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. 1995 Jan;177(2):312–319. doi: 10.1128/jb.177.2.312-319.1995

Nucleotide sequence and genetic analysis of the neuD and neuB genes in region 2 of the polysialic acid gene cluster of Escherichia coli K1.

P W Annunziato 1, L F Wright 1, W F Vann 1, R P Silver 1
PMCID: PMC176593  PMID: 7814319

Abstract

The K1 capsular polysaccharide, a polymer of sialic acid, is an important virulence determinant of extraintestinal pathogenic Escherichia coli. The genes responsible for the synthesis and expression of the polysialic acid capsule of E. coli K1 are located on the 17-kb kps gene cluster, which is functionally divided into three regions. Central region 2 encodes proteins necessary for the synthesis, activation, and polymerization of sialic acid, while flanking regions 1 and 3 are involved in polymer transport to the cell surface. In this study, we identified two genes at the proximal end of region 2, neuD and neuB, which encode proteins with predicted sizes of 22.7 and 38.7 kDa, respectively. Several observations suggest that the neuB gene encodes sialic acid synthase. EV24, a neuB chromosomal mutant that expresses a capsule when provided exogenous sialic acid, could be complemented in trans by the cloned neuB gene. In addition, NeuB has significant sequence similarity to the product of the cpsB gene of Neisseria meningitidis group B, which is postulated to encode sialic acid synthase. We also present data indicating that neuD has an essential role in K1 polymer production. Cells harboring pSR426, which contains all of region 2 but lacks region 1 and 3 genes, produce an intracellular polymer. In contrast, no polymer accumulated in cells carrying a derivative of pSR426 lacking a functional neuD gene. Unlike strains with mutations in neuB, however, neuD mutants are not complemented by exogenous sialic acid, suggesting that NeuD is not involved in sialic acid synthesis. Additionally, cells harboring a mutation in neuD accumulated sialic acid and CMP-sialic acid. We also found no significant differences between the endogenous and exogenous sialyltransferase activities of a neuD mutant and the wild-type organism. NeuD shows significant similarity to a family of bacterial acetyltransferases, leading to the theory that NeuD is an acetyltransferase which may exert its influences through modification of other region 2 proteins.

