The cobT gene of Salmonella typhimurium encodes the NaMN: 5,6-dimethylbenzimidazole phosphoribosyltransferase responsible for the synthesis of N1-(5-phospho-alpha-D-ribosyl)-5,6-dimethylbenzimidazole, an intermediate in the synthesis of the nucleotide loop of cobalamin (original) (raw)

Abstract

We present in vitro evidence which demonstrates that CobT is the nicotinate nucleotide:5,6-dimethylbenzimidazole (DMB) phosphoribosyltransferase (EC 2.4.2.21) that catalyzes the synthesis of N1-(5-phospho-alpha-D-ribosyl)-5,6-dimethylbenzimidazole, a biosynthetic intermediate of the pathway that assembles the nucleotide loop of cobalamin in Salmonella typhimurium. Mutants previously isolated as DMB auxotrophs are shown by physical and genetic mapping studies and complementation studies to carry lesions in cobT. Explanations for this unexpected phenotype of cobT mutants are discussed. The expected nucleotide loop assembly phenotype of cobT mutants can be observed only in a specific genetic background, i.e., cobB deficient, an observation that is consistent with the existence of an alternative CobT function (G. A. O'Toole, M. R. Rondon, and J. C. Escalante-Semerena, J. Bacteriol. 175:3317-3326, 1993). Computer analysis of CobT homologs showed that at the amino acid level, enteric CobT proteins were 80% identical whereas Pseudomonas denitrificans and Rhizobium meliloti CobT proteins were 95% identical. Interestingly, the degree of identity between enteric and nonenteric CobT homologs was only 30%. The same pattern of homologies was reported for the S. typhimurium CobA, Escherichia coli BtuR, and P. denitrificans CobO proteins (S.-J. Suh and J.C. Escalante-Semerena, Gene 129:93-97, 1993), suggesting evolutionary divergence between the cob genes found in the enteric bacteria E. coli and S. typhimurium and those found in P. denitrificans and R. meliloti.

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Selected References

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  1. Cameron B., Blanche F., Rouyez M. C., Bisch D., Famechon A., Couder M., Cauchois L., Thibaut D., Debussche L., Crouzet J. Genetic analysis, nucleotide sequence, and products of two Pseudomonas denitrificans cob genes encoding nicotinate-nucleotide: dimethylbenzimidazole phosphoribosyltransferase and cobalamin (5'-phosphate) synthase. J Bacteriol. 1991 Oct;173(19):6066–6073. doi: 10.1128/jb.173.19.6066-6073.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Castilho B. A., Olfson P., Casadaban M. J. Plasmid insertion mutagenesis and lac gene fusion with mini-mu bacteriophage transposons. J Bacteriol. 1984 May;158(2):488–495. doi: 10.1128/jb.158.2.488-495.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Escalante-Semerena J. C., Johnson M. G., Roth J. R. The CobII and CobIII regions of the cobalamin (vitamin B12) biosynthetic operon of Salmonella typhimurium. J Bacteriol. 1992 Jan;174(1):24–29. doi: 10.1128/jb.174.1.24-29.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Escalante-Semerena J. C., Suh S. J., Roth J. R. cobA function is required for both de novo cobalamin biosynthesis and assimilation of exogenous corrinoids in Salmonella typhimurium. J Bacteriol. 1990 Jan;172(1):273–280. doi: 10.1128/jb.172.1.273-280.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. FRIEDMANN H. C., HARRIS D. L. THE FORMATION OF ALPHA-GLYCOSIDIC 5'-NUCLEOTIDES BY A SINGLE DISPLACEMENT TRANS-N-GLYCOSIDASE. J Biol Chem. 1965 Jan;240:406–412. [PubMed] [Google Scholar]
  6. Hörig J. A., Renz P. Biosynthesis of vitamin B12. Some properties of the 5,6-dimethylbenzimidazole-forming system of Propionibacterium freudenreichii and Propionibacterium shermanii. Eur J Biochem. 1980 Apr;105(3):587–592. doi: 10.1111/j.1432-1033.1980.tb04536.x. [DOI] [PubMed] [Google Scholar]
  7. Jeter R. M., Olivera B. M., Roth J. R. Salmonella typhimurium synthesizes cobalamin (vitamin B12) de novo under anaerobic growth conditions. J Bacteriol. 1984 Jul;159(1):206–213. doi: 10.1128/jb.159.1.206-213.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Johnson M. G., Escalante-Semerena J. C. Identification of 5,6-dimethylbenzimidazole as the Co alpha ligand of the cobamide synthesized by Salmonella typhimurium. Nutritional characterization of mutants defective in biosynthesis of the imidazole ring. J Biol Chem. 1992 Jul 5;267(19):13302–13305. [PubMed] [Google Scholar]
  9. 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]
  10. Martinez E., Bartolomé B., de la Cruz F. pACYC184-derived cloning vectors containing the multiple cloning site and lacZ alpha reporter gene of pUC8/9 and pUC18/19 plasmids. Gene. 1988 Aug 15;68(1):159–162. doi: 10.1016/0378-1119(88)90608-7. [DOI] [PubMed] [Google Scholar]
  11. O'Toole G. A., Rondon M. R., Escalante-Semerena J. C. Analysis of mutants of Salmonella typhimurium defective in the synthesis of the nucleotide loop of cobalamin. J Bacteriol. 1993 Jun;175(11):3317–3326. doi: 10.1128/jb.175.11.3317-3326.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Roth J. R., Lawrence J. G., Rubenfield M., Kieffer-Higgins S., Church G. M. Characterization of the cobalamin (vitamin B12) biosynthetic genes of Salmonella typhimurium. J Bacteriol. 1993 Jun;175(11):3303–3316. doi: 10.1128/jb.175.11.3303-3316.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Schmieger H. A method for detection of phage mutants with altered transducing ability. Mol Gen Genet. 1971;110(4):378–381. doi: 10.1007/BF00438281. [DOI] [PubMed] [Google Scholar]
  14. Schmieger H., Backhaus H. The origin of DNA in transducing particles in P22-mutants with increased transduction-frequencies (HT-mutants). Mol Gen Genet. 1973 Jan 24;120(2):181–190. doi: 10.1007/BF00267246. [DOI] [PubMed] [Google Scholar]
  15. Smith P. K., Krohn R. I., Hermanson G. T., Mallia A. K., Gartner F. H., Provenzano M. D., Fujimoto E. K., Goeke N. M., Olson B. J., Klenk D. C. Measurement of protein using bicinchoninic acid. Anal Biochem. 1985 Oct;150(1):76–85. doi: 10.1016/0003-2697(85)90442-7. [DOI] [PubMed] [Google Scholar]
  16. Suh S. J., Escalante-Semerena J. C. Cloning, sequencing and overexpression of cobA which encodes ATP:corrinoid adenosyltransferase in Salmonella typhimurium. Gene. 1993 Jul 15;129(1):93–97. doi: 10.1016/0378-1119(93)90701-4. [DOI] [PubMed] [Google Scholar]
  17. VOGEL H. J., BONNER D. M. Acetylornithinase of Escherichia coli: partial purification and some properties. J Biol Chem. 1956 Jan;218(1):97–106. [PubMed] [Google Scholar]
  18. Way J. C., Davis M. A., Morisato D., Roberts D. E., Kleckner N. New Tn10 derivatives for transposon mutagenesis and for construction of lacZ operon fusions by transposition. Gene. 1984 Dec;32(3):369–379. doi: 10.1016/0378-1119(84)90012-x. [DOI] [PubMed] [Google Scholar]