Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1997 Jul;179(14):4493–4500. doi: 10.1128/jb.179.14.4493-4500.1997

Cloning and characterization of the region III flagellar operons of the four Shigella subgroups: genetic defects that cause loss of flagella of Shigella boydii and Shigella sonnei.

A A Al Mamun 1, A Tominaga 1, M Enomoto 1
PMCID: PMC179284  PMID: 9226258

Abstract

To detect genetic defects that might have caused loss of flagella in Shigella boydii and Shigella sonnei, the region III flagellar (fli) operons were cloned from certain strains and analyzed with reference to the restriction maps and genetic maps of Escherichia coli fli operons. S. boydii NCTC9733 (strain C5 in this paper) had the 988-bp internal deletion in the fliF gene that encodes a large substructural protein of the basal body. Two strains (C1 and C8) had deletions of the entire fliF operon, and the remaining three (C3, C4, and C9) differed in the size of the restriction fragments carrying the fliF and fliL operons. Loss of flagella in S. boydii appears to originate in some defect in the fliF operon. S. sonnei IID969 lacked the fliD gene and, in place of it, carried two IS600 elements as inverted repeats. Genes downstream from fliD were not detected in the cloned fragment despite its large size but did appear elsewhere in the chromosome. The fliD gene encodes a cap protein of the flagellar filament, and its deletion results in overexpression of class 3 operons by the increased amount of FliA (sigmaF) caused by the excess export of the anti-sigma factor FlgM. Three other strains also had the fliD deletion, and two of them had another deletion in the fliF-fliG-fliH region. The fliD deletion might be the primary cause of loss of flagella in S. sonnei. The lack of FliF or FliD in each subgroup is discussed in connection with the maintenance of virulence and bacterial growth. We also discuss the process of loss of flagella in relation to transposition of IS elements and alterations of the noncoding region, which were found to be common to at least three subgroups.

Full Text

The Full Text of this article is available as a PDF (548.8 KB).

Selected References

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

  1. Akerley B. J., Cotter P. A., Miller J. F. Ectopic expression of the flagellar regulon alters development of the Bordetella-host interaction. Cell. 1995 Feb 24;80(4):611–620. doi: 10.1016/0092-8674(95)90515-4. [DOI] [PubMed] [Google Scholar]
  2. Akerley B. J., Miller J. F. Flagellin gene transcription in Bordetella bronchiseptica is regulated by the BvgAS virulence control system. J Bacteriol. 1993 Jun;175(11):3468–3479. doi: 10.1128/jb.175.11.3468-3479.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Al Mamun A. A., Tominaga A., Enomoto M. Detection and characterization of the flagellar master operon in the four Shigella subgroups. J Bacteriol. 1996 Jul;178(13):3722–3726. doi: 10.1128/jb.178.13.3722-3726.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Albertini A. M., Hofer M., Calos M. P., Miller J. H. On the formation of spontaneous deletions: the importance of short sequence homologies in the generation of large deletions. Cell. 1982 Jun;29(2):319–328. doi: 10.1016/0092-8674(82)90148-9. [DOI] [PubMed] [Google Scholar]
  5. Bachmann B. J. Linkage map of Escherichia coli K-12, edition 8. Microbiol Rev. 1990 Jun;54(2):130–197. doi: 10.1128/mr.54.2.130-197.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bartlett D. H., Matsumura P. Identification of Escherichia coli region III flagellar gene products and description of two new flagellar genes. J Bacteriol. 1984 Nov;160(2):577–585. doi: 10.1128/jb.160.2.577-585.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. 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]
  8. Bernardi A., Bernardi F. Complete sequence of pSC101. Nucleic Acids Res. 1984 Dec 21;12(24):9415–9426. doi: 10.1093/nar/12.24.9415. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Bi X., Liu L. F. recA-independent and recA-dependent intramolecular plasmid recombination. Differential homology requirement and distance effect. J Mol Biol. 1994 Jan 14;235(2):414–423. doi: 10.1006/jmbi.1994.1002. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Chédin F., Dervyn E., Dervyn R., Ehrlich S. D., Noirot P. Frequency of deletion formation decreases exponentially with distance between short direct repeats. Mol Microbiol. 1994 May;12(4):561–569. doi: 10.1111/j.1365-2958.1994.tb01042.x. [DOI] [PubMed] [Google Scholar]
  12. DasGupta U., Weston-Hafer K., Berg D. E. Local DNA sequence control of deletion formation in Escherichia coli plasmid pBR322. Genetics. 1987 Jan;115(1):41–49. doi: 10.1093/genetics/115.1.41. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Eichelberg K., Ginocchio C. C., Galán J. E. Molecular and functional characterization of the Salmonella typhimurium invasion genes invB and invC: homology of InvC to the F0F1 ATPase family of proteins. J Bacteriol. 1994 Aug;176(15):4501–4510. doi: 10.1128/jb.176.15.4501-4510.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Enomoto M., Oosawa K., Momota H. Mapping of the pin locus coding for a site-specific recombinase that causes flagellar-phase variation in Escherichia coli K-12. J Bacteriol. 1983 Nov;156(2):663–668. doi: 10.1128/jb.156.2.663-668.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Ewing W. H. SHIGELLA NOMENCLATURE. J Bacteriol. 1949 Jun;57(6):633–638. doi: 10.1128/jb.57.6.633-638.1949. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. 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]
  19. Girón J. A. Expression of flagella and motility by Shigella. Mol Microbiol. 1995 Oct;18(1):63–75. doi: 10.1111/j.1365-2958.1995.mmi_18010063.x. [DOI] [PubMed] [Google Scholar]
  20. Glickman B. W., Ripley L. S. Structural intermediates of deletion mutagenesis: a role for palindromic DNA. Proc Natl Acad Sci U S A. 1984 Jan;81(2):512–516. doi: 10.1073/pnas.81.2.512. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hanahan D., Meselson M. Plasmid screening at high colony density. Methods Enzymol. 1983;100:333–342. doi: 10.1016/0076-6879(83)00066-x. [DOI] [PubMed] [Google Scholar]
  22. Hanahan D. Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 1983 Jun 5;166(4):557–580. doi: 10.1016/s0022-2836(83)80284-8. [DOI] [PubMed] [Google Scholar]
  23. Homma M., Iino T., Kutsukake K., Yamaguchi S. In vitro reconstitution of flagellar filaments onto hooks of filamentless mutants of Salmonella typhimurium by addition of hook-associated proteins. Proc Natl Acad Sci U S A. 1986 Aug;83(16):6169–6173. doi: 10.1073/pnas.83.16.6169. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Ikeda T., Asakura S., Kamiya R. "Cap" on the tip of Salmonella flagella. J Mol Biol. 1985 Aug 20;184(4):735–737. doi: 10.1016/0022-2836(85)90317-1. [DOI] [PubMed] [Google Scholar]
  25. Kawagishi I., Müller V., Williams A. W., Irikura V. M., Macnab R. M. Subdivision of flagellar region III of the Escherichia coli and Salmonella typhimurium chromosomes and identification of two additional flagellar genes. J Gen Microbiol. 1992 Jun;138(6):1051–1065. doi: 10.1099/00221287-138-6-1051. [DOI] [PubMed] [Google Scholar]
  26. Kihara M., Homma M., Kutsukake K., Macnab R. M. Flagellar switch of Salmonella typhimurium: gene sequences and deduced protein sequences. J Bacteriol. 1989 Jun;171(6):3247–3257. doi: 10.1128/jb.171.6.3247-3257.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Kleckner N. Transposable elements in prokaryotes. Annu Rev Genet. 1981;15:341–404. doi: 10.1146/annurev.ge.15.120181.002013. [DOI] [PubMed] [Google Scholar]
  28. Komeda Y., Kutsukake K., Iino T. Definition of additional flagellar genes in Escherichia coli K12. Genetics. 1980 Feb;94(2):277–290. doi: 10.1093/genetics/94.2.277. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. 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]
  30. Lederberg E M, Lederberg J. Genetic Studies of Lysogenicity in Escherichia Coli. Genetics. 1953 Jan;38(1):51–64. doi: 10.1093/genetics/38.1.51. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. 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]
