ANALYSIS OF MUTANTS OF SALMONELLA-TYPHIMURIUM DEFECTIVE IN THE SYNTHESIS OF THE NUCLEOTIDE LOOP OF COBALAMIN

The CobIII region of the cobalamin (CBL) biosynthetic (cob) operon of Salmonella typhimurium encodes functions necessary for the synthesis o f the nucleotide loop of CBL and comprises three genes, designated cob U, cobS, and cobT (26). Complementation studies identified two classes of CobIII mutants: (i) 34 mutants were complemented by a plasmid carr ying the cobU+ gene, and (ii) 27 mutants were complemented by a plasmi d carrying the cobS+ gene; none of the mutants tested was complemented by the cobT+ clone, a result suggesting that no cobT mutations were i solated. These data were consistent with those of complementation stud ies 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 su bset 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 synthas e activities proposed for CobU and CobS, respectively. The cobIII gene s under the control of the T7 promoter were overexpressed, and the res ulting polypeptides were analyzed by sodium dodecyl sulfate-polyacryla mide gel electrophoresis. Three polypeptides with apparent molecular m asses of 22, 26, and 39 kDa, consistent with the predicted masses for CobU, CobS, and CobT, respectively, were detected.