1887

Abstract

This study reports the organization of the gene cluster and characterization of a number of genes that are essential for B-band O antigen biosynthesis in the clinically prevalent serotype O6. Twelve genes were identified that share homology with other LPS and polysaccharide biosynthetic genes. This cluster contains homologues of (encoding the O antigen flippase/translocase) and (which modulates O antigen chain length distribution) genes, typical of a - dependent pathway. However, a complete gene (encoding the O-polymerase) was not found within the cluster. Four biosynthetic genes, , , and , and four putative glycosyltransferase genes, , , and , were identified in the cluster. To characterize their roles in LPS biosynthesis, null mutants of , , , and were generated using a gene-replacement strategy. Mutations in each of these genes caused deficiency in B-band synthesis. The mutant was deficient in both A-band and B-band LPS. WbpL is a bi-functional enzyme which could initiate B-band synthesis through the addition of QuiNAc to undecaprenol phosphate, and A-band synthesis by transferring either a GalNAc or a GlcNAc residue. Another approach used to assign function to the genes was by complementation analysis. Two genes from , and , responsible for the synthesis of a homopolymer of GalNAcA called Vi antigen were used in complementation experiments to verify the functions of and . and restored B-band synthesis in and mutants respectively, implying that and are involved in UDP-GalNAcA synthesis. Although has homology to of the serotype O5 B-band LPS synthesis cluster, complementation analysis using the respective null mutants showed that the genes are not interchangeable. A knockout mutation of (located downstream of ) did not abrogate LPS synthesis in either O5 or O6; therefore, it has been renamed . These results establish the organization of genes involved in B-band O antigen synthesis and provide the evidence to assign functions to a number of LPS biosynthetic genes.

Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-145-12-3505
1999-12-01
2021-06-20
Loading full text...

Full text loading...

/deliver/fulltext/micro/145/12/1453505a.html?itemId=/content/journal/micro/10.1099/00221287-145-12-3505&mimeType=html&fmt=ahah

