Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1994 Sep;176(17):5483–5493. doi: 10.1128/jb.176.17.5483-5493.1994

Studies of the biosynthesis of 3,6-dideoxyhexoses: molecular cloning and characterization of the asc (ascarylose) region from Yersinia pseudotuberculosis serogroup VA.

J S Thorson 1, S F Lo 1, O Ploux 1, X He 1, H W Liu 1
PMCID: PMC196737  PMID: 8071227

Abstract

The 3,6-dideoxyhexoses are found in the lipopolysaccharides of gram-negative bacteria, where they have been shown to be the dominant antigenic determinants. Of the five 3,6-dideoxyhexoses known to occur naturally, four have been found in various strains of Salmonella enterica (abequose, tyvelose, paratose, and colitose) and all five, including ascarylose, are present among the serotypes of Yersinia pseudotuberculosis. Although there exists one report of the cloning of the rfb region harboring the abequose biosynthetic genes from Y. pseudotuberculosis serogroup HA, the detailed genetic principles underlying a 3,6-dideoxyhexose polymorphism in Y. pseudotuberculosis have not been addressed. To extend the available information on the genes responsible for 3,6-dideoxyhexose formation in Yersinia spp. and facilitate a comparison with the established rfb (O antigen) cluster of Salmonella spp., we report the production of three overlapping clones containing the entire gene cluster required for CDP-ascarylose biosynthesis. On the basis of a detailed sequence analysis, the implications regarding 3,6-dideoxyhexose polymorphism among Salmonella and Yersinia spp. are discussed. In addition, the functional cloning of this region has allowed the expression of Ep (alpha-D-glucose cytidylyltransferase), Eod (CDP-D-glucose 4,6-dehydratase), E1 (CDP-6-deoxy-L-threo-D-glycero-4- hexulose-3-dehydrase), E3 (CDP-6-deoxy-delta 3,4-glucoseen reductase), Eep (CDP-3,6-dideoxy-D-glycero-D- glycero-4-hexulose-5-epimerase), and Ered (CDP-3,6-dideoxy-L-glycero-D-glycero-4-hexulose-4-reductase), facilitating future mechanistic studies of this intriguing biosynthetic pathway.

