Abstract
The O7-specific lipopolysaccharide (LPS) in strains of Escherichia coli consists of a repeating unit made of galactose, mannose, rhamnose, 4-acetamido-2,6-dideoxyglucose, and N-acetylglucosamine. We have recently cloned and characterized genetically the O7-specific LPS biosynthesis region (rfbEcO7) of the E. coli O7:K1 strain VW187 (C. L. Marolda, J. Welsh, L. Dafoe, and M. A. Valvano, J. Bacteriol. 172:3590-3599, 1990). In this study, we localized the gnd gene encoding gluconate-6-phosphate dehydrogenase at one end of the rfbEcO7 gene cluster and sequenced that end of the cluster. Three open reading frames (ORF) encoding polypeptides of 275, 464, and 453 amino acids were identified upstream of gndEcO7, all transcribed toward the gnd gene. ORF275 had 45% similarity at the protein level with ORF16.5, which occupies a similar position in the Salmonella enterica LT2 rfb region, and presumably encodes a nucleotide sugar transferase. The polypeptides encoded by ORFs 464 and 453 were expressed under the control of the ptac promoter and visualized in Coomassie blue-stained sodium dodecyl sulfate-polyacrylamide gels and by maxicell analysis. ORF464 expressed GDP-mannose pyrophosphorylase and ORF453 encoded a phosphomannomutase, the enzymes for the biosynthesis pathway of GDP-mannose, one of the nucleotide sugar precursors for the formation of the O7 repeating unit. They were designated rfbMEcO7 and rfbKEcO7, respectively. The RfbMEcO7 polypeptide was homologous to the corresponding protein in S. enterica LT2, XanB of Xanthomonas campestris, and AlgA of Pseudomonas aeruginosa, all GDP-mannose pyrophosphorylases. RfbKEcO7 was very similar to CpsG of S. enterica LT2, an enzyme presumably involved in the biosynthesis of the capsular polysaccharide colanic acid, but quite different from the corresponding RfbK protein of S. enterica LT2.
Full text
PDF![148](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/588a/196108/00eb446a1eff/jbacter00043-0172.png)
![149](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/588a/196108/96cfb8ae373c/jbacter00043-0173.png)
![150](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/588a/196108/89abfb88f0a5/jbacter00043-0174.png)
![151](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/588a/196108/67efdcf796f5/jbacter00043-0175.png)
![152](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/588a/196108/e988f79cfff5/jbacter00043-0176.png)
![153](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/588a/196108/134d82a55440/jbacter00043-0177.png)
![154](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/588a/196108/991e510cd301/jbacter00043-0178.png)
![155](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/588a/196108/5a49a6b20e19/jbacter00043-0179.png)
![156](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/588a/196108/1a54e2bf1189/jbacter00043-0180.png)
![157](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/588a/196108/02306ff5ad6b/jbacter00043-0181.png)
![158](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/588a/196108/2e291370923a/jbacter00043-0182.png)
Images in this article
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Bachmann B. J. Linkage map of Escherichia coli K-12, edition 8. Microbiol Rev. 1990 Jun;54(2):130–197. doi: 10.1128/mr.54.2.130-197.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bairoch A. PROSITE: a dictionary of sites and patterns in proteins. Nucleic Acids Res. 1991 Apr 25;19 (Suppl):2241–2245. doi: 10.1093/nar/19.suppl.2241. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Barcak G. J., Wolf R. E., Jr Comparative nucleotide sequence analysis of growth-rate-regulated gnd alleles from natural isolates of Escherichia coli and from Salmonella typhimurium LT-2. J Bacteriol. 1988 Jan;170(1):372–379. doi: 10.1128/jb.170.1.372-379.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Barcak G. J., Wolf R. E., Jr Growth-rate-dependent expression and cloning of gnd alleles from natural isolates of Escherichia coli. J Bacteriol. 1988 Jan;170(1):365–371. doi: 10.1128/jb.170.1.365-371.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Brown P. K., Romana L. K., Reeves P. R. Molecular analysis of the rfb gene cluster of Salmonella serovar muenchen (strain M67): the genetic basis of the polymorphism between groups C2 and B. Mol Microbiol. 1992 May;6(10):1385–1394. doi: 10.1111/j.1365-2958.1992.tb00859.x. [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]
