Abstract
The gene cluster of the capsular K5 polysaccharide, a representative of group II capsular antigens of Escherichia coli, has been cloned previously, and three regions responsible for polymerization and surface expression have been defined (I.S. Roberts, R. Mountford, R. Hodge, K. B. Jann, and G. J. Boulnois, J. Bacteriol. 170:1305-1330, 1988). Region 1 has now been sequenced, and five open reading frames (kpsEDUCS) have been defined (C. Pazzani, C. Rosenow, G. J. Boulnois, D. Bronner, K. Jann, and I. S. Roberts, J. Bacteriol. 175:5978-5983, 1993). In this study, we characterized region 1 mutants by immunoelectron microscopy, membrane-associated polymerization activity, cytoplasmic CMP-2-keto-3-deoxyoctonate (KDO) synthetase activity, and chemical analysis of their K5 polysaccharides. Certain mutations within region 1 not only effected polysaccharide transport (lack of region 1 gene products) but also impaired the polymerization capacity of the respective membranes, reflected in reduced amounts of polysaccharide but not in its chain length. KDO and phosphatidic acid (phosphatidyl-KDO) substitution was found with extracellular and periplasmic polysaccharide and not with cytoplasmic polysaccharide. This and the fact that the K5 polysaccharide is formed in a kpsU mutant (defective in capsule-specific K-CMP-KDO synthetase) showed that CMP-KDO is engaged not in initiation of polymerization but in translocation of the polysaccharide.
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Selected References
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- Acetarin J. D., Carlemalm E., Villiger W. Developments of new Lowicryl resins for embedding biological specimens at even lower temperatures. J Microsc. 1986 Jul;143(Pt 1):81–88. doi: 10.1111/j.1365-2818.1986.tb02766.x. [DOI] [PubMed] [Google Scholar]
- Ames G. F., Prody C., Kustu S. Simple, rapid, and quantitative release of periplasmic proteins by chloroform. J Bacteriol. 1984 Dec;160(3):1181–1183. doi: 10.1128/jb.160.3.1181-1183.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Armbruster B. L., Carlemalm E., Chiovetti R., Garavito R. M., Hobot J. A., Kellenberger E., Villiger W. Specimen preparation for electron microscopy using low temperature embedding resins. J Microsc. 1982 Apr;126(Pt 1):77–85. doi: 10.1111/j.1365-2818.1982.tb00358.x. [DOI] [PubMed] [Google Scholar]
- 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]
- 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]
- Chang A. C., Cohen S. N. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol. 1978 Jun;134(3):1141–1156. doi: 10.1128/jb.134.3.1141-1156.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- 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]
- 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]
- Fournet B., Strecker G., Leroy Y., Montreuil J. Gas--liquid chromatography and mass spectrometry of methylated and acetylated methyl glycosides. Application to the structural analysis of glycoprotein glycans. Anal Biochem. 1981 Sep 15;116(2):489–502. doi: 10.1016/0003-2697(81)90393-6. [DOI] [PubMed] [Google Scholar]
- 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]
- 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]
- Goldman R. C., Kohlbrenner W. E. Molecular cloning of the structural gene coding for CTP:CMP-3-deoxy-manno-octulosonate cytidylyltransferase from Escherichia coli K-12. J Bacteriol. 1985 Jul;163(1):256–261. doi: 10.1128/jb.163.1.256-261.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gotschlich E. C., Fraser B. A., Nishimura O., Robbins J. B., Liu T. Y. Lipid on capsular polysaccharides of gram-negative bacteria. J Biol Chem. 1981 Sep 10;256(17):8915–8921. [PubMed] [Google Scholar]
- 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]
- Karkhanis Y. D., Zeltner J. Y., Jackson J. J., Carlo D. J. A new and improved microassay to determine 2-keto-3-deoxyoctonate in lipopolysaccharide of Gram-negative bacteria. Anal Biochem. 1978 Apr;85(2):595–601. doi: 10.1016/0003-2697(78)90260-9. [DOI] [PubMed] [Google Scholar]
- 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]
- 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]