Full text

PDF
5493

Images in this article

5490Image
on p.5490

Selected References

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

  1. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Brahmbhatt H. N., Wyk P., Quigley N. B., Reeves P. R. Complete physical map of the rfb gene cluster encoding biosynthetic enzymes for the O antigen of Salmonella typhimurium LT2. J Bacteriol. 1988 Jan;170(1):98–102. doi: 10.1128/jb.170.1.98-102.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Brenner D. J. Characterization and clinical identification of Enterobacteriaceae by DNA hybridization. Prog Clin Pathol. 1978;7:71–117. [PubMed] [Google Scholar]
  4. Brutlag D. L., Dautricourt J. P., Maulik S., Relph J. Improved sensitivity of biological sequence database searches. Comput Appl Biosci. 1990 Jul;6(3):237–245. doi: 10.1093/bioinformatics/6.3.237. [DOI] [PubMed] [Google Scholar]
  5. 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]
  6. Hanessian S. Deoxy sugars. Adv Carbohydr Chem Biochem. 1966;21:143–207. doi: 10.1016/s0096-5332(08)60317-3. [DOI] [PubMed] [Google Scholar]
  7. Henikoff S. Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene. 1984 Jun;28(3):351–359. doi: 10.1016/0378-1119(84)90153-7. [DOI] [PubMed] [Google Scholar]
  8. Jiang X. M., Neal B., Santiago F., Lee S. J., Romana L. K., Reeves P. R. Structure and sequence of the rfb (O antigen) gene cluster of Salmonella serovar typhimurium (strain LT2). Mol Microbiol. 1991 Mar;5(3):695–713. doi: 10.1111/j.1365-2958.1991.tb00741.x. [DOI] [PubMed] [Google Scholar]
  9. Kessler A. C., Brown P. K., Romana L. K., Reeves P. R. Molecular cloning and genetic characterization of the rfb region from Yersinia pseudotuberculosis serogroup IIA, which determines the formation of the 3,6-dideoxyhexose abequose. J Gen Microbiol. 1991 Dec;137(12):2689–2695. doi: 10.1099/00221287-137-12-2689. [DOI] [PubMed] [Google Scholar]
  10. Kessler A. C., Haase A., Reeves P. R. Molecular analysis of the 3,6-dideoxyhexose pathway genes of Yersinia pseudotuberculosis serogroup IIA. J Bacteriol. 1993 Mar;175(5):1412–1422. doi: 10.1128/jb.175.5.1412-1422.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. Liu D., Verma N. K., Romana L. K., Reeves P. R. Relationships among the rfb regions of Salmonella serovars A, B, and D. J Bacteriol. 1991 Aug;173(15):4814–4819. doi: 10.1128/jb.173.15.4814-4819.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Lo S. F., Miller V. P., Lei Y., Thorson J. S., Liu H. W., Schottel J. L. CDP-6-deoxy-delta 3,4-glucoseen reductase from Yersinia pseudotuberculosis: enzyme purification and characterization of the cloned gene. J Bacteriol. 1994 Jan;176(2):460–468. doi: 10.1128/jb.176.2.460-468.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Lüderitz O., Staub A. M., Westphal O. Immunochemistry of O and R antigens of Salmonella and related Enterobacteriaceae. Bacteriol Rev. 1966 Mar;30(1):192–255. doi: 10.1128/br.30.1.192-255.1966. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Miller V. P., Thorson J. S., Ploux O., Lo S. F., Liu H. W. Cofactor characterization and mechanistic studies of CDP-6-deoxy-delta 3,4-glucoseen reductase: exploration into a novel enzymatic C-O bond cleavage event. Biochemistry. 1993 Nov 9;32(44):11934–11942. doi: 10.1021/bi00095a025. [DOI] [PubMed] [Google Scholar]
  16. Raetz C. R. Biochemistry of endotoxins. Annu Rev Biochem. 1990;59:129–170. doi: 10.1146/annurev.bi.59.070190.001021. [DOI] [PubMed] [Google Scholar]
  17. Reeves P. Evolution of Salmonella O antigen variation by interspecific gene transfer on a large scale. Trends Genet. 1993 Jan;9(1):17–22. doi: 10.1016/0168-9525(93)90067-R. [DOI] [PubMed] [Google Scholar]
  18. Roantree R. J. Salmonella O antigens and virulence. Annu Rev Microbiol. 1967;21:443–466. doi: 10.1146/annurev.mi.21.100167.002303. [DOI] [PubMed] [Google Scholar]
  19. Rubenstein P. A., Strominger J. L. Enzymatic synthesis of cytidine diphosphate 3,6-dideoxyhexosis. IX. Purification and properties of the cytidine diphosphate-D-glucose pyrophosphorylase from Pasteurella pseudotuberculosis. type V. J Biol Chem. 1974 Jun 25;249(12):3789–3796. [PubMed] [Google Scholar]
  20. Samuelsson K., Lindberg B., Brubaker R. R. Structure of O-specific side chains of lipopolysaccharides from Yersinia pseudotuberculosis. J Bacteriol. 1974 Mar;117(3):1010–1016. doi: 10.1128/jb.117.3.1010-1016.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Sharp P. M., Li W. H. The codon Adaptation Index--a measure of directional synonymous codon usage bias, and its potential applications. Nucleic Acids Res. 1987 Feb 11;15(3):1281–1295. doi: 10.1093/nar/15.3.1281. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Sueoka N. Directional mutation pressure and neutral molecular evolution. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2653–2657. doi: 10.1073/pnas.85.8.2653. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Thorson J. S., Kelly T. M., Liu H. W. Cloning, sequencing, and overexpression in Escherichia coli of the alpha-D-glucose-1-phosphate cytidylyltransferase gene isolated from Yersinia pseudotuberculosis. J Bacteriol. 1994 Apr;176(7):1840–1849. doi: 10.1128/jb.176.7.1840-1849.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Vesterberg O. Staining of protein zones after isoelectric focusing in polyacrylamide gels. Biochim Biophys Acta. 1971 Aug 27;243(2):345–348. doi: 10.1016/0005-2795(71)90094-8. [DOI] [PubMed] [Google Scholar]
  26. Weigel T. M., Liu L. D., Liu H. W. Mechanistic studies of the biosynthesis of 3,6-dideoxyhexoses in Yersinia pseudotuberculosis: purification and characterization of CDP-4-keto-6-deoxy-D-glucose-3-dehydrase. Biochemistry. 1992 Feb 25;31(7):2129–2139. doi: 10.1021/bi00122a034. [DOI] [PubMed] [Google Scholar]
  27. Wierenga R. K., Terpstra P., Hol W. G. Prediction of the occurrence of the ADP-binding beta alpha beta-fold in proteins, using an amino acid sequence fingerprint. J Mol Biol. 1986 Jan 5;187(1):101–107. doi: 10.1016/0022-2836(86)90409-2. [DOI] [PubMed] [Google Scholar]
  28. Wyk P., Reeves P. Identification and sequence of the gene for abequose synthase, which confers antigenic specificity on group B salmonellae: homology with galactose epimerase. J Bacteriol. 1989 Oct;171(10):5687–5693. doi: 10.1128/jb.171.10.5687-5693.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Yu Y., Russell R. N., Thorson J. S., Liu L. D., Liu H. W. Mechanistic studies of the biosynthesis of 3,6-dideoxyhexoses in Yersinia pseudotuberculosis. Purification and stereochemical analysis of CDP-D-glucose oxidoreductase. J Biol Chem. 1992 Mar 25;267(9):5868–5875. [PubMed] [Google Scholar]

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

RESOURCES