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Selected References

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  1. Bienroth S., Keller W., Wahle E. Assembly of a processive messenger RNA polyadenylation complex. EMBO J. 1993 Feb;12(2):585–594. doi: 10.1002/j.1460-2075.1993.tb05690.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. 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]
  3. 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]
  4. Boulnois G. J., Roberts I. S., Hodge R., Hardy K. R., Jann K. B., Timmis K. N. 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):242–246. doi: 10.1007/BF00330449. [DOI] [PubMed] [Google Scholar]
  5. Broome-Smith J. K., Tadayyon M., Zhang Y. Beta-lactamase as a probe of membrane protein assembly and protein export. Mol Microbiol. 1990 Oct;4(10):1637–1644. doi: 10.1111/j.1365-2958.1990.tb00540.x. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. Davis R. J., Czech M. P. Tumor-promoting phorbol diesters cause the phosphorylation of epidermal growth factor receptors in normal human fibroblasts at threonine-654. Proc Natl Acad Sci U S A. 1985 Apr;82(7):1974–1978. doi: 10.1073/pnas.82.7.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dicker I. B., Seetharam S. What is known about the structure and function of the Escherichia coli protein FirA? Mol Microbiol. 1992 Apr;6(7):817–823. doi: 10.1111/j.1365-2958.1992.tb01532.x. [DOI] [PubMed] [Google Scholar]
  10. Downie J. A. The nodL gene from Rhizobium leguminosarum is homologous to the acetyl transferases encoded by lacA and cysE. Mol Microbiol. 1989 Nov;3(11):1649–1651. doi: 10.1111/j.1365-2958.1989.tb00150.x. [DOI] [PubMed] [Google Scholar]
  11. Edelman G. M. Cell adhesion and the molecular processes of morphogenesis. Annu Rev Biochem. 1985;54:135–169. doi: 10.1146/annurev.bi.54.070185.001031. [DOI] [PubMed] [Google Scholar]
  12. Ehrmann M., Boyd D., Beckwith J. Genetic analysis of membrane protein topology by a sandwich gene fusion approach. Proc Natl Acad Sci U S A. 1990 Oct;87(19):7574–7578. doi: 10.1073/pnas.87.19.7574. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Fearon D. T., Austen K. F. Current concepts in immunology: the alternative pathway of complement--a system for host resistance to microbial infection. N Engl J Med. 1980 Jul 31;303(5):259–263. doi: 10.1056/NEJM198007313030505. [DOI] [PubMed] [Google Scholar]
  14. Finne J., Leinonen M., Mäkelä P. H. Antigenic similarities between brain components and bacteria causing meningitis. Implications for vaccine development and pathogenesis. Lancet. 1983 Aug 13;2(8346):355–357. doi: 10.1016/s0140-6736(83)90340-9. [DOI] [PubMed] [Google Scholar]
  15. Ganguli S., Zapata G., Wallis T., Reid C., Boulnois G., Vann W. F., Roberts I. S. Molecular cloning and analysis of genes for sialic acid synthesis in Neisseria meningitidis group B and purification of the meningococcal CMP-NeuNAc synthetase enzyme. J Bacteriol. 1994 Aug;176(15):4583–4589. doi: 10.1128/jb.176.15.4583-4589.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Gross R. J., Cheasty T., Rowe B. Isolation of bacteriophages specific for the K1 polysaccharide antigen of Escherichia coli. J Clin Microbiol. 1977 Dec;6(6):548–550. doi: 10.1128/jcm.6.6.548-550.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Güssow D., Clackson T. Direct clone characterization from plaques and colonies by the polymerase chain reaction. Nucleic Acids Res. 1989 May 25;17(10):4000–4000. doi: 10.1093/nar/17.10.4000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Higa H. H., Varki A. Acetyl-coenzyme A:polysialic acid O-acetyltransferase from K1-positive Escherichia coli. The enzyme responsible for the O-acetyl plus phenotype and for O-acetyl form variation. J Biol Chem. 1988 Jun 25;263(18):8872–8878. [PubMed] [Google Scholar]
  19. Horwitz M. A., Silverstein S. C. Influence of the Escherichia coli capsule on complement fixation and on phagocytosis and killing by human phagocytes. J Clin Invest. 1980 Jan;65(1):82–94. doi: 10.1172/JCI109663. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kaijser B. Immunology of Escherichia coli: K antigen and its relation to urinary-tract infection. J Infect Dis. 1973 Jun;127(6):670–677. doi: 10.1093/infdis/127.6.670. [DOI] [PubMed] [Google Scholar]
  21. Kasper D. L., Winkelhake J. L., Zollinger W. D., Brandt B. L., Artenstein M. S. Immunochemical similarity between polysaccharide antigens of Escherichia coli 07: K1(L):NM and group B Neisseria meningitidis. J Immunol. 1973 Jan;110(1):262–268. [PubMed] [Google Scholar]
  22. 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]
  23. Parent R., Roy P. H. The chloramphenicol acetyltransferase gene of Tn2424: a new breed of cat. J Bacteriol. 1992 May;174(9):2891–2897. doi: 10.1128/jb.174.9.2891-2897.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. 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]
  26. Prentki P., Krisch H. M. In vitro insertional mutagenesis with a selectable DNA fragment. Gene. 1984 Sep;29(3):303–313. doi: 10.1016/0378-1119(84)90059-3. [DOI] [PubMed] [Google Scholar]
