In Lactobacillus plantarum , Carbamoyl Phosphate Is Synthesized by Two Carbamoyl-Phosphate Synthetases (CPS): Carbon Dioxide Differentiates the Arginine-Repressed from the Pyrimidine-Regulated CPS

Abstract

Carbamoyl phosphate (CP) is an intermediate in pyrimidine and arginine biosynthesis. Carbamoyl-phosphate synthetase (CPS) contains a small amidotransferase subunit (GLN) that hydrolyzes glutamine and transfers ammonia to the large synthetase subunit (SYN), where CP biosynthesis occurs in the presence of ATP and CO 2 . Lactobacillus plantarum, a lactic acid bacterium, harbors a pyrimidine-inhibited CPS (CPS-P; Elagöz et al., Gene 182:37-43, 1996) and an arginine-repressed CPS (CPS-A). Sequencing has shown that CPS-A is encoded by carA (GLN) and carB (SYN). Transcriptional studies have demonstrated that carB is transcribed both monocistronically and in the carAB arginine-repressed operon. CP biosynthesis in L. plantarum was studied with three mutants (⌬CPS-P, ⌬CPS-A, and double deletion). In the absence of both CPSs, auxotrophy for pyrimidines and arginine was observed. CPS-P produced enough CP for both pathways. In CO 2 -enriched air but not in ordinary air, CPS-A provided CP only for arginine biosynthesis. Therefore, the uracil sensitivity observed in prototrophic wild-type L. plantarum without CO 2 enrichment may be due to the low affinity of CPS-A for its substrate CO 2 or to regulation of the CP pool by the cellular CO 2 /bicarbonate level.Lactobacilli are fastidious gram-positive bacteria with complex nutritional requirements resulting from numerous genetic lesions in metabolic pathways which often revert to prototrophy (24). Natural auxotrophies involved in carbamoyl phosphate (CP) biosynthesis were shown to be reversed in most cases by incubating lactobacilli in CO 2 -enriched air (2; F. Bringel, unpublished data). Metabolically, lactobacilli are at the threshold of the transition from anaerobic to aerobic life (16); Lactobacillus plantarum grows in aerobiosis but prefers microaerobiosis or increased CO 2 concentration.The arginine and pyrimidine biosynthetic pathways share CP as a common precursor. CP synthetase (CPS) catalyzes the synthesis of CP from bicarbonate, glutamine, and two molecules of ATP via a complex reaction mechanism that leads to several unstable intermediates. The X-ray crystal structure of CPS in Escherichia coli has revealed the location of three separate active sites connected by two molecular tunnels that run through the interior of the protein (33) and that would allow substrate channeling and subsequent protection of the reactive intermediates (reviewed in reference 22). Furthermore, CP biosynthesis in Pyrococcus abyssi also includes metabolic channeling, a process whereby the product of one enzyme is directly transferred to the next enzyme in the pathway without being released in the bulk solvent; CPS may interact with two CP-utilizing enzymes in the pyrimidine (aspartate carbamoyltransferase) and the arginine (anabolic ornithine carbamoyltransferase) biosynthetic pathways (29). The heterodimeric CPS enzyme is composed of a small subunit (GLN) which functions as a glutamine amidotransferase and a large synthetase subunit (SYN) that fulfills the other catalytic properties (for a re...

The relevance of carbon dioxide metabolism in Streptococcus thermophilus

“…In L. plantarum, carbamoyl phosphate, the precursor of both pyrimidine and arginine synthesis (Fig. 1), is synthesized by two CpSs, namely CpS-P and CpS-A, encoded by pyrAaAb and carAB, respectively, whose transcription is regulated by pyrimidine and arginine availability (Nicoloff et al, 2000(Nicoloff et al, , 2005. L. plantarum strains showing a high-CO 2 -requiring auxotroph are able to synthesize sufficient arginine and pyrimidines in CO 2 -enriched air only.…”

“…While a CO 2 -enriched atmosphere is used effectively in foodstuff preservation against bacterial spoilage, a growthstimulating effect of CO 2 has been reported for a limited number of lactic acid bacterial species. Detailed studies are available for Lactobacillus plantarum and Lactococcus lactis only (Wang et al, 1998(Wang et al, , 2000Nicoloff et al, 2000Nicoloff et al, , 2005Arsène-Ploetze et al, 2006). The high atmospheric concentration of CO 2 could have several effects on cellular physiology because it alters membrane properties, probably modifies the intracellular pH, and interferes with carboxylation reactions.…”

Article

“…In E. faecalis and L. mesenteroides, some auxotrophies were suppressed when strains were incubated in a CO 2 -enriched atmosphere [24,25]. In L. plantarum, auxotrophies for phenylalanine, arginine, tyrosine and pyrimidines were observed in several strains incubated under normal air but not when incubated in a CO 2 -enriched air [4,25,29]. CO 2 may be required in these organisms for carboxylation reactions like in E. coli.…”

“…nate stimulated L. plantarum growth in media supplemented with arginine and uracil IC stimulation of L. plantarum growth may be explained by the requirement of CO 2 as a substrate for the carboxylation reaction of the CPS (EC 6.3.5.5) involved in both arginine and pyrimidine biosynthesis pathways [29]. To test this hypothesis, we analysed the prototroph CCM 1904's growth with respect to IC supply, in the presence of arginine (A) and uracil (U) in different media: minimal medium, MM, or rich defined medium, DLA.…”

“…The authors attempted to relieve this growth inhibition by adding metabolites whose synthesis are known to depend on carboxylation reactions, but the growth inhibition could not be completely overcome. In L. plantarum, it was proposed that IC enrichment was required for efficient carbamoyl-phosphate synthetase (CPS) activity [29]. However, as demonstrated in E. coli, CO 2 could be important for LAB metabolism besides the sensu stricto carboxylation reactions.…”