1887

Abstract

strains PaW8 and PRS2000 produce flagellins with apparent molecular masses of 81 kDa and 50 kDa respectively. Two Tn5 insertion mutants of PaW8 lacking the ability to bind the flagellin-specific monoclonal antibody MLV1 were isolated. Mutant PaW8- contained a Tn5 insertion within a 2.6 kb RI fragment of the chromosome carrying putative basal body genes. DNA and deduced protein sequences suggested the presence on this fragment of two complete genes homologous to and from . The insertion of Tn5 occurred in the locus and appeared severely to reduce expression of the flagellin gene. A Tn5-containing fragment of DNA from a second mutant, PaW8-, was cloned and found to contain sequences that hybridized strongly with the flagellin gene. A 2.3 kb HindIII fragment containing all but 62 bp of the PaW8 flagellin gene was cloned and used as a probe to identify clones carrying the equivalent gene from PRS2000. Flagellin genes from both strains were sequenced and their amino acid sequences deduced. Both flagellins were found to contain conserved amino- and carboxy-terminal regions when compared to other flagellins, with the central region being more variable. The epitope for MLV1 is likely to lie within this central region of PaW8 flagellin. The deduced molecular mass of PaW8 flagellin (68 kDa) differed significantly from its apparent molecular mass estimated by PAGE, possibly as a consequence of post-translational modification. This was not the case with flagellin from PRS2000, where the predicted and apparent molecular masses were similar.

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1994-08-01
2024-12-08
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References

  1. Aitken A., Geisow M.J., Findlay J.B.C., Holmes C., Yarwood A. Peptide preparation and characterization. In Protein Sequencing 1989 Edited by Findlay J.B.C., Geisow M.J. Oxford: IRL Press; a Practical Approach, pp 43–68
    [Google Scholar]
  2. Albertini A.M., Caramori T., Crabb W.D., Scoffone F., Galizzi A. The fla A locus of Bacillus subtilis is part of a large operon coding for flagellar structures, motility functions, and an ATPaselike polypeptide. J Bacteriol 1991; 173:3573–3579
    [Google Scholar]
  3. Allison J.S., Dawson M., Drake D., Montie T.C. Electrophoretic separation and molecular weight characterization of Pseudomonas aeruginosa H-antigen flagellins. Infect lmmun 1985; 49:770–774
    [Google Scholar]
  4. Arnosti D.N., Chamberlin M.J. Secondary a factor controls transcription of flagellar and chemotaxis genes in Escherichia coli. Proc Natl Acad Sci USA 1989; 86:830–834
    [Google Scholar]
  5. Bartlett D.H., Frantz B.B., Matsumura P. Flagellar transcriptional activators FlbB and Flal: gene sequences and 5' consensus sequences of operons under FlbB and Flal control. J Bacteriol 1988; 170:1575–1581
    [Google Scholar]
  6. Bergman K., Nulty E., Su L.H. Mutations in the two flagellin genes of Rhizobium meliloti. J Bacteriol 1991; 173:3716–3723
    [Google Scholar]
  7. Bolivar F. Construction and characterisation of new cloning vehicles. III. Derivatives of plasmid pBR322 carrying unique EroRI sites for selection of EroRI generated recombinant DNA molecules. Gene 1978; 4:121–136
    [Google Scholar]
  8. Bolivar F., Rodriguez R.L., Green P.J., Belach H.C., Boyer H.W., Crosa J.J., Falkow S. Construction and characterization of new cloning vehicles. II. A multi-purpose cloning system. Gene 1977; 2:95–113