  27. Robbins J. B., McCracken G. H., Jr, Gotschlich E. C., Orskov F., Orskov I., Hanson L. A. Escherichia coli K1 capsular polysaccharide associated with neonatal meningitis. N Engl J Med. 1974 May 30;290(22):1216–1220. doi: 10.1056/NEJM197405302902202. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. Schmidt M. F. Fatty acylation of proteins. Biochim Biophys Acta. 1989 Dec 6;988(3):411–426. doi: 10.1016/0304-4157(89)90013-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Sheets M. D., Wickens M. Two phases in the addition of a poly(A) tail. Genes Dev. 1989 Sep;3(9):1401–1412. doi: 10.1101/gad.3.9.1401. [DOI] [PubMed] [Google Scholar]
  31. 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]
  32. 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]
  33. 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]
  34. Steenbergen S. M., Vimr E. R. Mechanism of polysialic acid chain elongation in Escherichia coli K1. Mol Microbiol. 1990 Apr;4(4):603–611. doi: 10.1111/j.1365-2958.1990.tb00629.x. [DOI] [PubMed] [Google Scholar]
  35. Steenbergen S. M., Wrona T. J., Vimr E. R. Functional analysis of the sialyltransferase complexes in Escherichia coli K1 and K92. J Bacteriol. 1992 Feb;174(4):1099–1108. doi: 10.1128/jb.174.4.1099-1108.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Troy F. A., 2nd Polysialylation: from bacteria to brains. Glycobiology. 1992 Feb;2(1):5–23. doi: 10.1093/glycob/2.1.5. [DOI] [PubMed] [Google Scholar]
  37. Troy F. A., McCloskey M. A. Role of a membranous sialyltransferase complex in the synthesis of surface polymers containing polysialic acid in Escherichia coli. Temperature-induced alteration in the assembly process. J Biol Chem. 1979 Aug 10;254(15):7377–7387. [PubMed] [Google Scholar]
  38. Troy F. A., Vijay I. K., McCloskey M. A., Rohr T. E. Synthesis of capsular polymers containing polysialic acid in Escherichia coli 07-K1. Methods Enzymol. 1982;83:540–548. doi: 10.1016/0076-6879(82)83050-4. [DOI] [PubMed] [Google Scholar]
  39. Vaara M. Eight bacterial proteins, including UDP-N-acetylglucosamine acyltransferase (LpxA) and three other transferases of Escherichia coli, consist of a six-residue periodicity theme. FEMS Microbiol Lett. 1992 Oct 15;76(3):249–254. doi: 10.1016/0378-1097(92)90344-n. [DOI] [PubMed] [Google Scholar]
  40. Vann W. F., Silver R. P., Abeijon C., Chang K., Aaronson W., Sutton A., Finn C. W., Lindner W., Kotsatos M. Purification, properties, and genetic location of Escherichia coli cytidine 5'-monophosphate N-acetylneuraminic acid synthetase. J Biol Chem. 1987 Dec 25;262(36):17556–17562. [PubMed] [Google Scholar]
  41. 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]
  42. Vimr E. R. Selective synthesis and labeling of the polysialic acid capsule in Escherichia coli K1 strains with mutations in nanA and neuB. J Bacteriol. 1992 Oct;174(19):6191–6197. doi: 10.1128/jb.174.19.6191-6197.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Vimr E. R., Troy F. A. Identification of an inducible catabolic system for sialic acids (nan) in Escherichia coli. J Bacteriol. 1985 Nov;164(2):845–853. doi: 10.1128/jb.164.2.845-853.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Vimr E. R., Troy F. A. Regulation of sialic acid metabolism in Escherichia coli: role of N-acylneuraminate pyruvate-lyase. J Bacteriol. 1985 Nov;164(2):854–860. doi: 10.1128/jb.164.2.854-860.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Vuorio R., Hirvas L., Vaara M. The Ssc protein of enteric bacteria has significant homology to the acyltransferase Lpxa of lipid A biosynthesis, and to three acetyltransferases. FEBS Lett. 1991 Nov 4;292(1-2):90–94. doi: 10.1016/0014-5793(91)80841-p. [DOI] [PubMed] [Google Scholar]
  46. Weisgerber C., Hansen A., Frosch M. Complete nucleotide and deduced protein sequence of CMP-NeuAc: poly-alpha-2,8 sialosyl sialyltransferase of Escherichia coli K1. Glycobiology. 1991 Sep;1(4):357–365. doi: 10.1093/glycob/1.4.357. [DOI] [PubMed] [Google Scholar]
  47. Weisgerber C., Troy F. A. Biosynthesis of the polysialic acid capsule in Escherichia coli K1. The endogenous acceptor of polysialic acid is a membrane protein of 20 kDa. J Biol Chem. 1990 Jan 25;265(3):1578–1587. [PubMed] [Google Scholar]
  48. Wunder D. E., Aaronson W., Hayes S. F., Bliss J. M., Silver R. P. Nucleotide sequence and mutational analysis of the gene encoding KpsD, a periplasmic protein involved in transport of polysialic acid in Escherichia coli K1. J Bacteriol. 1994 Jul;176(13):4025–4033. doi: 10.1128/jb.176.13.4025-4033.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Wyle F. A., Artenstein M. S., Brandt B. L., Tramont E. C., Kasper D. L., Altieri P. L., Berman S. L., Lowenthal J. P. Immunologic response of man to group B meningococcal polysaccharide vaccines. J Infect Dis. 1972 Nov;126(5):514–521. doi: 10.1093/infdis/126.5.514. [DOI] [PubMed] [Google Scholar]
  50. Zapata G., Crowley J. M., Vann W. F. Sequence and expression of the Escherichia coli K1 neuC gene product. J Bacteriol. 1992 Jan;174(1):315–319. doi: 10.1128/jb.174.1.315-319.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Zapata G., Vann W. F., Aaronson W., Lewis M. S., Moos M. Sequence of the cloned Escherichia coli K1 CMP-N-acetylneuraminic acid synthetase gene. J Biol Chem. 1989 Sep 5;264(25):14769–14774. [PubMed] [Google Scholar]

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