Genetic analysis of carbamoylphosphate synthesis in Rhizobium meliloti 104A14.

We have previously isolated ineffective (Fix-) mutants of Rhizobium meliloti 104A14 requiring both arginine and uracil, and thus probably defective in carbamoylphosphate synthetase. We describe here the molecular and genetic analysis of the R. meliloti genes coding for carbamoylphosphate synthetase. Plasmids that complement the mutations were isolated from a R. meliloti gene bank. Restriction analysis of these plasmids indicated that complementation involved two unlinked regions of the R. meliloti chromosome, carA and carB. Genetic complementation between the plasmids and mutants demonstrated a single complementation group for carA, but two overlapping complementation groups for carB. The cloned R. meliloti genes hybridize to the corresponding E. coli carA and carB genes which encode the two subunits of carbamoylphosphate synthetase. Transposon Tn5 mutagenesis was used to localize the carA and carB genes on the cloned R. meliloti DNA. The cloned R. meliloti carA and carB genes were unable to complement E. coli carA or carB mutants alone or in combination. We speculate on the mechanism of the unusual pattern of genetic complementation at the R. meliloti carB locus.