Low carbamoyl phosphate pools may drive Lactobacillus plantarum CO2-dependent growth phenotype

Lactobacillus plantarum is often found in nutrient-rich habitats with elevated levels of inorganic carbon (IC), and IC-dependent growth is commonly encountered in natural isolates of this species. High CO(2)-requiring (HCR) prototrophs are unable to grow under conditions of low IC unless arginine and pyrimidines are provided. Prototrophy is restored under high IC conditions, that is in 4% CO(2)-enriched air or bicarbonate-supplemented medium. Bicarbonate is required for the synthesis of carbamoyl phosphate (CP), a precursor of both arginine and pyrimidine biosynthesis. We hypothesize that at low IC levels, intracellular CP pools limit growth through the limitation of arginine and nucleotide supplies. HCR mutants obtained in the laboratory can be classified into 3 functional groups: mutants with impaired CP synthesis, increased CP consumption or increased CP requirements relative to wild type. This classification provides a framework for investigating the origin of the HCR phenotype in natural environmental isolates of Lactobacillus species, and to investigate the hypothesis that a low level of carbamoyl phosphate is a major determinant of the CO(2)-dependent growth phenotype often observed in L. plantarum isolates.     

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

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.     

Extent of Genetic Lesions of the Arginine and Pyrimidine Biosynthetic Pathways in Lactobacillus plantarum, L. paraplantarum, L. pentosus, and L. casei: Prevalence of CO2-Dependent Auxotrophs and Characterization of Deficient arg Genes in L. plantarum

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 CO₂. 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 CO₂ 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.     

Two arginine repressors regulate arginine biosynthesis in Lactobacillus plantarum

The repression of the carAB operon encoding carbamoyl phosphate synthase leads to Lactobacillus plantarum FB331 growth inhibition in the presence of arginine. This phenotype was used in a positive screening to select spontaneous mutants deregulated in the arginine biosynthesis pathway. Fourteen mutants were genetically characterized for constitutive arginine production. Mutations were located either in one of the arginine repressor genes (argR1 or argR2) present in L. plantarum or in a putative ARG operator in the intergenic region of the bipolar carAB-argCJBDF operons involved in arginine biosynthesis. Although the presence of two ArgR regulators is commonly found in gram-positive bacteria, only single arginine repressors have so far been well studied in Escherichia coli or Bacillus subtilis. In L. plantarum, arginine repression was abolished when ArgR1 or ArgR2 was mutated in the DNA binding domain, or in the oligomerization domain or when an A123D mutation occurred in ArgR1. A123, equivalent to the conserved residue A124 in E. coli ArgR involved in arginine binding, was different in the wild-type ArgR2. Thus, corepressor binding sites may be different in ArgR1 and ArgR2, which have only 35% identical residues. Other mutants harbored wild-type argR genes, and 20 mutants have lost their ability to grow in normal air without carbon dioxide enrichment; this revealed a link between arginine biosynthesis and a still-unknown CO2-dependent metabolic pathway. In many gram-positive bacteria, the expression and interaction of different ArgR-like proteins may imply a complex regulatory network in response to environmental stimuli.     

Salmonella typhimurium mutants with altered expression of the pyrA gene due to changes in RNA polymerase

Rifampicin-resistant mutants of Salmonella typhimurium were isolated and tested for pleiotropic defects in the regulation of pyr gene expression. Seven per cent of all the Rifr mutants were inhibited in growth by addition of uracil (uracil-sensitive). The uracil-sensitive phenotype ( UraS ) was reversed by arginine or citrulline, but not by ornithine, and it was suppressed by mutations in either argR or pyrH , which causes increased expression of pyrA . It was shown that the basal levels of carbamoylphosphate synthase (the pyrA gene product) was reduced to approximately 60% in the mutants, and that addition of arginine and/or uracil to the growth medium caused hyperrepression of pyrA expression. The expression of other genes of the arginine and pyrimidine biosynthetic pathways was not affected significantly in the mutants. The mutations were located in the rpoB gene coding for the beta-subunit of RNA polymerase, suggesting a regulatory function of RNA polymerase in the control of pyrA expression.     