References

  1. Altschul S. F., Madden T. L., Schaffer A. A., Zhang J., Zhang Z., Miller W., Lipman D. J. 1997; Gapped blast and psi- blast: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402 [CrossRef]
    [Google Scholar]
  2. Arsenault T. L., Hughes D. W., MacLean D. B., Szarek W. A., Kropinski A. M. B., Lam J. S. 1991; Structural studies on the polysaccharide portion of ‘A-band’ lipopolysaccharide from a mutant (AK1401) of Pseudomonas aeruginosa strain PAO1. . Can J Chem 69:1273–1280 [CrossRef]
    [Google Scholar]
  3. Bélanger M., Lam J. S. 1998; Molecular organization and characterization of genes required for the synthesis of Pseudomonas aeruginosa O6 B-band lipopolysaccharide. In 12th Annual North American Cystic Fibrosis ConferenceOct. 15–18Montreal, QC abstract no. 384
    [Google Scholar]
  4. Berry D., Kropinski A. M. 1986; Effect of lipopolysaccharide mutations and temperature on plasmid transformation efficiency in Pseudomonas aeruginosa. Can J Microbiol 32:436–438 [CrossRef]
    [Google Scholar]
  5. Bert F., Lambert-Zechovsky N. 1996; Comparative distribution of resistance patterns and serotypes in Pseudomonas aeruginosa isolates from intensive care units and other wards. J Antimicrob Chemother 37:809–813 [CrossRef]
    [Google Scholar]
  6. Birnboim H. C., Doly J. 1979; A rapid alkaline extraction procedure for screening recombinant plasmids. Nucleic Acids Res 7:1513–1523 [CrossRef]
    [Google Scholar]
  7. Burrows L. L., Lam J. S. 1999; Effect of wzx ( rfbX) mutations on A-band and B-band lipopolysaccharide biosynthesis in Pseudomonas aeruginosa O5. J Bacteriol 181:973–980
    [Google Scholar]
  8. Burrows L. L., Charter D. F., Lam J. S. 1996; Molecular characterization of the Pseudomonas aeruginosa serotype O5 (PAO1) B-band lipopolysaccharide gene cluster. Mol Microbiol 22:481–495 [CrossRef]
    [Google Scholar]
  9. Burrows L. L., Chow D., Lam J. S. 1997; Pseudomonas aeruginosa B-band O antigen chain length is modulated by Wzz (Rol). J Bacteriol 179:1482–1489
    [Google Scholar]
  10. Clarke B. R., Bronner D., Keenleyside W. J., Severn W. B., Richards J. C., Whitfield C. 1995; Role of Rfe and RfbF in the initiation of biosynthesis of d-galactan I, the lipopolysaccharide O antigen from Klebsiella pneumoniae serotype O1. . J Bacteriol 177:5411–5418
    [Google Scholar]
  11. Comstock L. E., Johnson J. A., Michalski J. M., Morris J. G. Jr, Kaper J. P. 1996; Cloning and sequence of a region encoding a surface polysaccharide of Vibrio cholerae O139 and characterization of the insertion site in the chromosome of Vibrio cholerae O1. Mol Microbiol 19:815–826 [CrossRef]
    [Google Scholar]
  12. Cryz S. Jr, Pitt T. L., Furer E., Gremanier R. 1984; Role of lipopolysaccharide in virulence of Pseudomonas aeruginosa. Infect Immun 44:508–513
    [Google Scholar]
  13. Curd H., Liu D., Reeves P. R. 1998; Relationships among the O antigen gene clusters of Salmonella enterica groups B, D1, D2, and D3. J Bacteriol 180:1002–1007
    [Google Scholar]
  14. Dean C. R., Franklund C. V., Retief J. D., Coyne M. J. Jr, Hatano K., Evans D. J., Pier G. B., Goldberg J. B. 1999; Characterization of the serogroup O11 O antigen locus of Pseudomonas aeruginosa PA103. J Bacteriol 181:4275–4284
    [Google Scholar]
  15. Dubray G., Bezard G. 1982; A highly sensitive periodic acid- silver stain for 1,2-diol groups of glycoproteins and polysaccharides in polyacrylamide gels. Anal Biochem 119:325–329 [CrossRef]
    [Google Scholar]
  16. Emara M. G., Tout, N. L., Kaushik A., Lam J. S. 1995; Diverse VH and VK genes encode antibodies to Pseudomonas aeruginosa LPS. J Immunol 155:3912–3921
    [Google Scholar]
  17. Ford N., Nolan C., Ferguson M. 1989; Transfer of DNA to nitrocellulose filters. In Molecular Cloning: a Laboratory Manual, 2nd edn. pp. 9.38–9.40Edited by Sambrook J. , Fritsch E. F., Maniatis T. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  18. Fotheringham I. G., Dacey S. A., Taylor P. P., Smith T. J., Hunter M. G., Finlay M. E., Primrose S. B., Parker D. M., Edwards R. M. 1986; The cloning and sequence analysis of the aspC and tyrB genes from Escherichia coli K12. Comparison of the primary structures of the aspartate aminotransferase and aromatic aminotransferase of Escherichia coli with those of the pig aspartate aminotransferase isoenzymes. Biochem J 234:593–604
    [Google Scholar]
  19. Geremia R. A., Petroni E. A., Ielpi L., Henrissat B. 1996; Towards a classification of glycosyltransferases based on amino acid sequence similarities: prokaryotic α-mannosyltransferases. Biochem J 318:133–138
    [Google Scholar]
