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PMID: 9244257 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

Identification and functional characterization of an ABC transport system involved in polysaccharide export of A-band lipopolysaccharide in Pseudomonas aeruginosa.

Journal of bacteriology ·Vol. 179 ·No. 15 ·1997-08-00 ·Pages 4713-24

Rocchetta HL, Lam JS

Abstract

Pseudomonas aeruginosa coexpresses two distinct lipopolysaccharide (LPS) molecules known as A band and B band. B band is the serospecific LPS, while A band is the common LPS antigen composed of a D-rhamnose O-polysaccharide region. An operon containing eight genes responsible for A-band polysaccharide biosynthesis and export has recently been identified and characterized (H. L. Rocchetta, L. L. Burrows, J. C. Pacan, and J. S. Lam, unpublished data; H. L. Rocchetta, J. C. Pacan, and J. S. Lam, unpublished data). In this study, we report the characterization of two genes within the cluster, designated wzm and wzt. The Wzm and Wzt proteins have predicted sizes of 29.5 and 47.2 kDa, respectively, and are homologous to a number of proteins that comprise ABC (ATP-binding cassette) transport systems. Wzm is an integral membrane protein with six potential membrane-spanning domains, while Wzt is an ATP-binding protein containing a highly conserved ATP-binding motif. Chromosomal wzm and wzt mutants were generated by using a gene replacement strategy in P. aeruginosa PAO1 (serotype 05). Western blot analysis and immunoelectron microscopy using A-band- and B-band-specific monoclonal antibodies demonstrated that the wzm and wzt mutants were able to synthesize A-band polysaccharide, although transport of the polymer to the cell surface was inhibited. The inability of the polymer to cross the inner membrane resulted in the accumulation of cytoplasmic A-band polysaccharide. This A-band polysaccharide is likely linked to a carrier lipid molecule with a phenol-labile linkage. Chromosomal mutations in wzm and wzt were found to have no effect on B-band LPS synthesis. Rather, immunoelectron microscopy revealed that the presence of A-band LPS may influence the arrangement of B-band LPS on the cell surface. These results demonstrate that A-band and B-band O-antigen assembly processes follow two distinct pathways, with the former requiring an ABC transport system for cell surface expression.

