Repression of the pyr Operon in Lactobacillus plantarum Prevents Its Ability To Grow at Low Carbon Dioxide Levels

Abstract

ABSTRACT Carbamoyl phosphate is a precursor for both arginine and pyrimidine biosynthesis. In Lactobacillus plantarum , carbamoyl phosphate is synthesized from glutamine, ATP, and carbon dioxide by two sets of identified genes encoding carbamoyl phosphate synthase (CPS). The expression of the carAB operon (encoding CPS-A) responds to arginine availability, whereas pyrAaAb (encoding CPS-P) is part of the pyrR1BCAaAbDFE operon coding for the de novo pyrimidine pathway repressed by exogenous uracil. The pyr operon is regulated by transcription attenuation mediated by a trans -acting repressor that binds to the pyr mRNA attenuation site in response to intracellular UMP/phosphoribosyl pyrophosphate pools. Intracellular pyrimidine triphosphate nucleoside pools were lower in mutant FB335 ( carAB deletion) harboring only CPS-P than in the wild-type strain harboring both CPS-A and CPS-P. Thus, CPS-P activity is the limiting step in pyrimidine synthesis. FB335 is unable to grow in the presence of uracil due to a lack of sufficient carbamoyl phosphate required for arginine biosynthesis. Forty independent spontaneous FB335-derived mutants that have lost regulation of the pyr operon were readily obtained by their ability to grow in the presence of uracil and absence of arginine; 26 harbored mutations in the pyrR1-pyrB loci. One was a prototroph with a deletion of both pyrR1 and the transcription attenuation site that resulted in large amounts of excreted pyrimidine nucleotides and increased intracellular UTP and CTP pools compared to wild-type levels. Low pyrimidine-independent expression of the pyr operon was obtained by antiterminator site-directed mutagenesis. The resulting AE1023 strain had reduced UTP and CTP pools and had the phenotype of a high-CO 2 -requiring auxotroph, since it was able to synthesize sufficient arginine and pyrimidines only in CO 2 -enriched air. Therefore, growth inhibition without CO 2 enrichment may be due to low carbamoyl phosphate pools from lack of CPS activity.