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biosynthesis were shown to be reversed in most cases by incubating lactobacilli in CO 2 -enriched air (2; F. Bringel, unpublished data).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...
IR was not limited to β-lactamase-producing bacteria, but was also caused by resistant bacteria carrying cytoplasmic antibiotic-modifying or -degrading enzymes that catalyse energy-consuming reactions requiring complex cellular cofactors. Our results suggest that IR is common and further emphasizes that coinfecting agents and the human microflora can have a negative impact during antimicrobial therapy.
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