Cloning and characterization of the pyruvate carboxylase from Sinorhizobium meliloti Rm1021

Abstract.

The gene encoding pyruvate carboxylase (pyc) was isolated from a Sinorhizobium meliloti Rm1021 cosmid bank by complementation of a Rhizobium tropici pyc mutant. PYC-negative mutants of S. meliloti Rm1021 were isolated by transposon mutagenesis and were unable to grow with glucose or pyruvate as sole carbon sources, but were symbiotically competent in combination with alfalfa plants. PYC activity assays, pyc::lacZ gene fusion studies and an in vivo biotinylation assay showed that PYC activity in S. meliloti was dependent mainly on biotin availability and not on changes in gene transcription. The subunit and holo-enzyme molecular masses of the S. meliloti PYC indicated that the enzyme was an α₄ homotetramer. The S. meliloti PYC had a high apparent K _a (0.23 mM) for the allosteric activator acetyl-CoA and was product-inhibited by sub-millimolar concentrations of oxaloacetate. In contrast to other bacterial α₄-PYCs which have been characterized, the S. meliloti enzyme was not strongly inhibited by L-aspartate.