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 (DeltaCPS-P, DeltaCPS-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.

FIG. 1

Sequencing strategy of the L. plantarum carAB operon. Relevant restriction sites are indicated on the top. The arrows indicate primer orientations and localizations. Primers P6 and P7 define the ends of the ΔCPS-A deletion constructed in strains FB335 and HN217. Steps in our sequencing strategy are marked with capital letters in rectangles as follows. Region A is part of a previously sequenced 7.2-kb fragment (3). Segment B, a 1.8-kb fragment, was PCR amplified with a universal primer (M13Rev) and the P9 (5′-CAAGTACTAACGGCTACC-3′) internal carA primer deduced from region A. The template was a ligation mixture of the SmaI-digested pGID023 vector (14) ligated with a complete HincII digest of the L. plantarum genomic DNA. Fragment C was PCR amplified with a universal primer (M13Rev) and the P10 primer (5′-AGATCAAGAATCCAGTCACGC-3′) deduced from segment B. The template was a ligation mixture of the HindIII-digested pUC19 vector (26) ligated with a complete HindIII digest of the L. plantarum DNA. Region D was sequenced on one strand directly from a 4-kb gel-purified BclI digestion of L. plantarum DNA, PCR amplified, and subsequently sequenced on the complementary strand.

FIG. 2

Prokaryote CPS-A and CPS-P comparisons of GLN subunit subdomain junctions. The framed 8-aa-long deletion (CPS-AΔ) was found when comparing the smaller GLN subunits of the gram-positive bacteria CPS-A with the larger gram-positive CPS-P and the single E. coli CPS. Numbers on the right indicate the studied sequence positions within the different whole GLN subunits. The underlined E. coli Leu¹⁵³ marks the border between the bilobal GLN subunit subdomains: the NH₂-terminal and catalytic COOH-terminal subdomains (33). Underneath the sequences, three characters (*, :, and .) indicate positions which harbor a single conserved residue having fully, strongly, and weakly conserved groups, respectively, as defined by the ClustalX multiple sequence alignment program (35).

FIG. 3

Transcriptional and translational elements of the L. plantarum carAB operon. Numbers correspond to the citrulline biosynthetic cluster sequence (accession number X99978). The +1 labels indicate transcriptional start sites, which are marked with thick arrows. Residues involved in the carA promoter are double underlined. A B. subtilis ARG box-like sequence (23) around the +1 site is indicated with a blackened box. The proposed translational initiation and stop codons are indicated with boxes for the CPS-A GLN (carA gene) and SYN (carB gene) subunits and for the adjacent divergently oriented ORF. Good putative ribosome binding sites which exhibit complementarity to the L. plantarum 3′-terminal and 5′-terminal 16S rRNA sequence (EMBL accession number M58827) are boldface and underlined, respectively. The free energy (ΔG [36]) characterizes the base pairing between the different RNAs. Two arrows indicate the inversely repeated (IR) sequences preceding a row of T residues, which suggests a transcriptional rho-independent terminator (estimated ΔG of −12 kcal/mol for IR pairing).

FIG. 4

Transcription studies and arginine repression with Northern hybridizations using mRNAs extracted from L. plantarum grown on defined medium without and with arginine (tracks 1 and 2, respectively) and probed with a carA (A) or carB (B) probe. The size of the transcript was determined using the Gibco BRL 0.24- to 9.5-kb RNA ladder (track M), which was revealed by hybridization with digoxigenin-labeled λ DNA.

FIG. 5

Primer extension mapping of the 5′ end of the carAB and carB mRNAs. RNAs were extracted from L. plantarum CCM1904 grown on defined medium without arginine and no agitation at 30°C. Sequencing ladders generated with the oligonucleotide (P11 [5′-GTCCGCTAAAGTTAAGTATTTG-3′] for carAB; P12 [5′-AGCAATCGAATGAATGGCATTG-3′] for carB) used for the primer extension were loaded next to the reaction mixture (track RT). On the DNA sequence of the sense strand, the first nucleotide in the transcript is indicated as +1 and the promoter −10 sequence is boxed.