T., Yokoseki T. Isolation and characterization of FliK-independent flagellation mutants from Salmonella typhimurium. J Bacteriol. 1994 Dec;176(24):7625–7629. doi: 10.1128/jb.176.24.7625-7629.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. 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]
  28. 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]
  29. Laemmli U. K., Favre M. Maturation of the head of bacteriophage T4. I. DNA packaging events. J Mol Biol. 1973 Nov 15;80(4):575–599. doi: 10.1016/0022-2836(73)90198-8. [DOI] [PubMed] [Google Scholar]
  30. Lederberg J. Detection of Fermentative Variants with Tetrazolium. J Bacteriol. 1948 Nov;56(5):695–695. doi: 10.1128/jb.56.5.695-695.1948. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Liu X., Fujita N., Ishihama A., Matsumura P. The C-terminal region of the alpha subunit of Escherichia coli RNA polymerase is required for transcriptional activation of the flagellar level II operons by the FlhD/FlhC complex. J Bacteriol. 1995 Sep;177(17):5186–5188. doi: 10.1128/jb.177.17.5186-5188.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Liu X., Matsumura P. Differential regulation of multiple overlapping promoters in flagellar class II operons in Escherichia coli. Mol Microbiol. 1996 Aug;21(3):613–620. doi: 10.1111/j.1365-2958.1996.tb02569.x. [DOI] [PubMed] [Google Scholar]
  33. Liu X., Matsumura P. The FlhD/FlhC complex, a transcriptional activator of the Escherichia coli flagellar class II operons. J Bacteriol. 1994 Dec;176(23):7345–7351. doi: 10.1128/jb.176.23.7345-7351.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Mizushima T., Koyanagi R., Suzuki E., Tomura A., Kutsukake K., Miki T., Sekimizu K. Control by phosphatidylglycerol of expression of the flhD gene in Escherichia coli. Biochim Biophys Acta. 1995 Dec 14;1245(3):397–401. doi: 10.1016/0304-4165(95)00114-x. [DOI] [PubMed] [Google Scholar]
  35. 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]
  36. Ohnishi K., Kutsukake K., Suzuki H., Lino T. A novel transcriptional regulation mechanism in the flagellar regulon of Salmonella typhimurium: an antisigma factor inhibits the activity of the flagellum-specific sigma factor, sigma F. Mol Microbiol. 1992 Nov;6(21):3149–3157. doi: 10.1111/j.1365-2958.1992.tb01771.x. [DOI] [PubMed] [Google Scholar]
  37. Ohnishi K., Ohto Y., Aizawa S., Macnab R. M., Iino T. FlgD is a scaffolding protein needed for flagellar hook assembly in Salmonella typhimurium. J Bacteriol. 1994 Apr;176(8):2272–2281. doi: 10.1128/jb.176.8.2272-2281.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Patterson-Delafield J., Martinez R. J., Stocker B. A., Yamaguchi S. A new fla gene in Salmonella typhimurium--flaR--and its mutant phenotype-superhooks. Arch Mikrobiol. 1973 Mar 26;90(2):107–120. doi: 10.1007/BF00414513. [DOI] [PubMed] [Google Scholar]
  39. Prentki P., Krisch H. M. In vitro insertional mutagenesis with a selectable DNA fragment. Gene. 1984 Sep;29(3):303–313. doi: 10.1016/0378-1119(84)90059-3. [DOI] [PubMed] [Google Scholar]
  40. Prüss B. M., Matsumura P. A regulator of the flagellar regulon of Escherichia coli, flhD, also affects cell division. J Bacteriol. 1996 Feb;178(3):668–674. doi: 10.1128/jb.178.3.668-674.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Schoenlein P. V., Roa B. B., Winkler M. E. Divergent transcription of pdxB and homology between the pdxB and serA gene products in Escherichia coli K-12. J Bacteriol. 1989 Nov;171(11):6084–6092. doi: 10.1128/jb.171.11.6084-6092.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Schägger H., von Jagow G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem. 1987 Nov 1;166(2):368–379. doi: 10.1016/0003-2697(87)90587-2. [DOI] [PubMed] [Google Scholar]
  43. Shi W., Bogdanov M., Dowhan W., Zusman D. R. The pss and psd genes are required for motility and chemotaxis in Escherichia coli. J Bacteriol. 1993 Dec;175(23):7711–7714. doi: 10.1128/jb.175.23.7711-7714.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Shi W., Zhou Y., Wild J., Adler J., Gross C. A. DnaK, DnaJ, and GrpE are required for flagellum synthesis in Escherichia coli. J Bacteriol. 1992 Oct;174(19):6256–6263. doi: 10.1128/jb.174.19.6256-6263.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Shin S., Park C. Modulation of flagellar expression in Escherichia coli by acetyl phosphate and the osmoregulator OmpR. J Bacteriol. 1995 Aug;177(16):4696–4702. doi: 10.1128/jb.177.16.4696-4702.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Silverman M. R., Simon M. I. Flagellar assembly mutants in Escherichia coli. J Bacteriol. 1972 Nov;112(2):986–993. doi: 10.1128/jb.112.2.986-993.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Silverman M., Simon M. Characterization of Escherichia coli flagellar mutants that are insensitive to catabolite repression. J Bacteriol. 1974 Dec;120(3):1196–1203. doi: 10.1128/jb.120.3.1196-1203.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Strathmann M., Hamilton B. A., Mayeda C. A., Simon M. I., Meyerowitz E. M., Palazzolo M. J. Transposon-facilitated DNA sequencing. Proc Natl Acad Sci U S A. 1991 Feb 15;88(4):1247–1250. doi: 10.1073/pnas.88.4.1247. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. 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]
  50. Suzuki T., Iino T. Role of the flaR gene in flagellar hook formation in Salmonella spp. J Bacteriol. 1981 Dec;148(3):973–979. doi: 10.1128/jb.148.3.973-979.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Tabor S., Richardson C. C. A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc Natl Acad Sci U S A. 1985 Feb;82(4):1074–1078. doi: 10.1073/pnas.82.4.1074. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Tsui H. C., Zhao G., Feng G., Leung H. C., Winkler M. E. The mutL repair gene of Escherichia coli K-12 forms a superoperon with a gene encoding a new cell-wall amidase. Mol Microbiol. 1994 Jan;11(1):189–202. doi: 10.1111/j.1365-2958.1994.tb00300.x. [DOI] [PubMed] [Google Scholar]
  53. Way J. C., Davis M. A., Morisato D., Roberts D. E., Kleckner N. New Tn10 derivatives for transposon mutagenesis and for construction of lacZ operon fusions by transposition. Gene. 1984 Dec;32(3):369–379. doi: 10.1016/0378-1119(84)90012-x. [DOI] [PubMed] [Google Scholar]
  54. Williams A. W., Yamaguchi S., Togashi F., Aizawa S. I., Kawagishi I., Macnab R. M. Mutations in fliK and flhB affecting flagellar hook and filament assembly in Salmonella typhimurium. J Bacteriol. 1996 May;178(10):2960–2970. doi: 10.1128/jb.178.10.2960-2970.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Wu T. T. A model for three-point analysis of random general transduction. Genetics. 1966 Aug;54(2):405–410. doi: 10.1093/genetics/54.2.405. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Youderian P., Sugiono P., Brewer K. L., Higgins N. P., Elliott T. Packaging specific segments of the Salmonella chromosome with locked-in Mud-P22 prophages. Genetics. 1988 Apr;118(4):581–592. doi: 10.1093/genetics/118.4.581. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Zhao G., Pease A. J., Bharani N., Winkler M. E. Biochemical characterization of gapB-encoded erythrose 4-phosphate dehydrogenase of Escherichia coli K-12 and its possible role in pyridoxal 5'-phosphate biosynthesis. J Bacteriol. 1995 May;177(10):2804–2812. doi: 10.1128/jb.177.10.2804-2812.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]

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