Extent of genetic lesions of the arginine and pyrimidine biosynthetic pathways in Lactobacillus plantarum, L. paraplantarum, L. pentosus, and L. casei: prevalence of CO(2)-dependent auxotrophs and characterization of deficient arg genes in L. plantarum

Abstract

Lactic acid bacteria require rich media since, due to mutations in their biosynthetic genes, they are unable to synthesize numerous amino acids and nucleobases. Arginine biosynthesis and pyrimidine biosynthesis have a common intermediate, carbamoyl phosphate (CP), whose synthesis requires CO(2). We investigated the extent of genetic lesions in both the arginine biosynthesis and pyrimidine biosynthesis pathways in a collection of lactobacilli, including 150 strains of Lactobacillus plantarum, 32 strains of L. pentosus, 15 strains of L. paraplantarum, and 10 strains of L. casei. The distribution of prototroph and auxotroph phenotypes varied between species. All L. casei strains, no L. paraplantarum strains, two L. pentosus strains, and seven L. plantarum strains required arginine for growth. Arginine auxotrophs were more frequently found in L. plantarum isolated from milk products than in L. plantarum isolated from fermented plant products or humans; association with dairy products might favor arginine auxotrophy. In L. plantarum the argCJBDF genes were functional in most strains, and when they were inactive, only one gene was mutated in more than one-half of the arginine auxotrophs. Random mutation may have generated these auxotrophs since different arg genes were inactivated (there were single point mutations in three auxotrophs and nonrevertible genetic lesions in four auxotrophs). These data support the hypothesis that lactic acid bacteria evolve by progressively loosing unnecessary genes upon adaptation to specific habitats, with genome evolution towards cumulative DNA degeneration. Although auxotrophy for only uracil was found in one L. pentosus strain, a high CO(2) requirement (HCR) for arginine and pyrimidine was common; it was found in 74 of 207 Lactobacillus strains tested. These HCR auxotrophs may have had their CP cellular pool-related genes altered or deregulated.

FIG. 1.

Arginine and pyrimidine biosynthesis in L. plantarum. Arginine synthesis and pyrimidine synthesis have a common intermediate, CP. CPS catalyzes the formation of CP from one molecule of glutamine, two molecules of Mg²⁺-ATP, and one molecule of bicarbonate, which is the dissolved form of CO₂. Two types of CPS are present in L. plantarum: CPS-A, the arginine-regulated CPS, and CPS-P, the pyrimidine-regulated CPS (28). (A) Summarized scheme of the arginine biosynthesis pathway (thick arrows) and the pyrimidine biosynthesis pathway (thin arrows). Ornithine and citrulline are precursors of arginine. The argCJBD genes are involved in ornithine synthesis from glutamate with the recycling of N-acetyl intermediates. The argF gene encodes OTCase responsible for citrulline synthesis from ornithine and CP. (B) Gene organization. The pyr operon (pyrRBCAaAbDFE) containing the de novo pyrimidine synthesis genes (13) is controlled by transcriptional attenuation by the regulatory protein PyrR. The genes involved in citrulline biosynthesis are organized in two divergently transcribed operons, carAB and argCJBDF, which are repressed by arginine (3). The genes associated with octagons were mutated in seven L. plantarum strains isolated from cheese or pickled vegetables.