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. 1995 Jun 6;92(12):5754–5758. doi: 10.1073/pnas.92.12.5754

Endogenous mutagenesis by an insertion sequence element identifies Aeromonas salmonicida AbcA as an ATP-binding cassette transport protein required for biogenesis of smooth lipopolysaccharide.

S Chu 1, B Noonan 1, S Cavaignac 1, T J Trust 1
PMCID: PMC41775  PMID: 7777581

Abstract

Analysis of an Aeromonas salmonicida A layer-deficient/O polysaccharide-deficient mutant carrying a Tn5 insertion in the structural gene for A protein (vapA) showed that the abcA gene immediately downstream of vapA had been interrupted by the endogenous insertion sequence element ISAS1. Immunoelectron microscopy showed that O polysaccharides did not accumulate at the inner membrane-cytoplasm interface of this mutant. abcA encodes an unusual protein; it carries both an amino-terminal ATP-binding cassette (ABC) domain showing high sequence similarity to ABC proteins implicated in the transport of certain capsular and O polysaccharides and a carboxyl-terminal potential DNA-binding domain, which distinguishes AbcA from other polysaccharide transport proteins in structural and evolutionary terms. The smooth lipopolysaccharide phenotype was restored by complementation with abcA but not by abcA carrying site-directed mutations in the sequence encoding the ATP-binding site of the protein. The genetic organization of the A. salmonicida ABC polysaccharide system differs from other bacteria. abcA also differs in apparently being required for both O-polysaccharide synthesis and in energizing the transport of O polysaccharides to the cell surface.

