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

Abstract

The CoblIl 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 CobI1 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 CobIli 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 CobIll 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:adenosylcobinamide-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.

The complete coenzyme B12 biosynthesis gene cluster of Lactobacillus reuteri CRL1098

“…reuteri's genomic DNA using Herculase II DNA polymerase (Stratagene), and primers LREf28196_28215 and LREr29724_29704 (Tables 1 and 2). Additionally, a fragment containing the native cob operon promoter (O'Toole et al, 1993) was amplified from Salmonella enterica strain TT25720 using primers 59-GACACCATTGTGGATGAGGTGGAG-39 and 59-GATGATCG-ATCATACCGGCTCCTGATGT-39 (ClaI cleavage site underlined). 39-A overhangs were added to both fragments by incubating the PCR reactions directly with 1 unit of Taq DNA polymerase for 3 min at 72 uC.…”

“…reuteri under control of the native cob operon promoter from Salmonella enterica (O'Toole et al, 1993). Growth experiments were then performed on minimal E plates using strains TT25720 and TT25722 as a positive and negative control, respectively.…”

The last step in coenzyme B12 synthesis is localized to the cell membrane in bacteria and archaea

“…Although the work reported here did not investigate the putative cobalamin synthase activity of the L18 protein in S. enterica, on the basis of previously reported genetic and biochemical evidence (Maggio-Hall & Escalante-Semerena, 1999;O'Toole et al, 1993) and this work, it is concluded that the cobalamin synthase activity of the CobS protein is essential for AdoCbl synthesis, and is associated with the inner membrane in this bacterium, and probably in all corrinoid-producing prokaryotes.…”

“…Plasmid pCOBS1 was derived from pJO21, a plasmid previously described (O'Toole et al, 1993). The HincII-HindIII fragment of pJO21 was cloned into cloning Fig.…”

“…In considering the physiological relevance of the putative cobalamin synthase activity of the L18 protein one must bear in mind that: (i) the L18 protein failed to use a-ribazole and a-ribazole phosphate, the documented substrates of cobalamin synthase (Walerych et al, 1968); (ii) the work on the L18 protein predated the identification of the cob genes in any prokaryote, and since the discovery of the cob genes, cobalamin synthase activity has been shown to be encoded by the cobS gene of S. enterica and the cobV gene of P. denitrificans (Cameron et al, 1991;Maggio-Hall & Escalante-Semerena, 1999); (iii) cobS mutant strains of S. enterica lack cobalamin synthase activity and are unable to convert AdoCbi to AdoCbl (Jeter et al, 1984;O'Toole et al, 1993). If the L18 protein had a physiologically relevant cobalamin synthase activity, no phenotype would be observed; (iv) in spite of the use of an extremely sensitive bioassay, cobalamin synthase activity was not detectable in cell-free extracts of strains carrying a deletion of the cobS gene (Maggio-Hall & EscalanteSemerena, 1999); and (v) wild-type chromosomal levels of cobalamin synthase activity in S. enterica were three orders of magnitude higher than the activity reported for purified E. coli ribosomes (Pezacka & Walerych, 1981).…”