- Cohen S. N., Chang A. C., Hsu L. Nonchromosomal antibiotic resistance in bacteria: genetic transformation of Escherichia coli by R-factor DNA. Proc Natl Acad Sci U S A. 1972 Aug;69(8):2110–2114. doi: 10.1073/pnas.69.8.2110. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Csonka L. N., Clark A. J. Deletions generated by the transposon Tn10 in the srl recA region of the Escherichia coli K-12 chromosome. Genetics. 1979 Oct;93(2):321–343. doi: 10.1093/genetics/93.2.321. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Dykhuizen D. E., Green L. Recombination in Escherichia coli and the definition of biological species. J Bacteriol. 1991 Nov;173(22):7257–7268. doi: 10.1128/jb.173.22.7257-7268.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
- Hanks S. K., Quinn A. M., Hunter T. The protein kinase family: conserved features and deduced phylogeny of the catalytic domains. Science. 1988 Jul 1;241(4861):42–52. doi: 10.1126/science.3291115. [DOI] [PubMed] [Google Scholar]
- 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]
- 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]
- Keenleyside W. J., Jayaratne P., MacLachlan P. R., Whitfield C. The rcsA gene of Escherichia coli O9:K30:H12 is involved in the expression of the serotype-specific group I K (capsular) antigen. J Bacteriol. 1992 Jan;174(1):8–16. doi: 10.1128/jb.174.1.8-16.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kyte J., Doolittle R. F. A simple method for displaying the hydropathic character of a protein. J Mol Biol. 1982 May 5;157(1):105–132. doi: 10.1016/0022-2836(82)90515-0. [DOI] [PubMed] [Google Scholar]
- Köplin R., Arnold W., Hötte B., Simon R., Wang G., Pühler A. Genetics of xanthan production in Xanthomonas campestris: the xanA and xanB genes are involved in UDP-glucose and GDP-mannose biosynthesis. J Bacteriol. 1992 Jan;174(1):191–199. doi: 10.1128/jb.174.1.191-199.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- L'vov V. L., Shashkov A. S., Dmitriev B. A., Kochetkov N. K., Jann B., Jann K. Structural studies of the O-specific side chain of the lipopolysaccharide from Escherichia coli O:7. Carbohydr Res. 1984 Mar 15;126(2):249–259. doi: 10.1016/0008-6215(84)85382-3. [DOI] [PubMed] [Google Scholar]
- LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
- 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]
- Lee S. J., Romana L. K., Reeves P. R. Cloning and structure of group C1 O antigen (rfb gene cluster) from Salmonella enterica serovar montevideo. J Gen Microbiol. 1992 Feb;138(2):305–312. doi: 10.1099/00221287-138-2-305. [DOI] [PubMed] [Google Scholar]
- Linn T., St Pierre R. Improved vector system for constructing transcriptional fusions that ensures independent translation of lacZ. J Bacteriol. 1990 Feb;172(2):1077–1084. doi: 10.1128/jb.172.2.1077-1084.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lipman D. J., Pearson W. R. Rapid and sensitive protein similarity searches. Science. 1985 Mar 22;227(4693):1435–1441. doi: 10.1126/science.2983426. [DOI] [PubMed] [Google Scholar]
- 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]
- Lugtenberg B., Van Alphen L. Molecular architecture and functioning of the outer membrane of Escherichia coli and other gram-negative bacteria. Biochim Biophys Acta. 1983 Mar 21;737(1):51–115. doi: 10.1016/0304-4157(83)90014-x. [DOI] [PubMed] [Google Scholar]
- Maloy S. R., Nunn W. D. Selection for loss of tetracycline resistance by Escherichia coli. J Bacteriol. 1981 Feb;145(2):1110–1111. doi: 10.1128/jb.145.2.1110-1111.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Markovitz A., Sydiskis R. J., Lieberman M. M. Genetic and biochemical studies on mannose-negative mutants that are deficient in phosphomannose isomerase in Escherichia coli K-12. J Bacteriol. 1967 Nov;94(5):1492–1496. doi: 10.1128/jb.94.5.1492-1496.1967. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Marolda C. L., Welsh J., Dafoe L., Valvano M. A. Genetic analysis of the O7-polysaccharide biosynthesis region from the Escherichia coli O7:K1 strain VW187. J Bacteriol. 1990 Jul;172(7):3590–3599. doi: 10.1128/jb.172.7.3590-3599.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Nasoff M. S., Wolf R. E., Jr Molecular cloning, correlation of genetic and restriction maps, and determination of the direction of transcription of gnd of Escherichia coli. J Bacteriol. 1980 Aug;143(2):731–741. doi: 10.1128/jb.143.2.731-741.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Neuhard J., Thomassen E. Altered deoxyribonucleotide pools in P2 eductants of Escherichia coli K-12 due to deletion of the dcd gene. J Bacteriol. 1976 May;126(2):999–1001. doi: 10.1128/jb.126.2.999-1001.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Nikaido H., Nikaido K., Mäkelä P. H. Genetic determination of enzymes synthesizing O-specific sugars of Salmonella lipopolysaccharides. J Bacteriol. 1966 Mar;91(3):1126–1135. doi: 10.1128/jb.91.3.1126-1135.1966. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Novel G., Novel M. Mutants d'Escherichia coli K 12 affectés pour leur croissance sur méthyl-beta-D-glucuronide: localisation of gène de structure de la beta-D-glucuronidase (uid A. Mol Gen Genet. 1973;120(4):319–335. [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]