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Selected References

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

  1. Belland R. J., Trust T. J. Synthesis, export, and assembly of Aeromonas salmonicida A-layer analyzed by transposon mutagenesis. J Bacteriol. 1985 Sep;163(3):877–881. doi: 10.1128/jb.163.3.877-881.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Boeke J. D., Model P. A prokaryotic membrane anchor sequence: carboxyl terminus of bacteriophage f1 gene III protein retains it in the membrane. Proc Natl Acad Sci U S A. 1982 Sep;79(17):5200–5204. doi: 10.1073/pnas.79.17.5200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chart H., Shaw D. H., Ishiguro E. E., Trust T. J. Structural and immunochemical homogeneity of Aeromonas salmonicida lipopolysaccharide. J Bacteriol. 1984 Apr;158(1):16–22. doi: 10.1128/jb.158.1.16-22.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chu S., Cavaignac S., Feutrier J., Phipps B. M., Kostrzynska M., Kay W. W., Trust T. J. Structure of the tetragonal surface virulence array protein and gene of Aeromonas salmonicida. J Biol Chem. 1991 Aug 15;266(23):15258–15265. [PubMed] [Google Scholar]
  5. Chu S., Trust T. J. An Aeromonas salmonicida gene which influences a-protein expression in Escherichia coli encodes a protein containing an ATP-binding cassette and maps beside the surface array protein gene. J Bacteriol. 1993 May;175(10):3105–3114. doi: 10.1128/jb.175.10.3105-3114.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Dooley J. S., Engelhardt H., Baumeister W., Kay W. W., Trust T. J. Three-dimensional structure of an open form of the surface layer from the fish pathogen Aeromonas salmonicida. J Bacteriol. 1989 Jan;171(1):190–197. doi: 10.1128/jb.171.1.190-197.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dooley J. S., Lallier R., Shaw D. H., Trust T. J. Electrophoretic and immunochemical analyses of the lipopolysaccharides from various strains of Aeromonas hydrophila. J Bacteriol. 1985 Oct;164(1):263–269. doi: 10.1128/jb.164.1.263-269.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dooley J. S., Trust T. J. Surface protein composition of Aeromonas hydrophila strains virulent for fish: identification of a surface array protein. J Bacteriol. 1988 Feb;170(2):499–506. doi: 10.1128/jb.170.2.499-506.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Filip C., Fletcher G., Wulff J. L., Earhart C. F. Solubilization of the cytoplasmic membrane of Escherichia coli by the ionic detergent sodium-lauryl sarcosinate. J Bacteriol. 1973 Sep;115(3):717–722. doi: 10.1128/jb.115.3.717-722.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Frosch M., Edwards U., Bousset K., Krausse B., Weisgerber C. Evidence for a common molecular origin of the capsule gene loci in gram-negative bacteria expressing group II capsular polysaccharides. Mol Microbiol. 1991 May;5(5):1251–1263. doi: 10.1111/j.1365-2958.1991.tb01899.x. [DOI] [PubMed] [Google Scholar]
  11. Gustafson C. E., Chu S., Trust T. J. Mutagenesis of the paracrystalline surface protein array of Aeromonas salmonicida by endogenous insertion elements. J Mol Biol. 1994 Apr 8;237(4):452–463. doi: 10.1006/jmbi.1994.1247. [DOI] [PubMed] [Google Scholar]
  12. Hashimoto Y., Orellana A., Gil G., Hirschberg C. B. Molecular cloning and expression of rat liver N-heparan sulfate sulfotransferase. J Biol Chem. 1992 Aug 5;267(22):15744–15750. [PubMed] [Google Scholar]
  13. Higgins D. G., Sharp P. M. CLUSTAL: a package for performing multiple sequence alignment on a microcomputer. Gene. 1988 Dec 15;73(1):237–244. doi: 10.1016/0378-1119(88)90330-7. [DOI] [PubMed] [Google Scholar]
  14. Ishiguro E. E., Ainsworth T., Shaw D. H., Kay W. W., Trust T. J. A lipopolysaccharide-specific bacteriophage for Aeromonas salmonicida. Can J Microbiol. 1983 Oct;29(10):1458–1461. doi: 10.1139/m83-223. [DOI] [PubMed] [Google Scholar]
  15. Ishiguro E. E., Kay W. W., Ainsworth T., Chamberlain J. B., Austen R. A., Buckley J. T., Trust T. J. Loss of virulence during culture of Aeromonas salmonicida at high temperature. J Bacteriol. 1981 Oct;148(1):333–340. doi: 10.1128/jb.148.1.333-340.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Jann K., Jann B. Capsules of Escherichia coli, expression and biological significance. Can J Microbiol. 1992 Jul;38(7):705–710. doi: 10.1139/m92-116. [DOI] [PubMed] [Google Scholar]
