Skip to main content
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1992 Jul;174(14):4736–4745. doi: 10.1128/jb.174.14.4736-4745.1992

Structures of the rfaB, rfaI, rfaJ, and rfaS genes of Escherichia coli K-12 and their roles in assembly of the lipopolysaccharide core.

E Pradel 1, C T Parker 1, C A Schnaitman 1
PMCID: PMC206270  PMID: 1624461

Abstract

Analysis of the sequence of a 4.1-kb rfa region downstream from rfaP revealed four genes. The first of these encodes a basic protein of 36,730 Da and does not correspond to any known rfa gene. It has been designated rfaS. The second gene was identified as rfaB on the basis of its ability to complement a Salmonella typhimurium rfaB mutant and encodes a 42,060-Da protein. The third and fourth genes encode proteins of 39,423 and 36,046 Da which are strongly homologous to the RfaI and RfaJ proteins of S. typhimurium. Escherichia coli K-12 restriction fragments carrying these genes complement an S. typhimurium rfaI mutant and, at lower efficiency, an rfaJ mutant. The difference in complementation efficiency suggests that the rfaI and rfaJ genes of E. coli K-12 have sugar and acceptor specificities different from those of S. typhimurium, as predicted from the different lipopolysaccharide (LPS) core structures of the two organisms. Defined mutations affecting all four genes were constructed in vitro and crossed onto the chromosome. The phenotypes of these mutations suggest that extension of the core may require protein-protein interactions between the enzymes involved in core completion as well as the interaction of these enzymes with their specific acceptor molecules. Mutants blocked at rfaI or genes encoding earlier steps in core biosynthesis exhibited a single predominant LPS band on gels while mutants blocked at rfaJ or genes encoding later steps produced multiple strong bands, indicating that one of the processes generating core heterogeneity requires a functional rfaI gene.