MeSH Terms
ATP-Binding Cassette Transporters/genetics,metabolism Amino Acid Sequence Bacterial Proteins Base Sequence Biological Transport Blotting, Southern Chromosomes, Bacterial Cloning, Molecular Cosmids DNA, Bacterial Gene Expression Lipopolysaccharides/metabolism Membrane Proteins/genetics,metabolism Microscopy, Immunoelectron Molecular Sequence Data Mutation Pseudomonas aeruginosa/genetics,metabolism,ultrastructure Sequence Homology, Amino Acid Serotyping
Chemicals
ATP-Binding Cassette Transporters Bacterial Proteins DNA, Bacterial Lipopolysaccharides Membrane Proteins Wzt protein, bacteria lipopolysaccharide A
Authors & Affiliations
2 authors, click to expand affiliations / ORCID
Rocchetta H L
Department of Microbiology and Canadian Bacterial Diseases Network, University of Guelph, Ontario.
Lam J S
References (58)
58 references, click to expand
  1. Properties of the O-specific hapten formed in vivo by mutant strains of Salmonella typhimurium.
    Biochemistry. 1968 Dec;7(12):4396-408 PMID: 4882709
  2. Periplasmic binding protein-dependent transport systems: the membrane-associated components.
    Philos Trans R Soc Lond B Biol Sci. 1990 Jan 30;326(1236):353-64; discussion 364-5 PMID: 1970642
  3. A rapid alkaline extraction procedure for screening recombinant plasmid DNA.
    Nucleic Acids Res. 1979 Nov 24;7(6):1513-23 PMID: 388356
  4. Outer membrane of Pseudomonas aeruginosa: heat- 2-mercaptoethanol-modifiable proteins.
    J Bacteriol. 1979 Dec;140(3):902-10 PMID: 118160
  5. "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A.
    Anal Biochem. 1981 Apr;112(2):195-203 PMID: 6266278
  6. A simple method for displaying the hydropathic character of a protein.
    J Mol Biol. 1982 May 5;157(1):105-32 PMID: 7108955
  7. The helical hydrophobic moment: a measure of the amphiphilicity of a helix.
    Nature. 1982 Sep 23;299(5881):371-4 PMID: 7110359
  8. Morphological heterogeneity among Salmonella lipopolysaccharide chemotypes in silver-stained polyacrylamide gels.
    J Bacteriol. 1983 Apr;154(1):269-77 PMID: 6187729
  9. Pseudomonas aeruginosa isolates from patients with cystic fibrosis: a class of serum-sensitive, nontypable strains deficient in lipopolysaccharide O side chains.
    Infect Immun. 1983 Oct;42(1):170-7 PMID: 6413410
  10. Distantly related sequences in the alpha- and beta-subunits of ATP synthase, myosin, kinases and other ATP-requiring enzymes and a common nucleotide binding fold.
    EMBO J. 1982;1(8):945-51 PMID: 6329717
  11. Cloning of genes controlling alginate biosynthesis from a mucoid cystic fibrosis isolate of Pseudomonas aeruginosa.
    J Bacteriol. 1984 Jul;159(1):9-18 PMID: 6330052
  12. High state of order of isolated bacterial lipopolysaccharide and its possible contribution to the permeation barrier property of the outer membrane.
    J Bacteriol. 1985 Apr;162(1):9-20 PMID: 3980449
  13. The detection and classification of membrane-spanning proteins.
    Biochim Biophys Acta. 1985 May 28;815(3):468-76 PMID: 3838905
  14. Analysis of the K1 capsule biosynthesis genes of Escherichia coli: definition of three functional regions for capsule production.
    Mol Gen Genet. 1987 Jun;208(1-2):242-6 PMID: 3302608
  15. Molecular characterization of the oligopeptide permease of Salmonella typhimurium.
    J Mol Biol. 1987 May 5;195(1):125-42 PMID: 2821267
  16. Optimal alignments in linear space.
    Comput Appl Biosci. 1988 Mar;4(1):11-7 PMID: 3382986
  17. Genetic analysis of chromosomal mutations in the polysialic acid gene cluster of Escherichia coli K1.
    J Bacteriol. 1989 Feb;171(2):1106-17 PMID: 2644224
  18. Three new major somatic antigens of Pseudomonas aeruginosa.
    J Clin Microbiol. 1990 May;28(5):922-5 PMID: 2112563
  19. Polysaccharide antigens of Pseudomonas aeruginosa.
    Crit Rev Microbiol. 1990;17(4):273-304 PMID: 1698385
  20. High efficiency electroporation of Pseudomonas aeruginosa using frozen cell suspensions.
    FEMS Microbiol Lett. 1990 Jul;58(2):221-5 PMID: 2121587
  21. Basic local alignment search tool.
    J Mol Biol. 1990 Oct 5;215(3):403-10 PMID: 2231712
  22. The bex locus in encapsulated Haemophilus influenzae: a chromosomal region involved in capsule polysaccharide export.
    Mol Microbiol. 1990 Nov;4(11):1853-62 PMID: 2082145
  23. Molecular analysis of the Escherichia coli K5 kps locus: identification and characterization of an inner-membrane capsular polysaccharide transport system.
    Mol Microbiol. 1990 Nov;4(11):1863-9 PMID: 2082146
  24. The membrane-bound proteins of periplasmic permeases form a complex. Identification of the histidine permease HisQMP complex.
    J Biol Chem. 1991 May 25;266(15):9857-65 PMID: 2033074
  25. 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):4603-10 PMID: 1856162
  26. Molecular cloning of genes involved with expression of A-band lipopolysaccharide, an antigenically conserved form, in Pseudomonas aeruginosa.
    J Bacteriol. 1991 Sep;173(18):5624-30 PMID: 1909320
  27. Electrotransformation in Salmonella typhimurium LT2.
    Can J Microbiol. 1991 Jun;37(6):474-7 PMID: 1913351
  28. Molecular cloning of the rfb region of Klebsiella pneumoniae serotype O1:K20: the rfb gene cluster is responsible for synthesis of the D-galactan I O polysaccharide.
    J Bacteriol. 1992 Jul;174(14):4614-21 PMID: 1378055
  29. Molecular modelling of the three-dimensional structure and conformational flexibility of bacterial lipopolysaccharide.