  20. Goldberg J. B., Ohman D. E. 1984; Cloning and expression in Pseudomonas aeruginosa of a gene involved with the production of alginate. J Bacteriol 158:1115–1121
    [Google Scholar]
  21. Goldberg J. B., Pier G. B. 1996; Pseudomonas aeruginosa lipopolysaccharides and pathogenesis. Trends Microbiol 4:490–494 [CrossRef]
    [Google Scholar]
  22. Goldberg J. B., Hatano K., Meluleni G. S., Pier G. B. 1992; Cloning and surface expression of P. aeruginosa O antigen in E. coli. Proc Natl Acad Sci USA 89:10716–10720 [CrossRef]
    [Google Scholar]
  23. Hancock R. E. W., Carey A. M. 1979; Outer membrane of Pseudomonas aeruginosa: heat- and 2-mercaptoethanol-modifiable proteins. J Bacteriol 158:115–1121
    [Google Scholar]
  24. Hashimoto Y., Li N., Yokoyama H., Ezaki T. 1993; Complete nucleotide sequence and molecular characterization of viaB region encoding Vi antigen in Salmonella typhi. J Bacteriol 175:4456–4465
    [Google Scholar]
  25. Hitchcock P. J., Brown T. M. 1983; Morphological heterogeneity among Salmonella lipopolysaccharide chemotypes in silver-stained polyacrylamide gels. J Bacteriol 154:269–277
    [Google Scholar]
  26. Hoang T. T., Karkhoff-Schweizer R. R., Kutchma A. J., Schweizer H. P. 1998; A broad-host-range Flp–FRT recombination system for site-specific excision of chromosomally-located DNA sequences: application for isolation of unmarked Pseudomonas aeruginosa mutants. Gene 212:77–86 [CrossRef]
    [Google Scholar]
  27. Houng H.-S. H., Venkatesan M. M. 1998; Genetic analysis of Shigella sonnei form I antigen: identification of a novel IS 630 as an essential element for the form I antigen expression. . Microb Pathog 25:165–173 [CrossRef]
    [Google Scholar]
  28. Huff J. P., Grant B. J., Penning C. A., Sullivan K. F. 1990; Optimization of routine transformation of Escherichia coli with plasmid DNA. . BioTechniques 9:570–577
    [Google Scholar]
  29. de Kievit T. R., Dasgupta T., Schweizer H., Lam J. S. 1995; Molecular cloning and characterization of the rfc gene of Pseudomonas aeruginosa (serotype O5). Mol Microbiol 16:565–574 [CrossRef]
    [Google Scholar]
  30. Knirel Y. A. 1990; Polysaccharide antigens of Pseudomonas aeruginosa. Crit Rev Microbiol 17:273–304 [CrossRef]
    [Google Scholar]
  31. Knirel Y. A., Kochetkov N. K. 1994; The structure of lipopolysaccharides of gram-negative bacteria. III. The structure of O antigens: a review. Biochemistry 59:1325–1383
    [Google Scholar]
  32. Lightfoot J. L., Lam J. S. 1993; Chromosomal mapping, expression and synthesis of lipopolysaccharide in Pseudomonas aeruginosa: a role for guanosine diphospho (GDP)-d-mannose. Mol Microbiol 8:771–782 [CrossRef]
    [Google Scholar]
  33. Liu P. V., Wang S. P. 1990; Three new major somatic antigens of Pseudomonas aeruginosa. J Clin Microbiol 28:922–925
    [Google Scholar]
  34. Liu D., Cole R. A., Reeves P. R. 1996; An O antigen processing function for Wzx (RfbX): a promising candidate for O-unit flippase. J Bacteriol 178:2102–2107
    [Google Scholar]
  35. Liu P. V., Matsumoto H., Kusama H., Bergan T. 1983; Survey of heat-stable major somatic antigens of Pseudomonas aeruginosa. Int J Syst Bacteriol 33:256–264 [CrossRef]
    [Google Scholar]
  36. Ma S., Selvaraj U., Ohman D. E., Quarless R., Hassett D. J., Wozniak D. J. 1998; Phosphorylation- independent activity of the response regulators AlgB and AlgR in promoting alginate biosynthesis in mucoid Pseudomonas aeruginosa. J Bacteriol 180:956–968
    [Google Scholar]
  37. MacIntyre S., Lucken R., Owen P. 1986; Smooth lipopolysaccharide is the major protective antigen for mice in the surface extract from IATS serotype 6 contributing to the polyvalent Pseudomonas aeruginosa vaccine PEV. Infect Immun 52:76–84
    [Google Scholar]
  38. Macpherson D. F., Manning P. A., Morona R. 1994; Characterization of the dTDP rhamnose bioynthetic genes encoded in the rfb locus of Shigella flexneri. Mol Microbiol 11:281–292 [CrossRef]
    [Google Scholar]
  39. Masoud H., Sadovskaya I., De Kievit T., Altman E., Richards J. C., Lam J. S. 1995; Structural elucidation of the lipopolysaccharide core region of the O-chain-deficient mutant strain A28 from Pseudomonas aeruginosa serotype O6 (International Antigenic Typing Scheme). J Bacteriol 177:6718–6726
    [Google Scholar]
  40. May T. B., Shinabarger D., Maharaj R.9 other authors 1991; Alginate synthesis by Pseudomonas aeruginosa: a key pathogenic factor in chronic pulmonary infections of cystic fibrosis patients. Clin Microbiol Rev 4:191–206
    [Google Scholar]
  41. Naide Y., Nikaido H., Mäkelä P. H., Wilkinson R. G., Stocker B. A. D. 1965; Semirough strains of Salmonella. Proc Natl Acad Sci USA 53:147–153 [CrossRef]
    [Google Scholar]
  42. Nakai Y., Hayashi H., Kagamiyama H. 1996; Cloning and characterization of the tyrB gene from Salmonella typhimurium. . Biochim Biophys Acta 1308:189–192 [CrossRef]
    [Google Scholar]
  43. Pitt T. L. 1988; Epidemiological typing of Pseudomonas aeruginosa. Eur J Clin Microbiol Infect Dis 7:238–247 [CrossRef]
    [Google Scholar]
  44. Reeves P. 1993; Evolution of Salmonella O antigen variation by interspecific gene transfer on a large scale. Trends Genet 9:17–22 [CrossRef]
    [Google Scholar]
  45. Reeves P. R., Hobbs M., Valvano M. A.8 other authors 1996; Bacterial polysaccharide synthesis and gene nomenclature. Trends Microbiol 4:495–503 [CrossRef]
    [Google Scholar]
  46. Rocchetta H. L., Burrows L. L., Pacan J. C., Lam J. S. 1998; Three rhamnosyltransferases responsible for assembly of the A-band d-rhamnan polysaccharide in Pseudomonas aeruginosa: a fourth transferase, WbpL, is required for initiation of both A-band and B-band LPS synthesis. Mol Microbiol 28:1103–1119 [CrossRef]
    [Google Scholar]
  47. Sambrook J., Fritsch E. F., Maniatis T. 1989 Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  48. Schnaitman C. A., Klena J. D. 1993; Genetics of lipopolysaccharide biosynthesis in enteric bacteria. Microbiol Rev 57:655–682
    [Google Scholar]
  49. Schweizer H. P. 1993; Small broad-host- range gentamicin resistance gene cassettes for site-specific insertion and deletion mutagenesis. BioTechniques 15:831–833
    [Google Scholar]
  50. Schweizer H. P. 1994; A method for construction of bacterial hosts for lac-based cloning and expression vectors: α-complementation and regulated expression. . BioTechniques 17:452–456
    [Google Scholar]
  51. Schweizer H. P., Hoang T. T. 1995; An improved system for gene replacement and xylE fusion analysis in Pseudomonas aeruginosa. Gene 158:15–22 [CrossRef]
    [Google Scholar]
  52. Simon R., Priefer U., Pühler A. 1983; A broad-host-range mobilization system for in vivo genetic engineering: transposon mutagenesis in Gram-negative bacteria. . Bio/Technology 1:784–791 [CrossRef]
    [Google Scholar]
  53. Skurnik M., Venho R., Toivanen P., Al-Hendy A. 1995; A novel locus of Yersinia enterocolitica serotype O:3 involved in lipopolysaccharide outer core biosynthesis. Mol Microbiol 17:575–594 [CrossRef]
    [Google Scholar]
  54. Stroeher U. H., Parasivam G., Dredge B. K., Manning P. A. 1997; Novel Vibrio cholerae O139 genes involved in lipopolysaccharide biosynthesis. . J Bacteriol 179:2740–2747
    [Google Scholar]
  55. Vachee A., Scheftel J. M., Husson M. O., Izard, D., Ross P., Monteil H. 1997; Tricentric study of the sensitivity of Pseudomonas aeruginosa serotyping to beta-lactams and aminoglycosides. Pathol Biol 45:357–362
    [Google Scholar]
  56. Virlogeux I., Waxin H., Ecobichon C., Popoff M. Y. 1995; Role of the viaB locus in synthesis, transport and expression of Salmonella typhi Vi antigen. Microbiology 141:3039–3047 [CrossRef]
    [Google Scholar]
  57. Waxin H., Virlogeux I., Kolyva S., Popoff M. Y. 1993; Identification of six open reading frames in the Salmonella enterica subsp. enterica ser. Typhi viaB locus involved in Vi antigen production. Res Microbiol 144:363–371 [CrossRef]
    [Google Scholar]
  58. West S. E. H., Schweizer H. P., Dall C., Sample A. K., Runyen-Janecky L. J. 1994; Construction of improved Escherichia–Pseudomonas shuttle vectors derived from pUC18/19 and the sequence of the region required for their replication in Pseudomonas aeruginosa. Gene 128:81–86
    [Google Scholar]
  59. Woods D. E., Lam J. S., Paranchych W., Speert D. P., Campbell M., Godfrey A. J. 1997; Correlation of Pseudomonas aeruginosa virulence factors from clinical and environmental isolates with pathogenicity in the neutropenic mouse. Can J Microbiol 43:541–551 [CrossRef]
    [Google Scholar]
  60. Wyk P., Reeves P. R. 1989; Identification and sequence of the gene for abequose synthetase which confers antigenic specificity on group B Salmonellae: homology with galactose epimerase. . J Bacteriol 171:5687–5693
    [Google Scholar]
  61. Zhang L., Radziejewska-Lebrecht J., Krajewska-Pietrasik D., Toivanen P., Skurnik M. 1997; Molecular and chemical characterization of the lipopolysaccharide O antigen and its role in the virulence of Yersinia enterocolitica serotype O:8. Mol Microbiol 23:63–76 [CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/00221287-145-12-3505
Loading
/content/journal/micro/10.1099/00221287-145-12-3505
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error