Polyphasic investigation of the diversity within Lactobacillus plantarum related strains revealed two L. plantarum subgroups

The diversity of 140 strains related to Lactobacillus plantarum was investigated using a polyphasic approach combining two molecular techniques: randomly amplified polymorphic DNA fingerprinting (RAPD) and Southern hybridisation with a pyr probe on BglI digests of chromosomal DNA, as well as phenotypic characterization. The RAPD technique allowed us to classify a subset of 60 representative strains into four groups. One group belonged to Lactobacillus paraplantarum, the second to Lactobacillus pentosus and the two remaining groups to L. plantarum (G(L)p1 and G(L)p2). The Southern hybridisation technique (F. Bringel, M.-C. Curk and J.-C. Hubert, Int. J. Syst. Bacteriol. 46: 588-594, 1996) revealed nine groups of profiles (I to IX). Results indicated an excellent convergence between RAPD and hybridisation classifications for more than 93% (56/60) of the strains studied. When we compared the fermentation patterns of the L. plantarum strains, three differences were found. Melezitose fermentation was not fermented by the G(L)p2 RAPD group, unlike the G(L)p1 RAPD group which included L. plantarum type strain NCIMB11974T. Second, alpha-methyl-D-mannoside was fermented by a majority of the strains of the G(L)p1 RAPD group but by none of the strains in the G(L)p2 RAPD group. Third, dulcitol was catabolized by nearly half of the strains of the G(L)p2 RAPD group but by none of the strains in the G(L)p1 RAPD group. Molecular diversity within L. plantarum was confirmed using Southern profiles, PCR amplification and subsequent sequencing of these PCR products. A 773 bp sequence overlapping the pyrDF genes showed high homology: at least 97% identical in L. plantarum strains (V to IX) and 99.9% identical in hybridisation groups VII and VIII. The same G-T transversion which destroyed the pyrF BglI site was found in 11 strains (hybridisation groups VI, VII and VIII). DNA rearrangements were identified downstream from the pyr genes, by PCR amplification and Southern hybridisation profile analysis in three strains of hybridisation groups VIII and IX, two of which also harboured the G-T transversion.     

Repression and derepression of the enzymes of the pyrimidine biosynthetic pathway in Salmonella typhimurium

Activities of five enzymes of the pyrimidine biosynthetic pathway and one enzyme involved in arginine synthesis were measured during batch culture of Salmonella typhimurium. Aspartate carbamoyltransferase, dihydroorotase, and the arginine pathway enzyme, ornithine carbamoyltransferase, remained constant during the growth cycle but showed a sharp decrease in activity after entering the stationary phase. Dihydroorotate dehydrogenase, orotate phosphoribosyltransferase and orotidine-5'-monophosphate (OMP) decarboxylase showed peaks of activity corresponding to the mid-point of the exponential phase of growth while remaining comparatively stable in the stationary phase. Derepression studies carried out by starving individual pyrimidine (Pyr-) deletion mutants for uracil showed that the extent of derepression obtained for aspartate carbamoyltransferase, dihydroorotase and dihydroorotate dehydrogenase depended on the location of the pyr gene mutation. Orotate phosphoribosyltransferase and OMP decarboxylase derepression levels were independent of the location of the pyr mutation. Aspartate carbamoyltransferase showed the greatest degree of derepression of the six enzymes studied, with pyrA strains (blocked in the first step of the pathway) showing about twice as much derepression as pyrF strains (blocked in the sixth step of the pathway). A study of the kinetics of repression on derepressed levels of the pyrimidine enzymes produced data that were compatible with dilution of specific activity by cell division when repressive amounts of uracil were added to the derepression medium.     

Two genes encoding uracil phosphoribosyltransferase are present in Bacillus subtilis.

Uracil phosphoribosyltransferase (UPRTase) catalyzes the key reaction in the salvage of uracil in many microorganisms. Surprisingly, two genes encoding UPRTase activity were cloned from Bacillus subtilis by complementation of an Escherichia coli mutant. The genes were sequenced, and the putative amino acid sequences were deduced. One gene showed a high level of homology to UPRTases from other organisms, whereas the other gene with a low level of homology to other UPRTases turned out to be the pyrR gene--the repressor of the pyr operon. The role of these genes in uracil metabolism was established by an analysis of the phenotypes of upp and pyrR mutants.     

pyrR identical to pyrH in Salmonella typhimurium: control of expression of the pyr genes.

Mutants of Salmonella typhimurium showing constitutive synthesis of the pyrimidine biosynthetic enzymes coded for by the pyrA-F genes (G. A. O'Donavan and J. C. Gerhart, 1972) have been reinvestigated. The high rate of expression of the pyrB-F genes in these mutants as well as their pyrimidine excretion is shown to be due to mutations in the gene pyrH encoding uridine 5'-monophosphate kinase. Thus, the term pyrR used for these mutants should be replaced by the designation pyrH.