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. 1993 Jun;175(11):3317–3326. doi: 10.1128/jb.175.11.3317-3326.1993

Analysis of mutants of Salmonella typhimurium defective in the synthesis of the nucleotide loop of cobalamin.

G A O'Toole 1, M R Rondon 1, J C Escalante-Semerena 1
PMCID: PMC204728  PMID: 8501035

Abstract

The CobIII region of the cobalamin (CBL) biosynthetic (cob) operon of Salmonella typhimurium encodes functions necessary for the synthesis of the nucleotide loop of CBL and comprises three genes, designated cobU, cobS, and cobT (26). Complementation studies identified two classes of CobIII mutants: (i) 34 mutants were complemented by a plasmid carrying the cobU+ gene, and (ii) 27 mutants were complemented by a plasmid carrying the cobS+ gene; none of the mutants tested was complemented by the cobT+ clone, a result suggesting that no cobT mutations were isolated. These data were consistent with those of complementation studies done with F' cobUST plasmids, which also suggested that the CobIII region comprises two complementation groups. A plasmid carrying cobUS+ was sufficient to complement a deletion of the entire CobIII region, a result suggesting that CobT was not required for CBL biosynthesis. Nutritional studies done with synthetic putative intermediates of the CobIII pathway were performed to further classify cobIII mutants. A subset of cobU mutants were found to be responsive to exogenous dicyano-cobinamide-GDP, while cobS mutants were found to be responsive only to CBL. These results are consistent with the adenosyl-cobinamide kinase-GTP:adenosyl-cobinamide-phosphate guanylyltransferase and CBL synthase activities proposed for CobU and CobS, respectively. The cobIII genes under the control of the T7 promoter were overexpressed, and the resulting polypeptides were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Three polypeptides with apparent molecular masses of 22, 26 and 39 kDa, consistent with the predicted masses for CobU, CobS, and CobT, respectively, were detected.

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Selected References

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  1. Balch W. E., Fox G. E., Magrum L. J., Woese C. R., Wolfe R. S. Methanogens: reevaluation of a unique biological group. Microbiol Rev. 1979 Jun;43(2):260–296. doi: 10.1128/mr.43.2.260-296.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bassford P. J., Jr, kadner R. J. Genetic analysis of components involved in vitamin B12 uptake in Escherichia coli. J Bacteriol. 1977 Dec;132(3):796–805. doi: 10.1128/jb.132.3.796-805.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Blanche F., Debussche L., Famechon A., Thibaut D., Cameron B., Crouzet J. A bifunctional protein from Pseudomonas denitrificans carries cobinamide kinase and cobinamide phosphate guanylyltransferase activities. J Bacteriol. 1991 Oct;173(19):6052–6057. doi: 10.1128/jb.173.19.6052-6057.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Blanche F., Thibaut D., Couder M., Muller J. C. Identification and quantitation of corrinoid precursors of cobalamin from Pseudomonas denitrificans by high-performance liquid chromatography. Anal Biochem. 1990 Aug 15;189(1):24–29. doi: 10.1016/0003-2697(90)90038-b. [DOI] [PubMed] [Google Scholar]
  5. 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]
  6. Elliott T., Roth J. R. Heme-deficient mutants of Salmonella typhimurium: two genes required for ALA synthesis. Mol Gen Genet. 1989 Apr;216(2-3):303–314. doi: 10.1007/BF00334369. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. 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]
  9. Grabau C., Roth J. R. A Salmonella typhimurium cobalamin-deficient mutant blocked in 1-amino-2-propanol synthesis. J Bacteriol. 1992 Apr;174(7):2138–2144. doi: 10.1128/jb.174.7.2138-2144.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. Jeter R. M., Roth J. R. Cobalamin (vitamin B12) biosynthetic genes of Salmonella typhimurium. J Bacteriol. 1987 Jul;169(7):3189–3198. doi: 10.1128/jb.169.7.3189-3198.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. 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]
  13. KENNEDY E. P. The synthesis of cytidine diphosphate choline, cytidine diphosphate ethanolamine, and related compounds. J Biol Chem. 1956 Sep;222(1):185–191. [PubMed] [Google Scholar]
  14. 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]
  15. Ronzio R. A., Barker H. A. Enzymic synthesis of guanosine diphosphate cobinamide by extracts of propionic acid bacteria. Biochemistry. 1967 Aug;6(8):2344–2354. doi: 10.1021/bi00860a009. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. 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]
  18. 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]
  19. 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]
  20. Tabor S., Richardson C. C. A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc Natl Acad Sci U S A. 1985 Feb;82(4):1074–1078. doi: 10.1073/pnas.82.4.1074. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Tabor S., Richardson C. C. DNA sequence analysis with a modified bacteriophage T7 DNA polymerase. Proc Natl Acad Sci U S A. 1987 Jul;84(14):4767–4771. doi: 10.1073/pnas.84.14.4767. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. WAGNER F. [Syntheses in the vitamin B12 field. XII. Cobinamide phosphate and cobalamine-5'-phosphate]. Biochem Z. 1962;336:99–101. [PubMed] [Google Scholar]
  24. Xu K., Delling J., Elliott T. The genes required for heme synthesis in Salmonella typhimurium include those encoding alternative functions for aerobic and anaerobic coproporphyrinogen oxidation. J Bacteriol. 1992 Jun;174(12):3953–3963. doi: 10.1128/jb.174.12.3953-3963.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]

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