- 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]
- LUFT J. H. Improvements in epoxy resin embedding methods. J Biophys Biochem Cytol. 1961 Feb;9:409–414. doi: 10.1083/jcb.9.2.409. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Min H., Cowman M. K. Combined alcian blue and silver staining of glycosaminoglycans in polyacrylamide gels: application to electrophoretic analysis of molecular weight distribution. Anal Biochem. 1986 Jun;155(2):275–285. doi: 10.1016/0003-2697(86)90437-9. [DOI] [PubMed] [Google Scholar]
- Orskov F., Orskov I., Jann B., Jann K. Immunoelectrophoretic patterns of extracts from all Escherichia coli O and K antigen test strains: correlation with pathogenicity. Acta Pathol Microbiol Scand B Microbiol Immunol. 1971;79(2):142–152. doi: 10.1111/j.1699-0463.1971.tb02141.x. [DOI] [PubMed] [Google Scholar]
- 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]
- Orskov I., Nyman K. Genetic mapping of the antigenic determinants of two polysaccharide K antigens, K10 and K54, in Escherichia coli. J Bacteriol. 1974 Oct;120(1):43–51. doi: 10.1128/jb.120.1.43-51.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Orskov I., Orskov F., Jann B., Jann K. Serology, chemistry, and genetics of O and K antigens of Escherichia coli. Bacteriol Rev. 1977 Sep;41(3):667–710. doi: 10.1128/br.41.3.667-710.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Osborn M. J., Rick P. D., Rasmussen N. S. Mechanism of assembly of the outer membrane of Salmonella typhimurium. Translocation and integration of an incomplete mutant lipid A into the outer membrane. J Biol Chem. 1980 May 10;255(9):4246–4251. [PubMed] [Google Scholar]
- Paakkanen J., Gotschlich E. C., Mäkelä P. H. Protein K: a new major outer membrane protein found in encapsulated Escherichia coli. J Bacteriol. 1979 Sep;139(3):835–841. doi: 10.1128/jb.139.3.835-841.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- 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]
- Pelkonen S., Häyrinen J., Finne J. Polyacrylamide gel electrophoresis of the capsular polysaccharides of Escherichia coli K1 and other bacteria. J Bacteriol. 1988 Jun;170(6):2646–2653. doi: 10.1128/jb.170.6.2646-2653.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Raetz C. R., Purcell S., Meyer M. V., Qureshi N., Takayama K. Isolation and characterization of eight lipid A precursors from a 3-deoxy-D-manno-octylosonic acid-deficient mutant of Salmonella typhimurium. J Biol Chem. 1985 Dec 25;260(30):16080–16088. [PubMed] [Google Scholar]
- Ray P. H., Benedict C. D. CTP:CMP-3-deoxy-D-manno-octulosonate cytidylyltransferase (CMP-KDO synthetase). Methods Enzymol. 1982;83:535–540. doi: 10.1016/0076-6879(82)83049-8. [DOI] [PubMed] [Google Scholar]
- Rick P. D., Young D. A. Isolation and characterization of a temperature-sensitive lethal mutant of Salmonella typhimurium that is conditionally defective in 3-deoxy-D-manno-octulosonate-8-phosphate synthesis. J Bacteriol. 1982 May;150(2):447–455. doi: 10.1128/jb.150.2.447-455.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- 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]
- 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]
- 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]
- Smith A. N., Boulnois G. J., Roberts I. S. 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):1863–1869. doi: 10.1111/j.1365-2958.1990.tb02035.x. [DOI] [PubMed] [Google Scholar]
- Vann W. F., Jann K. Structure and serological specificity of the K13-antigenic polysaccharide (K13 antigen) of urinary tract-infective Escherichia coli. Infect Immun. 1979 Jul;25(1):85–92. doi: 10.1128/iai.25.1.85-92.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- 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]
- Vimr E. R. Map position and genomic organization of the kps cluster for polysialic acid synthesis in Escherichia coli K1. J Bacteriol. 1991 Feb;173(3):1335–1338. doi: 10.1128/jb.173.3.1335-1338.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- WEISSBACH A., HURWITZ J. The formation of 2-keto-3-deoxyheptonic acid in extracts of Escherichia coli B. I. Identification. J Biol Chem. 1959 Apr;234(4):705–709. [PubMed] [Google Scholar]