  17. Kay W. W., Buckley J. T., Ishiguro E. E., Phipps B. M., Monette J. P., Trust T. J. Purification and disposition of a surface protein associated with virulence of Aeromonas salmonicida. J Bacteriol. 1981 Sep;147(3):1077–1084. doi: 10.1128/jb.147.3.1077-1084.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kimura A., Patrick C. C., Miller E. E., Cope L. D., McCracken G. H., Jr, Hansen E. J. Haemophilus influenzae type b lipooligosaccharide: stability of expression and association with virulence. Infect Immun. 1987 Sep;55(9):1979–1986. doi: 10.1128/iai.55.9.1979-1986.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kroll J. S., Hopkins I., Moxon E. R. Capsule loss in H. influenzae type b occurs by recombination-mediated disruption of a gene essential for polysaccharide export. Cell. 1988 May 6;53(3):347–356. doi: 10.1016/0092-8674(88)90155-9. [DOI] [PubMed] [Google Scholar]
  20. Kroll J. S., Loynds B., Brophy L. N., Moxon E. R. The bex locus in encapsulated Haemophilus influenzae: a chromosomal region involved in capsule polysaccharide export. Mol Microbiol. 1990 Nov;4(11):1853–1862. doi: 10.1111/j.1365-2958.1990.tb02034.x. [DOI] [PubMed] [Google Scholar]
  21. Kyte J., Doolittle R. F. A simple method for displaying the hydropathic character of a protein. J Mol Biol. 1982 May 5;157(1):105–132. doi: 10.1016/0022-2836(82)90515-0. [DOI] [PubMed] [Google Scholar]
  22. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  23. Logan S. M., Trust T. J. Structural and antigenic heterogeneity of lipopolysaccharides of Campylobacter jejuni and Campylobacter coli. Infect Immun. 1984 Jul;45(1):210–216. doi: 10.1128/iai.45.1.210-216.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Munn C. B., Ishiguro E. E., Kay W. W., Trust T. J. Role of surface components in serum resistance of virulent Aeromonas salmonicida. Infect Immun. 1982 Jun;36(3):1069–1075. doi: 10.1128/iai.36.3.1069-1075.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Myers E. W., Miller W. Optimal alignments in linear space. Comput Appl Biosci. 1988 Mar;4(1):11–17. doi: 10.1093/bioinformatics/4.1.11. [DOI] [PubMed] [Google Scholar]
  26. Pavelka M. S., Jr, Wright L. F., Silver R. P. Identification of two genes, kpsM and kpsT, in region 3 of the polysialic acid gene cluster of Escherichia coli K1. J Bacteriol. 1991 Aug;173(15):4603–4610. doi: 10.1128/jb.173.15.4603-4610.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Reizer J., Reizer A., Saier M. H., Jr A new subfamily of bacterial ABC-type transport systems catalyzing export of drugs and carbohydrates. Protein Sci. 1992 Oct;1(10):1326–1332. doi: 10.1002/pro.5560011012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Rosenberg A. H., Lade B. N., Chui D. S., Lin S. W., Dunn J. J., Studier F. W. Vectors for selective expression of cloned DNAs by T7 RNA polymerase. Gene. 1987;56(1):125–135. doi: 10.1016/0378-1119(87)90165-x. [DOI] [PubMed] [Google Scholar]
  29. Shaw D. H., Lee Y. Z., Squires M. J., Lüderitz O. Structural studies on the O-antigen of Aeromonas salmonicida. Eur J Biochem. 1983 Apr 5;131(3):633–638. doi: 10.1111/j.1432-1033.1983.tb07310.x. [DOI] [PubMed] [Google Scholar]
  30. Stroeher U. H., Karageorgos L. E., Morona R., Manning P. A. Serotype conversion in Vibrio cholerae O1. Proc Natl Acad Sci U S A. 1992 Apr 1;89(7):2566–2570. doi: 10.1073/pnas.89.7.2566. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Thomas S., Austin J. W., McCubbin W. D., Kay C. M., Trust T. J. Roles of structural domains in the morphology and surface anchoring of the tetragonal paracrystalline array of Aeromonas hydrophila. Biochemical characterization of the major structural domain. J Mol Biol. 1992 Nov 20;228(2):652–661. doi: 10.1016/0022-2836(92)90847-d. [DOI] [PubMed] [Google Scholar]
  32. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Trust T. J., Klotz F. W., Miller L. H. Pathogenesis of infectious diseases of fish. Annu Rev Microbiol. 1986;40:479–502. doi: 10.1146/annurev.mi.40.100186.002403. [DOI] [PubMed] [Google Scholar]
  34. Tsai C. M., Frasch C. E. A sensitive silver stain for detecting lipopolysaccharides in polyacrylamide gels. Anal Biochem. 1982 Jan 1;119(1):115–119. doi: 10.1016/0003-2697(82)90673-x. [DOI] [PubMed] [Google Scholar]
  35. Whitfield C., Valvano M. A. Biosynthesis and expression of cell-surface polysaccharides in gram-negative bacteria. Adv Microb Physiol. 1993;35:135–246. doi: 10.1016/s0065-2911(08)60099-5. [DOI] [PubMed] [Google Scholar]
  36. Yang L. Y., Pei Z. H., Fujimoto S., Blaser M. J. Reattachment of surface array proteins to Campylobacter fetus cells. J Bacteriol. 1992 Feb;174(4):1258–1267. doi: 10.1128/jb.174.4.1258-1267.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Zhang L., al-Hendy A., Toivanen P., Skurnik M. Genetic organization and sequence of the rfb gene cluster of Yersinia enterocolitica serotype O:3: similarities to the dTDP-L-rhamnose biosynthesis pathway of Salmonella and to the bacterial polysaccharide transport systems. Mol Microbiol. 1993 Jul;9(2):309–321. doi: 10.1111/j.1365-2958.1993.tb01692.x. [DOI] [PubMed] [Google Scholar]

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