- Passador L., Linn T. Autogenous regulation of the RNA polymerase beta subunit of Escherichia coli occurs at the translational level in vivo. J Bacteriol. 1989 Nov;171(11):6234–6242. doi: 10.1128/jb.171.11.6234-6242.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Peyru G., Fraenkel D. G. Genetic mapping of loci for glucose-6-phosphate dehydrogenase, gluconate-6-phosphate dehydrogenase, and gluconate-6-phosphate dehydrase in Escherichia coli. J Bacteriol. 1968 Apr;95(4):1272–1278. doi: 10.1128/jb.95.4.1272-1278.1968. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ray W. J., Jr, Hermodson M. A., Puvathingal J. M., Mahoney W. C. The complete amino acid sequence of rabbit muscle phosphoglucomutase. J Biol Chem. 1983 Aug 10;258(15):9166–9174. [PubMed] [Google Scholar]
- Sanderson K. E., Roth J. R. Linkage map of Salmonella typhimurium, Edition VI. Microbiol Rev. 1983 Sep;47(3):410–453. doi: 10.1128/mr.47.3.410-453.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- 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]
- Shinabarger D., Berry A., May T. B., Rothmel R., Fialho A., Chakrabarty A. M. Purification and characterization of phosphomannose isomerase-guanosine diphospho-D-mannose pyrophosphorylase. A bifunctional enzyme in the alginate biosynthetic pathway of Pseudomonas aeruginosa. J Biol Chem. 1991 Feb 5;266(4):2080–2088. [PubMed] [Google Scholar]
- Stachel S. E., An G., Flores C., Nester E. W. A Tn3 lacZ transposon for the random generation of beta-galactosidase gene fusions: application to the analysis of gene expression in Agrobacterium. EMBO J. 1985 Apr;4(4):891–898. doi: 10.1002/j.1460-2075.1985.tb03715.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Stevenson G., Lee S. J., Romana L. K., Reeves P. R. The cps gene cluster of Salmonella strain LT2 includes a second mannose pathway: sequence of two genes and relationship to genes in the rfb gene cluster. Mol Gen Genet. 1991 Jun;227(2):173–180. doi: 10.1007/BF00259668. [DOI] [PubMed] [Google Scholar]
- Sá-Correia I., Darzins A., Wang S. K., Berry A., Chakrabarty A. M. Alginate biosynthetic enzymes in mucoid and nonmucoid Pseudomonas aeruginosa: overproduction of phosphomannose isomerase, phosphomannomutase, and GDP-mannose pyrophosphorylase by overexpression of the phosphomannose isomerase (pmi) gene. J Bacteriol. 1987 Jul;169(7):3224–3231. doi: 10.1128/jb.169.7.3224-3231.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Valvano M. A., Crosa J. H. Aerobactin iron transport genes commonly encoded by certain ColV plasmids occur in the chromosome of a human invasive strain of Escherichia coli K1. Infect Immun. 1984 Oct;46(1):159–167. doi: 10.1128/iai.46.1.159-167.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Valvano M. A., Crosa J. H. Molecular cloning and expression in Escherichia coli K-12 of chromosomal genes determining the O7 lipopolysaccharide antigen of a human invasive strain of E. coli O7:K1. Infect Immun. 1989 Mar;57(3):937–943. doi: 10.1128/iai.57.3.937-943.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Valvano M. A. Pathogenicity and molecular genetics of O-specific side-chain lipopolysaccharides of Escherichia coli. Can J Microbiol. 1992 Jul;38(7):711–719. doi: 10.1139/m92-117. [DOI] [PubMed] [Google Scholar]
- Valvano M. A., Silver R. P., Crosa J. H. Occurrence of chromosome- or plasmid-mediated aerobactin iron transport systems and hemolysin production among clonal groups of human invasive strains of Escherichia coli K1. Infect Immun. 1986 Apr;52(1):192–199. doi: 10.1128/iai.52.1.192-199.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wang L., Romana L. K., Reeves P. R. Molecular analysis of a Salmonella enterica group E1 rfb gene cluster: O antigen and the genetic basis of the major polymorphism. Genetics. 1992 Mar;130(3):429–443. doi: 10.1093/genetics/130.3.429. [DOI] [PMC free article] [PubMed] [Google Scholar]