Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1991 Mar;173(6):2077–2085. doi: 10.1128/jb.173.6.2077-2085.1991

Expression of two Rhizobium meliloti flagellin genes and their contribution to the complex filament structure.

E Pleier 1, R Schmitt 1
PMCID: PMC207743  PMID: 2002009

Abstract

The complex flagellar filaments of Rhizobium meliloti are composed of two related (87% identical) flagellins that are encoded by closely linked, separately transcribed genes, flaA and flaB (E. Pleier and R. Schmitt, J. Bacteriol. 171:1467-1475, 1989). To elucidate the role of the subunits, A and B, in assembling the complex filament, the wild-type alleles were replaced with defective ones containing a 2,249-bp deletion (accompanied by substitution of a kanamycin resistance cartridge), which eliminates 74% of flaA (3' end) and 85% of flaB (5' end). The resulting nonmotile, filamentless mutant, RU11011, was tested for complementation with wild-type flaA, flaB, and flaA flaB genes provided on the multiple-copy vector pRK290. Whereas flaA alone did not restore motility and filament production, both flaB and flaA flaB restored 20 to 30% of wild-type motility. Apparent causes of this reduced motility were fewer flagella per cell and/or shortened filaments sometimes ending in unusually thin, fragile structures. Tests with enzyme-linked antiflagellin antibodies indicated that flaA is expressed at higher levels than flaB and that multiple copies of flaA lead to reduced flagellin export. We conclude that the proximal portion of the complex filament is assembled from B subunits (not produced sufficiently to form full-length flagella) and that the distal portion is made from A subunits. Multiple copies of the strong flaA promoter may offset transcriptional controls that regulate the synthesis of flagellar structures required for flagellin export.