    [Google Scholar]
  9. Boulnois G.J., Varley J.M., Sharpe G.S., Franklin F.C.H. Transposon donor plasmids, based on ColIb-P9, for use in Pseudomonas putida and a variety of other Gram-negative bacteria. Mol & Gen Genet 1985; 200:65–67
    [Google Scholar]
  10. Champer R., Dingwall A., Shapiro L. Cascade regulation of Caulobacter flagellar and chemotaxis genes. J Mol Biol 1987; 194:71–80
    [Google Scholar]
  11. Chou P.Y., Fasman G.D. Prediction of the secondary structure of proteins from their amino acid sequences. Adv Enzymol 1978; 47:45–148
    [Google Scholar]
  12. Cohen S.N., Chang A.C.Y., Hsu C.L. Non-chromosomal antibiotic resistance in bacteria: genetic transformation of Escherichia coli by R factor DNA. Proc Natl Acad Sci USA 1972; 69:2110–2114
    [Google Scholar]
  13. Dower W.J., Miller J.F., Ragsdale C.W. High efficiency transformation of E coli by high voltage electroporation. Nucleic Acids Res 1988; 16:6127–6145
    [Google Scholar]
  14. Gamier J., Osguthorpe D.J., Robson B. Analysis of the accuracy and implications of simple methods for predicting the secondary structure of globular proteins. J Mol Biol 1978; 120:97–120
    [Google Scholar]
  15. Gassmann G.S., Jacobs E., Deutzmann R., Gobel U.B. Analysis of the Borrelia burgdorferi GeHo fla gene and antigenic characterization of its product. J Bacteriol 1991; 173:1452–1459
    [Google Scholar]
  16. Gill P.R., Agabian N. The nucleotide sequence of the Mr = 28, 500 flagellin gene of Caulobacter crescentus. J Biol Chem 1983; 258:7395–7401
    [Google Scholar]
  17. Girvitz S.C., Bacchetti S., Rainbow A.J., Graham F.W. A rapid and efficient procedure for the purification of DNA from agarose gels. Anal Biochem 1980; 106:492–496
    [Google Scholar]
  18. Harlow E., Lane D. Antibodies: a Eaboratory Manual 1988 Cold Spring, Harbor NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  19. Harshey R.M., Estepa G., Yanagi H. Cloning and nucleotide sequence of a flagellin-coding gene (hag) from Serratia marcescens 274. Gene 1989; 79:1–8
    [Google Scholar]
  20. Holmes D.S., Quigley M. A rapid boiling method for the preparation of bacterial plasmids. Anal Biochem 1981; 114:193–197
    [Google Scholar]
  21. Homma M., Fujita H., Yamaguchi S., Lino T. Regions of Salmonella typhimurium flagellin essential for its polymerization and excretion. J Bacteriol 1987a; 169:291–296
    [Google Scholar]
  22. Homma M., Komeda Y., Lino 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 1987b; 169:1493–1498
    [Google Scholar]
  23. Homma M., Ohnishi K., Lino T., Macnab R.M. Identification of flagellar hook and basal body gene products (FlaV, FlaVI, FlaVII, and FlaVIII) in Salmonella typhimurium. J Bacteriol 1987c; 169:3617–3624
    [Google Scholar]
  24. Homma M., Kutsukake K., Hasebe M., Lino T., Macnab R.M. A family of structurally related proteins in the flagellar basal body of Salmonella typhimurium. J Mol Biol 1990; 211:465–477
    [Google Scholar]
  25. Inouye S., Kimoto M., Nakazawa A., Nakazawa T. Presence of flagella in Pseudomonas putida is dependent on the ntrA (rpoN) gene. Mol & Gen Genet 1990; 221:295–298
    [Google Scholar]
  26. 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; 171:3890–3900
    [Google Scholar]
  27. Joys T.M. The covalent structure of phase-1 flagellar filament protein of Salmonella typhimurium and its comparison with other flagellins. J Biol Chem 1985; 260:15758–15761
