The gene encoding pyruvate carboxylase (pyc) was isolated from a Sinorhizob
ium meliloti Rm1021 cosmid bank by complementation of a Rhizobium tropici p
yc mutant. PYC-negative mutants of S. meliloti Rm1021 were isolated by tran
sposon mutagenesis and were unable to grow with glucose or pyruvate as sole
carbon sources, but were symbiotically competent in combination with alfal
fa plants. PYC activity assays, pyc::lacZ gene fusion studies and an in viv
o biotinylation assay showed that PYC activity in S. meliloti was dependent
mainly on biotin availability and not on changes in gene transcription. Th
e subunit and holo-enzyme molecular masses of the S. meliloti PYC indicated
that the enzyme was an alpha (4) homotetramer. The S. meliloti PYC had a h
igh apparent Ka (0.23 mM) for the allosteric activator acetyl-CoA and was p
roduct-inhibited by sub-millimolar concentrations of oxaloacetate. In contr
ast to other bacterial alpha (4)-PYCS which have been characterized, the S.
meliloti enzyme was not strongly inhibited by L-aspartate.