Expression of the pyr operon of Lactobacillus plantarum is regulated by inorganic carbon availability through a second regulator, pyr[R.sub.2], homologous to the pyrimidine-dependent regulator pyr[R.sub.1]

Inorganic carbon (IC), such as bicarbonate or carbon dioxide, stimulates the growth of Lactobacillus plantarum. At low IC levels, one-third of natural isolated L. plantarum strains are nutritionally dependent on exogenous arginine and pyrimidine, a phenotype previously defined as high-C[O.sub.2]-requiring (HCR) prototrophy. IC enrichment significantly decreased the amounts of the enzymes in the pyrimidine biosynthetic pathway encoded by the pyr[R.sub.1]BC[Aa.sub.1][Ab.sub.1]DFE operon, as demonstrated by proteomic analysis. Northern blot and reverse transcription-PCR experiments demonstrated that IC levels regulated pyr genes mainly at the level of transcription or RNA stability. Two putative PyrR regulators with 62% amino acid identity are present in the L. plantarum genome. Pyr[R.sub.1] is an RNA-binding protein that regulates the pyr genes in response to pyrimidine availability by a mechanism of transcriptional attenuation. In this work, the role of Pyr[R.sub.2] was investigated by allelic gene replacement. Unlike the pyr[R.sub.1] mutant, the [DELTA]pyr[R.sub.2] strain acquired a demand for both pyrimidines and arginine unless bicarbonate or C[O.sub.2] was present at high concentrations, which is known as an HCR phenotype. Analysis of the IC- and pyrimidine-mediated regulation in [pyrR.sub.1] and [pyrR.sub.2] mutants suggested that only Pyr[R.sub.2] positively regulates the expression levels of the pyr genes in response to IC levels but had no effect on pyrimidine-mediated repression. A model is proposed for the respective roles of Pyr[R.sub.1] and Pyr[R.sub.2] in the pyr regulon expression.