    [Google Scholar]
  28. Joys T.M., Kim H. Identification of e-N-methyllysine residues in the phase-1 flagellar protein of Salmonella typhimurium. Microbios Lett 1978; 76:5–68
    [Google Scholar]
  29. Kelly-Wintenberg K., Anderson T., Montie T.C. Phosphorylated tyrosine in the flagellum filament protein of Pseudomonas aeruginosa. J Bacteriol 1990; 172:5135–5139
    [Google Scholar]
  30. Kelly-Wintenberg K., Montie T.C. Cloning and expression of Pseudomonas aeruginosa flagellin in Escherichia coli. J Bacteriol 1989; 171:6357–6362
    [Google Scholar]
  31. Khambaty F.M., Ely B. Molecular genetics of the flgl region and its role in flagellum biosynthesis in Caulobacter crescentus. J Bacteriol 1992; 174:4101–4109
    [Google Scholar]
  32. Komeda Y. Transcriptional control of flagellar genes in Escherichia coli K-12. J Bacteriol 1986; 168:1315–1318
    [Google Scholar]
  33. Kutsukake K., Ohya Y., Lino T. Transcriptional analysis of the flagellar regulon on Salmonella typhimurium. J Bacteriol 1990; 172:741–747
    [Google Scholar]
  34. Kuwajima G., Asaka J.I., Fujiwara T., Node K., Kondo E. Nucleotide-sequence of the hag gene encoding flagellin of Escherichia coli. J Bacteriol 1986; 168:1479–1483
    [Google Scholar]
  35. Kuwajima G., Kawagishi I., Homma M., Asaka J.I., Kondo E., Macnab R.M. Export of an N-terminal fragment of Escherichia coli flagellin by a flagellum-specific pathway. Proc Natl Acad Sci USA 1989; 86:4953–4957
    [Google Scholar]
  36. Kyte J., Doolittle R.F. A simple method for displaying the hydropathic character of a protein. J Mol Biol 1982; 157:105–132
    [Google Scholar]
  37. Laemmli U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227:680–685
    [Google Scholar]
  38. Logan S.M., Trust T.J., Guerry P. Evidence for posttranslational modification and gene duplication of Campylobacter flagellin. J Bacteriol 1989; 171:3031–3038
    [Google Scholar]
  39. Macnab R.M. Genetics and biogenesis of bacterial flagella. Annu Rev Genet 1992; 26:131–158
    [Google Scholar]
  40. McDonough M.W., Smith S.E. Molecular weight variation among bacterial flagellins. Microbios 1976; 16:49–53
    [Google Scholar]
  41. Martin J.H., Savage D.C. Cloning, nucleotide sequence, and taxonomic implications of the flagellin gene of Roseburia cecicola. J Bacterial 1988; 170:2612–2617
    [Google Scholar]
  42. Morgan J.A.W., Winstanley C., Pickup R.W., Saunders J.R. Rapid immunocapture of Pseudomonas putida cells from lake water by using bacterial flagella. Appl Environ Microbiol 1991; 57:503–509
    [Google Scholar]
  43. Newton S.M.G., Jacob C.O., Stocker B.A.D. Immune-response to cholera-toxin epitope inserted in Salmonella flagellin. Science 1989; 244:70–72
    [Google Scholar]
  44. Nuijten P.J.M., Vanasten F.J.A.M., Gaastra W., Vanderzeijst B.A.M. Structural and functional analysis of two Campylobacter jejuni flagellin genes. J Biol Chem 1990; 265:17798–17804
    [Google Scholar]
  45. Nuijten P.J.M., Vanderzeijst B.A.M., Newell D.G. Localization of immunogenic regions on the flagellin proteins of Campylobacter jejuni 81116. Infect lmmun 1991; 59:1100–1105
    [Google Scholar]
  46. Oliver D. Protein secretion in Escherichia coli. Annu Rev Microbiol 1985; 39:615–648
    [Google Scholar]
  47. Pearson W.R., Lipman D.J. Improved tools for biological sequence comparison. Proc Natl Acad Sci USA 1988; 85:2444–2448
    [Google Scholar]