Full text

PDF
4745

Images in this article

Selected References

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

  1. Austin E. A., Graves J. F., Hite L. A., Parker C. T., Schnaitman C. A. Genetic analysis of lipopolysaccharide core biosynthesis by Escherichia coli K-12: insertion mutagenesis of the rfa locus. J Bacteriol. 1990 Sep;172(9):5312–5325. doi: 10.1128/jb.172.9.5312-5325.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Carstenius P., Flock J. I., Lindberg A. Nucleotide sequence of rfaI and rfaJ genes encoding lipopolysaccharide glycosyl transferases from Salmonella typhimurium. Nucleic Acids Res. 1990 Oct 25;18(20):6128–6128. doi: 10.1093/nar/18.20.6128. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Clementz T., Raetz C. R. A gene coding for 3-deoxy-D-manno-octulosonic-acid transferase in Escherichia coli. Identification, mapping, cloning, and sequencing. J Biol Chem. 1991 May 25;266(15):9687–9696. [PubMed] [Google Scholar]
  4. Creeger E. S., Chen J. F., Rothfield L. I. Cloning of genes for bacterial glycosyltransferases. II. Selection of a hybrid plasmid carrying the rfah gene. J Biol Chem. 1979 Feb 10;254(3):811–815. [PubMed] [Google Scholar]
  5. Creeger E. S., Rothfield L. I. Cloning of genes for bacterial glycosyltransferases. I. Selection of hybrid plasmids carrying genes for two glucosyltransferases. J Biol Chem. 1979 Feb 10;254(3):804–810. [PubMed] [Google Scholar]
  6. Creeger E. S., Schulte T., Rothfield L. I. Regulation of membrane glycosyltransferases by the sfrB and rfaH genes of Escherichia coli and Salmonella typhimurium. J Biol Chem. 1984 Mar 10;259(5):3064–3069. [PubMed] [Google Scholar]
  7. Hodgson A. L., Bird P., Nisbet I. T. Cloning, nucleotide sequence, and expression in Escherichia coli of the phospholipase D gene from Corynebacterium pseudotuberculosis. J Bacteriol. 1990 Mar;172(3):1256–1261. doi: 10.1128/jb.172.3.1256-1261.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Holst O., Röhrscheidt-Andrzejewski E., Brade H. Isolation and characterisation of 3-deoxy-D-manno-2-octulopyranosonate 7-(2-aminoethyl phosphate) from the inner core region of Escherichia coli K-12 and Salmonella minnesota lipopolysaccharides. Carbohydr Res. 1990 Sep 5;204:93–102. doi: 10.1016/0008-6215(90)84024-o. [DOI] [PubMed] [Google Scholar]
  9. Holst O., Zähringer U., Brade H., Zamojski A. Structural analysis of the heptose/hexose region of the lipopolysaccharide from Escherichia coli K-12 strain W3100. Carbohydr Res. 1991 Aug 20;215(2):323–335. doi: 10.1016/0008-6215(91)84031-9. [DOI] [PubMed] [Google Scholar]
  10. Jansson P. E., Lindberg A. A., Lindberg B., Wollin R. Structural studies on the hexose region of the core in lipopolysaccharides from Enterobacteriaceae. Eur J Biochem. 1981 Apr;115(3):571–577. doi: 10.1111/j.1432-1033.1981.tb06241.x. [DOI] [PubMed] [Google Scholar]
  11. Koplow J., Goldfine H. Alterations in the outer membrane of the cell envelope of heptose-deficient mutants of Escherichia coli. J Bacteriol. 1974 Feb;117(2):527–543. doi: 10.1128/jb.117.2.527-543.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Parker C. T., Kloser A. W., Schnaitman C. A., Stein M. A., Gottesman S., Gibson B. W. Role of the rfaG and rfaP genes in determining the lipopolysaccharide core structure and cell surface properties of Escherichia coli K-12. J Bacteriol. 1992 Apr;174(8):2525–2538. doi: 10.1128/jb.174.8.2525-2538.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Parker C. T., Pradel E., Schnaitman C. A. Identification and sequences of the lipopolysaccharide core biosynthetic genes rfaQ, rfaP, and rfaG of Escherichia coli K-12. J Bacteriol. 1992 Feb;174(3):930–934. doi: 10.1128/jb.174.3.930-934.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Pradel E., Schnaitman C. A. Effect of rfaH (sfrB) and temperature on expression of rfa genes of Escherichia coli K-12. J Bacteriol. 1991 Oct;173(20):6428–6431. doi: 10.1128/jb.173.20.6428-6431.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Prehm P., Stirm S., Jann B., Jann K. Cell-wall lipopolysaccharide from Escherichia coli B. Eur J Biochem. 1975 Aug 1;56(1):41–55. doi: 10.1111/j.1432-1033.1975.tb02205.x. [DOI] [PubMed] [Google Scholar]
  16. Roncero C., Casadaban M. J. Genetic analysis of the genes involved in synthesis of the lipopolysaccharide core in Escherichia coli K-12: three operons in the rfa locus. J Bacteriol. 1992 May;174(10):3250–3260. doi: 10.1128/jb.174.10.3250-3260.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Sandulache R., Prehm P., Kamp D. Cell wall receptor for bacteriophage Mu G(+). J Bacteriol. 1984 Oct;160(1):299–303. doi: 10.1128/jb.160.1.299-303.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Schnaitman C. A., Parker C. T., Klena J. D., Pradel E. L., Pearson N. B., Sanderson K. E., MacClachlan P. R. Physical maps of the rfa loci of Escherichia coli K-12 and Salmonella typhimurium. J Bacteriol. 1991 Dec;173(23):7410–7411. doi: 10.1128/jb.173.23.7410-7411.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Wollin R., Creeger E. S., Rothfield L. I., Stocker B. A., Lindberg A. A. Salmonella typhimurium mutants defective in UDP-D-galactose:lipopolysaccharide alpha 1,6-D-galactosyltransferase. Structural, immunochemical, and enzymologic studies of rfaB mutants. J Biol Chem. 1983 Mar 25;258(6):3769–3774. [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES