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. 1995 Mar;177(5):1137–1143. doi: 10.1128/jb.177.5.1137-1143.1995

Characterization and localization of the KpsE protein of Escherichia coli K5, which is involved in polysaccharide export.

C Rosenow 1, F Esumeh 1, I S Roberts 1, K Jann 1
PMCID: PMC176716  PMID: 7868584

Abstract

In Escherichia coli with group II capsules, the synthesis and cellular expression of capsular polysaccharide are encoded by the kps gene cluster. This gene cluster is composed of three regions. The central region 2 encodes proteins involved in polysaccharide synthesis, and the flanking regions 1 and 3 direct the translocation of the finished polysaccharide across the cytoplasmic membrane and its surface expression. The kps genes of the K5 polysaccharide, which is a group II capsular polysaccharide, have been cloned and sequenced. Region 1 contains the kpsE, -D, -U, -C, and -S genes. In this communication we describe the KpsE protein, the product of the kpsE gene. A truncated kpsE gene was fused with a truncated beta-galactosidase gene to generate a fusion protein containing the first 375 amino acids of beta-galactosidase and amino acids 67 to 382 of KpsE (KpsE'). This fusion protein was isolated and cleaved with factor Xa, and the purified KpsE' was used to immunize rabbits. Intact KpsE was extracted from the membranes of a KpsE-overexpressing recombinant strain with octyl-beta-glucoside. It was purified by affinity chromatography with immobilized anti-KpsE antibodies. Cytofluorometric analysis using the anti-KpsE antibodies with whole cells and spheroplasts, as well as sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting (immunoblotting) of proteins from spheroplasts and membranes before and after treatment with proteinase K, indicated that the KpsE protein is associated with the cytoplasmic membrane and has an exposed periplasmic domain. By TnphoA mutagenesis and by constructing beta-lactamase fusions to the KpseE protein, it was possible to determine the topology of the KpsE protein within the cytoplasmic membrane.

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Selected References

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  1. Akiyama Y., Ito K. The SecY membrane component of the bacterial protein export machinery: analysis by new electrophoretic methods for integral membrane proteins. EMBO J. 1985 Dec 1;4(12):3351–3356. doi: 10.1002/j.1460-2075.1985.tb04088.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bayer M. H., Costello G. P., Bayer M. E. Isolation and partial characterization of membrane vesicles carrying markers of the membrane adhesion sites. J Bacteriol. 1982 Feb;149(2):758–767. doi: 10.1128/jb.149.2.758-767.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Boulnois G. J., Jann K. Bacterial polysaccharide capsule synthesis, export and evolution of structural diversity. Mol Microbiol. 1989 Dec;3(12):1819–1823. doi: 10.1111/j.1365-2958.1989.tb00168.x. [DOI] [PubMed] [Google Scholar]
  4. Boulnois G. J., Roberts I. S. Genetics of capsular polysaccharide production in bacteria. Curr Top Microbiol Immunol. 1990;150:1–18. doi: 10.1007/978-3-642-74694-9_1. [DOI] [PubMed] [Google Scholar]
  5. Bronner D., Sieberth V., Pazzani C., Roberts I. S., Boulnois G. J., Jann B., Jann K. Expression of the capsular K5 polysaccharide of Escherichia coli: biochemical and electron microscopic analyses of mutants with defects in region 1 of the K5 gene cluster. J Bacteriol. 1993 Sep;175(18):5984–5992. doi: 10.1128/jb.175.18.5984-5992.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bronner D., Sieberth V., Pazzani C., Smith A., Boulnois G., Roberts I., Jann B., Jann K. Synthesis of the K5 (group II) capsular polysaccharide in transport-deficient recombinant Escherichia coli. FEMS Microbiol Lett. 1993 Nov 1;113(3):279–284. doi: 10.1111/j.1574-6968.1993.tb06527.x. [DOI] [PubMed] [Google Scholar]
  7. Cieslewicz M. J., Steenbergen S. M., Vimr E. R. Cloning, sequencing, expression, and complementation analysis of the Escherichia coli K1 kps region 1 gene, kpsE, and identification of an upstream open reading frame encoding a protein with homology to GutQ. J Bacteriol. 1993 Dec;175(24):8018–8023. doi: 10.1128/jb.175.24.8018-8023.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cross A. S. The biologic significance of bacterial encapsulation. Curr Top Microbiol Immunol. 1990;150:87–95. doi: 10.1007/978-3-642-74694-9_5. [DOI] [PubMed] [Google Scholar]
  9. Dalbey R. E. Positively charged residues are important determinants of membrane protein topology. Trends Biochem Sci. 1990 Jul;15(7):253–257. doi: 10.1016/0968-0004(90)90047-f. [DOI] [PubMed] [Google Scholar]
  10. Dower W. J., Miller J. F., Ragsdale C. W. High efficiency transformation of E. coli by high voltage electroporation. Nucleic Acids Res. 1988 Jul 11;16(13):6127–6145. doi: 10.1093/nar/16.13.6127. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Ellinger S., Glockshuber R., Jahn G., Plückthun A. Cleavage of procaryotically expressed human immunodeficiency virus fusion proteins by factor Xa and application in western blot (immunoblot) assays. J Clin Microbiol. 1989 May;27(5):971–976. doi: 10.1128/jcm.27.5.971-976.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Elvin C. M., Thompson P. R., Argall M. E., Hendry P., Stamford N. P., Lilley P. E., Dixon N. E. Modified bacteriophage lambda promoter vectors for overproduction of proteins in Escherichia coli. Gene. 1990 Mar 1;87(1):123–126. doi: 10.1016/0378-1119(90)90503-j. [DOI] [PubMed] [Google Scholar]
  13. Fernandez-Patron C., Castellanos-Serra L., Rodriguez P. Reverse staining of sodium dodecyl sulfate polyacrylamide gels by imidazole-zinc salts: sensitive detection of unmodified proteins. Biotechniques. 1992 Apr;12(4):564–573. [PubMed] [Google Scholar]
  14. Finke A., Bronner D., Nikolaev A. V., Jann B., Jann K. Biosynthesis of the Escherichia coli K5 polysaccharide, a representative of group II capsular polysaccharides: polymerization in vitro and characterization of the product. J Bacteriol. 1991 Jul;173(13):4088–4094. doi: 10.1128/jb.173.13.4088-4094.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Finke A., Jann B., Jann K. CMP-KDO-synthetase activity in Escherichia coli expressing capsular polysaccharides. FEMS Microbiol Lett. 1990 May;57(1-2):129–133. doi: 10.1016/0378-1097(90)90426-q. [DOI] [PubMed] [Google Scholar]
  16. Finke A., Roberts I., Boulnois G., Pzzani C., Jann K. Activity of CMP-2-keto-3-deoxyoctulosonic acid synthetase in Escherichia coli strains expressing the capsular K5 polysaccharide implication for K5 polysaccharide biosynthesis. J Bacteriol. 1989 Jun;171(6):3074–3079. doi: 10.1128/jb.171.6.3074-3079.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. Frosch M., Müller D., Bousset K., Müller A. Conserved outer membrane protein of Neisseria meningitidis involved in capsule expression. Infect Immun. 1992 Mar;60(3):798–803. doi: 10.1128/iai.60.3.798-803.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Higgins C. F., Hyde S. C., Mimmack M. M., Gileadi U., Gill D. R., Gallagher M. P. Binding protein-dependent transport systems. J Bioenerg Biomembr. 1990 Aug;22(4):571–592. doi: 10.1007/BF00762962. [DOI] [PubMed] [Google Scholar]
  20. Ishidate K., Creeger E. S., Zrike J., Deb S., Glauner B., MacAlister T. J., Rothfield L. I. Isolation of differentiated membrane domains from Escherichia coli and Salmonella typhimurium, including a fraction containing attachment sites between the inner and outer membranes and the murein skeleton of the cell envelope. J Biol Chem. 1986 Jan 5;261(1):428–443. [PubMed] [Google Scholar]
  21. Jann B., Jann K. Structure and biosynthesis of the capsular antigens of Escherichia coli. Curr Top Microbiol Immunol. 1990;150:19–42. doi: 10.1007/978-3-642-74694-9_2. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Koenen M., Rüther U., Müller-Hill B. Immunoenzymatic detection of expressed gene fragments cloned in the lac Z gene of E. coli. EMBO J. 1982;1(4):509–512. doi: 10.1002/j.1460-2075.1982.tb01199.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. 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]
  26. Kröncke K. D., Boulnois G., Roberts I., Bitter-Suermann D., Golecki J. R., Jann B., Jann K. Expression of the Escherichia coli K5 capsular antigen: immunoelectron microscopic and biochemical studies with recombinant E. coli. J Bacteriol. 1990 Feb;172(2):1085–1091. doi: 10.1128/jb.172.2.1085-1091.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Kröncke K. D., Golecki J. R., Jann K. Further electron microscopic studies on the expression of Escherichia coli group II capsules. J Bacteriol. 1990 Jun;172(6):3469–3472. doi: 10.1128/jb.172.6.3469-3472.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. 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]
  29. Levengood S. K., Webster R. E. Nucleotide sequences of the tolA and tolB genes and localization of their products, components of a multistep translocation system in Escherichia coli. J Bacteriol. 1989 Dec;171(12):6600–6609. doi: 10.1128/jb.171.12.6600-6609.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Lewis K. Multidrug resistance pumps in bacteria: variations on a theme. Trends Biochem Sci. 1994 Mar;19(3):119–123. doi: 10.1016/0968-0004(94)90204-6. [DOI] [PubMed] [Google Scholar]
  31. Navia J. L., Riesenfeld J., Vann W. F., Lindahl U., Rodén L. Assay of N-acetylheparosan deacetylase with a capsular polysaccharide from Escherichia coli K5 as substrate. Anal Biochem. 1983 Nov;135(1):134–140. doi: 10.1016/0003-2697(83)90741-8. [DOI] [PubMed] [Google Scholar]
  32. Orskov F., Sharma V., Orskov I. Influence of growth temperature on the development of Escherichia coli polysaccharide K antigens. J Gen Microbiol. 1984 Oct;130(10):2681–2684. doi: 10.1099/00221287-130-10-2681. [DOI] [PubMed] [Google Scholar]
  33. Pazzani C., Rosenow C., Boulnois G. J., Bronner D., Jann K., Roberts I. S. Molecular analysis of region 1 of the Escherichia coli K5 antigen gene cluster: a region encoding proteins involved in cell surface expression of capsular polysaccharide. J Bacteriol. 1993 Sep;175(18):5978–5983. doi: 10.1128/jb.175.18.5978-5983.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. 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]
  35. Roberts I. S., Mountford R., Hodge R., Jann K. B., Boulnois G. J. Common organization of gene clusters for production of different capsular polysaccharides (K antigens) in Escherichia coli. J Bacteriol. 1988 Mar;170(3):1305–1310. doi: 10.1128/jb.170.3.1305-1310.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Roberts I., Mountford R., High N., Bitter-Suermann D., Jann K., Timmis K., Boulnois G. Molecular cloning and analysis of genes for production of K5, K7, K12, and K92 capsular polysaccharides in Escherichia coli. J Bacteriol. 1986 Dec;168(3):1228–1233. doi: 10.1128/jb.168.3.1228-1233.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Saier M. H., Jr Computer-aided analyses of transport protein sequences: gleaning evidence concerning function, structure, biogenesis, and evolution. Microbiol Rev. 1994 Mar;58(1):71–93. doi: 10.1128/mr.58.1.71-93.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Silver R. P., Aaronson W., Vann W. F. Translocation of capsular polysaccharides in pathogenic strains of Escherichia coli requires a 60-kilodalton periplasmic protein. J Bacteriol. 1987 Dec;169(12):5489–5495. doi: 10.1128/jb.169.12.5489-5495.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Silver R. P., Vann W. F., Aaronson W. Genetic and molecular analyses of Escherichia coli K1 antigen genes. J Bacteriol. 1984 Feb;157(2):568–575. doi: 10.1128/jb.157.2.568-575.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. 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]
  41. Vann W. F., Schmidt M. A., Jann B., Jann K. The structure of the capsular polysaccharide (K5 antigen) of urinary-tract-infective Escherichia coli 010:K5:H4. A polymer similar to desulfo-heparin. Eur J Biochem. 1981 May 15;116(2):359–364. doi: 10.1111/j.1432-1033.1981.tb05343.x. [DOI] [PubMed] [Google Scholar]
  42. Vimr E. R., Aaronson W., Silver R. P. Genetic analysis of chromosomal mutations in the polysialic acid gene cluster of Escherichia coli K1. J Bacteriol. 1989 Feb;171(2):1106–1117. doi: 10.1128/jb.171.2.1106-1117.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Wilkins B. M., Boulnois G. J., Lanka E. A plasmid DNA primase active in discontinuous bacterial DNA replication. Nature. 1981 Mar 19;290(5803):217–221. doi: 10.1038/290217a0. [DOI] [PubMed] [Google Scholar]
  44. Zhang Y. B., Broome-Smith J. K. Correct insertion of a simple eukaryotic plasma-membrane protein into the cytoplasmic membrane of Escherichia coli. Gene. 1990 Nov 30;96(1):51–57. doi: 10.1016/0378-1119(90)90340-w. [DOI] [PubMed] [Google Scholar]

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