  48. Rasiah G., Schiltz E., Reichert J., Voft A. Purification and characterization of a tryptic peptide of Borrelia burgdorferi flagellin which reduces cross-reactivity in immunoblots and ELISA. J Gen Microbiol 1992; 138:147–154
    [Google Scholar]
  49. Sanger F., Nicklen S., Coulson R.A. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 1977; 74:5463–5467
    [Google Scholar]
  50. Shine J., Dalgarno L. The S'-terminal sequence of Escherichia coli 16S ribosomal RNA: complementary to nonsense triplets and ribosome binding sites. Proc Natl Acad Sci USA 1974; 71:1342–1346
    [Google Scholar]
  51. Simon M.I., Emerson S.U., Shaper J.H., Bernard P.D., Glazer A.N. Classification of Bacillus subtilis flagellins. J Bacteriol 1977; 130:200–204
    [Google Scholar]
  52. Smith N.H., Selander R.K. Sequence invariance of the antigen-coding central region of the phase 1 flagellar filament gene (fliC) among strains of Salmonella typhimurium. J Bacteriol 1990; 172:603–609
    [Google Scholar]
  53. Smith N.H., Beltran P., Selander R.K. Recombination of Salmonella phase-1 flagellin genes generates new serovars. J Bacteriol 1990; 172:2209–2216
    [Google Scholar]
  54. Stallmeyer M.J.B., Hahnenberger K.M., Sosinsky G.E., Shapiro L., Derosier D.J. Image reconstruction of the flagellar basal body of Caulobacter crescentus. J Mol Biol 1989a; 205:511–518
    [Google Scholar]
  55. Stallmeyer M.J.B., Aizawa S.-I., Macnab R.M., Derosier D.J. Image reconstruction of the flagellar basal body of Salmonella typhimurium. J Mol Biol 1989b; 205:519–528
    [Google Scholar]
  56. Totten P.A., Lory S. Characterization of the type a flagellin gene from Pseudomonas aeruginosa PAK. J Bacteriol 1990; 172:7188–7199
    [Google Scholar]
  57. Totten P.A., Lara J.C., Lory S. The rpoN gene product of Pseudomonas aeruginosa is required for expression of diverse genes, including the flagellin gene. J Bacteriol 1990; 172:389–396
    [Google Scholar]
  58. Towbin H., Staehelin J., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 1979; 76:4350–4352
    [Google Scholar]
  59. Wei L.N., Joys T.M. The nucleotide sequence of the H-lr gene of Salmonella rubislaw. Nucleic Acids Res 1986; 14:8227
    [Google Scholar]
  60. Wieland F., Paul G., Sumper M. Halobacterial flagellins are sulfated glycoproteins. J Biol Chem 1985; 260:15180–15185
    [Google Scholar]
  61. Winstanley G., Morgan J.A.W., Pickup R.W., Jones J.G., Saunders J.R. Differential regulation of lambda pL and lambda pR promoters by a cl repressor in a broad-host-range thermoregulated plasmid marker system. Appl Environ Microbio 1989; l55:771–777
    [Google Scholar]
  62. Winstanley G., Carter J.P., Seasman M., Morgan J.A.W., Pickup R.W., Saunders J.R. A comparison of the survival of stable and unstable chromosomally-located xylE marker cassettes as an indicator of cell division within populations of Pseudomonas putida released into lake water and soil. Microb Releases 1993; 2:97–107
    [Google Scholar]
  63. Worsey M.J., Williams P.A. Metabolism of toluene and xylenes by Pseudomonas putida (arvilla) mt-2: evidence for a new function of the TOL plasmid. J Bacteriol 1975; 124:7–13
    [Google Scholar]
  64. Yanisch-Perron C., Vieira J., Messing J. Improved M13 cloning vectors and host strains: nucleotide sequences of the M13mpl8 and pUC19 vectors. Gene 1985; 33:103–119
    [Google Scholar]
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