    J Bacteriol. 1992 Jul;174(14):4798-806 PMID: 1624466
  30. Ultrastructural examination of the lipopolysaccharides of Pseudomonas aeruginosa strains and their isogenic rough mutants by freeze-substitution.
    J Bacteriol. 1992 Nov;174(22):7159-67 PMID: 1429438
  31. ABC transporters: from microorganisms to man.
    Annu Rev Cell Biol. 1992;8:67-113 PMID: 1282354
  32. Identification of protein coding regions by database similarity search.
    Nat Genet. 1993 Mar;3(3):266-72 PMID: 8485583
  33. 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):3105-14 PMID: 8491726
  34. Chromosomal mapping, expression and synthesis of lipopolysaccharide in Pseudomonas aeruginosa: a role for guanosine diphospho (GDP)-D-mannose.
    Mol Microbiol. 1993 May;8(4):771-82 PMID: 7687320
  35. 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):309-21 PMID: 7692217
  36. Function of the rfb gene cluster and the rfe gene in the synthesis of O antigen by Shigella dysenteriae 1.
    Mol Microbiol. 1993 Jul;9(2):393-402 PMID: 7692219
  37. Small broad-host-range gentamycin resistance gene cassettes for site-specific insertion and deletion mutagenesis.
    Biotechniques. 1993 Nov;15(5):831-4 PMID: 8267974
  38. Synthesis of the K5 (group II) capsular polysaccharide in transport-deficient recombinant Escherichia coli.
    FEMS Microbiol Lett. 1993 Nov 1;113(3):279-84 PMID: 8270192
  39. ABC transporters: bacterial exporters.
    Microbiol Rev. 1993 Dec;57(4):995-1017 PMID: 8302219
  40. Role of the rfe gene in the synthesis of the O8 antigen in Escherichia coli K-12.
    J Bacteriol. 1994 May;176(10):2877-84 PMID: 7514591
  41. Construction of improved Escherichia-Pseudomonas shuttle vectors derived from pUC18/19 and sequence of the region required for their replication in Pseudomonas aeruginosa.
    Gene. 1994 Oct 11;148(1):81-6 PMID: 7926843
  42. Relationships between rfb gene clusters required for biosynthesis of identical D-galactose-containing O antigens in Klebsiella pneumoniae serotype O1 and Serratia marcescens serotype O16.
    J Bacteriol. 1995 Mar;177(6):1544-53 PMID: 7533758
  43. Identification of an ATP-binding cassette transport system required for translocation of lipopolysaccharide O-antigen side-chains across the cytoplasmic membrane of Klebsiella pneumoniae serotype O1.
    Mol Microbiol. 1994 Nov;14(3):505-19 PMID: 7533882
  44. Expression of the O9 polysaccharide of Escherichia coli: sequencing of the E. coli O9 rfb gene cluster, characterization of mannosyl transferases, and evidence for an ATP-binding cassette transport system.
    J Bacteriol. 1995 Apr;177(8):2178-87 PMID: 7536735
  45. Putative O-antigen transport genes within the rfb region of Vibrio cholerae O1 are homologous to those for capsule transport.
    Gene. 1995 May 26;158(1):1-7 PMID: 7540582
  46. An improved system for gene replacement and xylE fusion analysis in Pseudomonas aeruginosa.
    Gene. 1995 May 26;158(1):15-22 PMID: 7789804
  47. Virulence factors are released from Pseudomonas aeruginosa in association with membrane vesicles during normal growth and exposure to gentamicin: a novel mechanism of enzyme secretion.
    J Bacteriol. 1995 Jul;177(14):3998-4008 PMID: 7608073
  48. Biosynthesis of lipopolysaccharide O antigens.
    Trends Microbiol. 1995 May;3(5):178-85 PMID: 7542987
  49. The tagGH operon of Bacillus subtilis 168 encodes a two-component ABC transporter involved in the metabolism of two wall teichoic acids.
    Mol Microbiol. 1995 Apr;16(2):345-55 PMID: 7565096
  50. Molecular cloning and characterization of the rfc gene of Pseudomonas aeruginosa (serotype O5).
    Mol Microbiol. 1995 May;16(3):565-74 PMID: 7565115
  51. The Myxococcus xanthus rfbABC operon encodes an ATP-binding cassette transporter homolog required for O-antigen biosynthesis and multicellular development.
    J Bacteriol. 1996 Mar;178(6):1631-9 PMID: 8626291
  52. A novel pathway for O-polysaccharide biosynthesis in Salmonella enterica serovar Borreze.
    J Biol Chem. 1996 Nov 8;271(45):28581-92 PMID: 8910488
  53. Molecular and chemical characterization of the lipopolysaccharide O-antigen and its role in the virulence of Yersinia enterocolitica serotype O:8.
    Mol Microbiol. 1997 Jan;23(1):63-76 PMID: 9004221
  54. Bacterial polysaccharide synthesis and gene nomenclature.
    Trends Microbiol. 1996 Dec;4(12):495-503 PMID: 9004408
  55. Pseudomonas aeruginosa B-band O-antigen chain length is modulated by Wzz (Ro1).
    J Bacteriol. 1997 Mar;179(5):1482-9 PMID: 9045803
  56. Occurrence of a common lipopolysaccharide antigen in standard and clinical strains of Pseudomonas aeruginosa.
    J Clin Microbiol. 1989 May;27(5):962-7 PMID: 2501356
  57. Expression of the Escherichia coli K5 capsular antigen: immunoelectron microscopic and biochemical studies with recombinant E. coli.
    J Bacteriol. 1990 Feb;172(2):1085-91 PMID: 2404935
  58. Simple method for identification of plasmid-coded proteins.
    J Bacteriol. 1979 Jan;137(1):692-3 PMID: 368040
Article Info
Journal
Journal of bacteriology
Abbr.
J Bacteriol
ISSN
0021-9193
DOI
10.1128/jb.179.15.4713-4724.1997
Published
1997-08-00
Pages
4713-24
Language
English
Region
United States
NLM ID
2985120R
PMCID
PMC179316
Subset
IM
Databases
GENBANK
U63722, U63723
External Links
PubMed source DOI: 10.1128/jb.179.15.4713-4724.1997 PMC Full Text View journal details
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