Full text

PDF
2078

Images in this article

2082Image
on p.2082
2083Image
on p.2083
2084Image
on p.2084

Selected References

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

  1. Bachmann B. J. Linkage map of Escherichia coli K-12, edition 7. Microbiol Rev. 1983 Jun;47(2):180–230. doi: 10.1128/mr.47.2.180-230.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Berg H. C., Tedesco P. M. Transient response to chemotactic stimuli in Escherichia coli. Proc Natl Acad Sci U S A. 1975 Aug;72(8):3235–3239. doi: 10.1073/pnas.72.8.3235. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Beringer J. E. R factor transfer in Rhizobium leguminosarum. J Gen Microbiol. 1974 Sep;84(1):188–198. doi: 10.1099/00221287-84-1-188. [DOI] [PubMed] [Google Scholar]
  4. Clewell D. B., Helinski D. R. Supercoiled circular DNA-protein complex in Escherichia coli: purification and induced conversion to an opern circular DNA form. Proc Natl Acad Sci U S A. 1969 Apr;62(4):1159–1166. doi: 10.1073/pnas.62.4.1159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Ditta G., Stanfield S., Corbin D., Helinski D. R. Broad host range DNA cloning system for gram-negative bacteria: construction of a gene bank of Rhizobium meliloti. Proc Natl Acad Sci U S A. 1980 Dec;77(12):7347–7351. doi: 10.1073/pnas.77.12.7347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Driks A., Bryan R., Shapiro L., DeRosier D. J. The organization of the Caulobacter crescentus flagellar filament. J Mol Biol. 1989 Apr 20;206(4):627–636. doi: 10.1016/0022-2836(89)90571-8. [DOI] [PubMed] [Google Scholar]
  7. Figurski D. H., Helinski D. R. Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc Natl Acad Sci U S A. 1979 Apr;76(4):1648–1652. doi: 10.1073/pnas.76.4.1648. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gerl L., Sumper M. Halobacterial flagellins are encoded by a multigene family. Characterization of five flagellin genes. J Biol Chem. 1988 Sep 15;263(26):13246–13251. [PubMed] [Google Scholar]
  9. 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]
  10. 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]
  11. 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]
  12. Homma M., Fujita H., Yamaguchi S., Iino T. Regions of Salmonella typhimurium flagellin essential for its polymerization and excretion. J Bacteriol. 1987 Jan;169(1):291–296. doi: 10.1128/jb.169.1.291-296.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. Huet J., Sentenac A., Fromageot P. Spot-immunodetection of conserved determinants in eukaryotic RNA polymerases. Study with antibodies to yeast RNA polymerases subunits. J Biol Chem. 1982 Mar 10;257(5):2613–2618. [PubMed] [Google Scholar]
  15. Iino T. Genetics of structure and function of bacterial flagella. Annu Rev Genet. 1977;11:161–182. doi: 10.1146/annurev.ge.11.120177.001113. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. Joys T. M. The covalent structure of the phase-1 flagellar filament protein of Salmonella typhimurium and its comparison with other flagellins. J Biol Chem. 1985 Dec 15;260(29):15758–15761. [PubMed] [Google Scholar]
  18. Krupski G., Götz R., Ober K., Pleier E., Schmitt R. Structure of complex flagellar filaments in Rhizobium meliloti. J Bacteriol. 1985 Apr;162(1):361–366. doi: 10.1128/jb.162.1.361-366.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kuwajima G., Asaka J., Fujiwara T., Fujiwara T., Node K., Kondo E. Nucleotide sequence of the hag gene encoding flagellin of Escherichia coli. J Bacteriol. 1986 Dec;168(3):1479–1483. doi: 10.1128/jb.168.3.1479-1483.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. LaVallie E. R., Stahl M. L. Cloning of the flagellin gene from Bacillus subtilis and complementation studies of an in vitro-derived deletion mutation. J Bacteriol. 1989 Jun;171(6):3085–3094. doi: 10.1128/jb.171.6.3085-3094.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Macnab R. M., DeRosier D. J. Bacterial flagellar structure and function. Can J Microbiol. 1988 Apr;34(4):442–451. doi: 10.1139/m88-077. [DOI] [PubMed] [Google Scholar]
  22. Maruyama M., Lodderstaedt G., Schmitt R. Purification and biochemical properties of complex flagella isolated from Rhizobium lupini H13-3. Biochim Biophys Acta. 1978 Jul 21;535(1):110–124. doi: 10.1016/0005-2795(78)90038-7. [DOI] [PubMed] [Google Scholar]
  23. McKenney K., Shimatake H., Court D., Schmeissner U., Brady C., Rosenberg M. A system to study promoter and terminator signals recognized by Escherichia coli RNA polymerase. Gene Amplif Anal. 1981;2:383–415. [PubMed] [Google Scholar]
  24. Novick R. P., Clowes R. C., Cohen S. N., Curtiss R., 3rd, Datta N., Falkow S. Uniform nomenclature for bacterial plasmids: a proposal. Bacteriol Rev. 1976 Mar;40(1):168–189. doi: 10.1128/br.40.1.168-189.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]
  26. Schmitt R., Raska I., Mayer F. Plain and complex flagella of Pseudomonas rhodos: analysis of fine structure and composition. J Bacteriol. 1974 Feb;117(2):844–857. doi: 10.1128/jb.117.2.844-857.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Simon R., O'Connell M., Labes M., Pühler A. Plasmid vectors for the genetic analysis and manipulation of rhizobia and other gram-negative bacteria. Methods Enzymol. 1986;118:640–659. doi: 10.1016/0076-6879(86)18106-7. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. Tautz D., Renz M. An optimized freeze-squeeze method for the recovery of DNA fragments from agarose gels. Anal Biochem. 1983 Jul 1;132(1):14–19. doi: 10.1016/0003-2697(83)90419-0. [DOI] [PubMed] [Google Scholar]
  30. Thomashow L. S., Rittenberg S. C. Waveform analysis and structure of flagella and basal complexes from Bdellovibrio bacteriovorus 109J. J Bacteriol. 1985 Sep;163(3):1038–1046. doi: 10.1128/jb.163.3.1038-1046.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Trachtenberg S., DeRosier D. J., Aizawa S., Macnab R. M. Pairwise perturbation of flagellin subunits. The structural basis for the differences between plain and complex bacterial flagellar filaments. J Mol Biol. 1986 Aug 20;190(4):569–576. doi: 10.1016/0022-2836(86)90242-1. [DOI] [PubMed] [Google Scholar]
  32. Trachtenberg S., DeRosier D. J., Macnab R. M. Three-dimensional structure of the complex flagellar filament of Rhizobium lupini and its relation to the structure of the plain filament. J Mol Biol. 1987 Jun 5;195(3):603–620. doi: 10.1016/0022-2836(87)90185-9. [DOI] [PubMed] [Google Scholar]
  33. Ubben D., Schmitt R. A transposable promoter and transposable promoter probes derived from Tn1721. Gene. 1987;53(1):127–134. doi: 10.1016/0378-1119(87)90100-4. [DOI] [PubMed] [Google Scholar]
  34. Wei L. N., Joys T. M. Covalent structure of three phase-1 flagellar filament proteins of Salmonella. J Mol Biol. 1985 Dec 20;186(4):791–803. doi: 10.1016/0022-2836(85)90397-3. [DOI] [PubMed] [Google Scholar]
  35. 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