  32. Lovett S. T., Gluckman T. J., Simon P. J., Sutera V. A., Jr, Drapkin P. T. Recombination between repeats in Escherichia coli by a recA-independent, proximity-sensitive mechanism. Mol Gen Genet. 1994 Nov 1;245(3):294–300. doi: 10.1007/BF00290109. [DOI] [PubMed] [Google Scholar]
  33. 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]
  34. Matsutani S., Ohtsubo E. Distribution of the Shigella sonnei insertion elements in Enterobacteriaceae. Gene. 1993 May 15;127(1):111–115. doi: 10.1016/0378-1119(93)90624-c. [DOI] [PubMed] [Google Scholar]
  35. Matsutani S., Ohtsubo H., Maeda Y., Ohtsubo E. Isolation and characterization of IS elements repeated in the bacterial chromosome. J Mol Biol. 1987 Aug 5;196(3):445–455. doi: 10.1016/0022-2836(87)90023-4. [DOI] [PubMed] [Google Scholar]
  36. Maurelli A. T., Sansonetti P. J. Genetic determinants of Shigella pathogenicity. Annu Rev Microbiol. 1988;42:127–150. doi: 10.1146/annurev.mi.42.100188.001015. [DOI] [PubMed] [Google Scholar]
  37. Mytelka D. S., Chamberlin M. J. Escherichia coli fliAZY operon. J Bacteriol. 1996 Jan;178(1):24–34. doi: 10.1128/jb.178.1.24-34.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Müller V., Jones C. J., Kawagishi I., Aizawa S., Macnab R. M. Characterization of the fliE genes of Escherichia coli and Salmonella typhimurium and identification of the FliE protein as a component of the flagellar hook-basal body complex. J Bacteriol. 1992 Apr;174(7):2298–2304. doi: 10.1128/jb.174.7.2298-2304.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. 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]
  40. Okazaki N., Matsuo S., Saito K., Tominaga A., Enomoto M. Conversion of the Salmonella phase 1 flagellin gene fliC to the phase 2 gene fljB on the Escherichia coli K-12 chromosome. J Bacteriol. 1993 Feb;175(3):758–766. doi: 10.1128/jb.175.3.758-766.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Raha M., Kawagishi I., Müller V., Kihara M., Macnab R. M. Escherichia coli produces a cytoplasmic alpha-amylase, AmyA. J Bacteriol. 1992 Oct;174(20):6644–6652. doi: 10.1128/jb.174.20.6644-6652.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Raha M., Kihara M., Kawagishi I., Macnab R. M. Organization of the Escherichia coli and Salmonella typhimurium chromosomes between flagellar regions IIIa and IIIb, including a large non-coding region. J Gen Microbiol. 1993 Jul;139(7):1401–1407. doi: 10.1099/00221287-139-7-1401. [DOI] [PubMed] [Google Scholar]
  43. Roman S. J., Frantz B. B., Matsumura P. Gene sequence, overproduction, purification and determination of the wild-type level of the Escherichia coli flagellar switch protein FliG. Gene. 1993 Oct 29;133(1):103–108. doi: 10.1016/0378-1119(93)90232-r. [DOI] [PubMed] [Google Scholar]
  44. 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]
  45. Schmitt C. K., Darnell S. C., O'Brien A. D. The attenuated phenotype of a Salmonella typhimurium flgM mutant is related to expression of FliC flagellin. J Bacteriol. 1996 May;178(10):2911–2915. doi: 10.1128/jb.178.10.2911-2915.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Schmitt C. K., Darnell S. C., Tesh V. L., Stocker B. A., O'Brien A. D. Mutation of flgM attenuates virulence of Salmonella typhimurium, and mutation of fliA represses the attenuated phenotype. J Bacteriol. 1994 Jan;176(2):368–377. doi: 10.1128/jb.176.2.368-377.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Scott J. R. Genetic studies on bacteriophage P1. Virology. 1968 Dec;36(4):564–574. doi: 10.1016/0042-6822(68)90188-8. [DOI] [PubMed] [Google Scholar]
  48. Shapiro L. The bacterial flagellum: from genetic network to complex architecture. Cell. 1995 Feb 24;80(4):525–527. doi: 10.1016/0092-8674(95)90505-7. [DOI] [PubMed] [Google Scholar]
  49. 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]
  50. Tominaga A., Enomoto M. Magnesium-dependent plaque formation by bacteriophage P1cinC(-) on Escherichia coli C and Shigella sonnei. Virology. 1986 Nov;155(1):284–288. doi: 10.1016/0042-6822(86)90190-x. [DOI] [PubMed] [Google Scholar]
  51. Tominaga A., Ikemizu S., Enomoto M. Site-specific recombinase genes in three Shigella subgroups and nucleotide sequences of a pinB gene and an invertible B segment from Shigella boydii. J Bacteriol. 1991 Jul;173(13):4079–4087. doi: 10.1128/jb.173.13.4079-4087.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Tominaga A., Mahmoud M. A., Mukaihara T., Enomoto M. Molecular characterization of intact, but cryptic, flagellin genes in the genus Shigella. Mol Microbiol. 1994 Apr;12(2):277–285. doi: 10.1111/j.1365-2958.1994.tb01016.x. [DOI] [PubMed] [Google Scholar]
  53. Trinh T. Q., Sinden R. R. Preferential DNA secondary structure mutagenesis in the lagging strand of replication in E. coli. Nature. 1991 Aug 8;352(6335):544–547. doi: 10.1038/352544a0. [DOI] [PubMed] [Google Scholar]
  54. Ueno T., Oosawa K., Aizawa S. Domain structures of the MS ring component protein (FliF) of the flagellar basal body of Salmonella typhimurium. J Mol Biol. 1994 Feb 18;236(2):546–555. doi: 10.1006/jmbi.1994.1164. [DOI] [PubMed] [Google Scholar]
  55. Ueno T., Oosawa K., Aizawa S. M ring, S ring and proximal rod of the flagellar basal body of Salmonella typhimurium are composed of subunits of a single protein, FliF. J Mol Biol. 1992 Oct 5;227(3):672–677. doi: 10.1016/0022-2836(92)90216-7. [DOI] [PubMed] [Google Scholar]
  56. 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]
  57. 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]
  58. Yamaguchi K., Masamune Y. Autogenous regulation of synthesis of the replication protein in plasmid pSC101. Mol Gen Genet. 1985;200(3):362–367. doi: 10.1007/BF00425718. [DOI] [PubMed] [Google Scholar]
  59. Yokoseki T., Iino T., Kutsukake K. Negative regulation by fliD, fliS, and fliT of the export of the flagellum-specific anti-sigma factor, FlgM, in Salmonella typhimurium. J Bacteriol. 1996 Feb;178(3):899–901. doi: 10.1128/jb.178.3.899-901.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES