Repression of the pyr operon in Lactobacillus plantarum prevents its ability to grow at low carbon dioxide levels

Abstract

Carbamoyl phosphate is a precursor for both arginine and pyrimidine biosynthesis. In Lactobacillus plantarum, carbamoyl phosphate is synthesized from glutamine, ATP, and carbon dioxide by two sets of identified genes encoding carbamoyl phosphate synthase (CPS). The expression of the carAB operon (encoding CPS-A) responds to arginine availability, whereas pyrAaAb (encoding CPS-P) is part of the pyrR1BCAaAbDFE operon coding for the de novo pyrimidine pathway repressed by exogenous uracil. The pyr operon is regulated by transcription attenuation mediated by a trans-acting repressor that binds to the pyr mRNA attenuation site in response to intracellular UMP/phosphoribosyl pyrophosphate pools. Intracellular pyrimidine triphosphate nucleoside pools were lower in mutant FB335 (carAB deletion) harboring only CPS-P than in the wild-type strain harboring both CPS-A and CPS-P. Thus, CPS-P activity is the limiting step in pyrimidine synthesis. FB335 is unable to grow in the presence of uracil due to a lack of sufficient carbamoyl phosphate required for arginine biosynthesis. Forty independent spontaneous FB335-derived mutants that have lost regulation of the pyr operon were readily obtained by their ability to grow in the presence of uracil and absence of arginine; 26 harbored mutations in the pyrR1-pyrB loci. One was a prototroph with a deletion of both pyrR1 and the transcription attenuation site that resulted in large amounts of excreted pyrimidine nucleotides and increased intracellular UTP and CTP pools compared to wild-type levels. Low pyrimidine-independent expression of the pyr operon was obtained by antiterminator site-directed mutagenesis. The resulting AE1023 strain had reduced UTP and CTP pools and had the phenotype of a high-CO2-requiring auxotroph, since it was able to synthesize sufficient arginine and pyrimidines only in CO2-enriched air. Therefore, growth inhibition without CO2 enrichment may be due to low carbamoyl phosphate pools from lack of CPS activity.

FIG. 1.

Simplified pyrimidine biosynthesis and link with arginine biosynthesis in an L. plantarum ΔcarAB mutant. Carbamoyl phosphate (CP) is a common precursor in arginine and pyrimidine biosynthesis. The wild-type strain CCM 1904 has two functional carbamoyl phosphate synthases (CPSs) (19). The carAB operon codes for the two subunits of the arginine-repressed CPS, CPS-A. The pyrAaAb genes present on the pyr operon (6) code for the pyrimidine-regulated CPS, CPS-P (19). Exogenous uracil enters the cell and is metabolized to UMP, which negatively controls the pyr operon, including the pyrAaAb genes. The carAB genes have been deleted in strain FB335, so that CPS-P is the only source of carbamoyl phosphate for pyrimidine and arginine biosynthesis (19). In the presence of added uracil, FB335 is unable to grow for lack of carbamoyl phosphate for arginine synthesis. For this reason, FB335 is sensitive to uracil (Ura^s).

FIG. 2.

Gene organization of the pyr genes. A, Scheme of the transcription attenuation mechanism of the biosynthetic pyrimidine operon pyrR1BCAaAbDFE (EMBL database accession no. Z54240). Two transcription attenuators are found in the 5′ leader sequence of the pyr operon (6) which consists of overlapping repeated sequences that form exclusive RNA loops such as the anti-antiterminator loop (aat), the antiterminator loop (at), and terminator loop (t). Terminators t1, t2, and t3 are indicated. B, pyrR2pyrAa2Ab2 cluster (EMBL database accession no. AJ617795) with the pseudogene pyrAb2.

FIG. 3.

Pyrimidine-regulated transcription of the L. plantarum pyr operon. RNA was extracted from cells cultivated without agitation in defined medium without (−U) and with added uracil at a concentration of 200 μg/ml (+U). A, Primer extension by reverse transcription with primer 2071. The same primer was used for the sequencing reaction (A, C, G, and T tracks). The transcription start site is indicated (+1). B, Northern hybridization on 13 μg of RNA probed with two DNA fragments hybridizing to pyrR1 and to pyrB. The sizes of the bands detected were deduced from molecular size markers (lane M), and relevant bands are marked: a, 0.8 kb; b, 9.7 kb.

FIG. 4.

L. plantarum mutations in the pyr operon 5′ leader. Mutations are indicated with arrows. Mutant names are indicated in parentheses except for AE1023, whose mutations are marked with an asterisk. The deleted nucleotides are surrounded in U1 and highlighted with a gray background in mutant U25. Sequence coordinates refer to EMBL accession no. Z54240. A, Proposed hairpin structures of the anti-antitermination (aat2) and termination attenuator (t2) that favor transcription termination. Underlined, nucleotides (CAGAGA) in the repressor RNA binding aat2 hexaloop. B, Proposed hairpin structure of the antitermination attenuator at2 that allows transcription of the pyr operon.

FIG. 5.

Deletions and insertions found in the spontaneous Ura^r mutants. Rectangles schematize the open reading frame that encodes wild-type PyrR1 protein (white) or chimeric peptides (gray). Insertion and deletion events are delimited by brackets and are oriented outwards and inwards, respectively. The point mutation in mutant U40 is localized with an arrow. The RNA loop binding the PyrR repressor is indicated as anti-antiterminator (aat2) and is mutated in mutants U25, U1, U8, and U28.

FIG. 6.

Sequence alignment of PyrR homologs and localization of L. plantarum PyrR1 mutations. Four functional gram-positive bacteria repressors were aligned, L. plantarum (accession no. Z54240), Bacillus subtilis (accession no. M59757), Enterococcus faecalis V583 (accession no. AF044978), and Lactococcus lactis subsp. lactis (accession no. Q9L4N8), and compared to the gram-negative Haemophilus influenzae Rd KW20 (accession no. U32728) homolog. Identical and similar amino acids are highlighted with black and gray backgrounds, respectively. The secondary structure of the B. subtilis protein is indicated; arrows and cylinders represent β strands and α helices, respectively (23). A basic concave surface at the dimer interface was proposed to be required for B. subtilis RNA binding and comprised two distinct regions (black lines) (20). The conserved phosphoribosyltransferase (PRTase) structural elements of substrate and product binding involve three regions marked with double lines: the phosphoribosyltransferase domain, the flexible loop which is disordered in the dimer structure, and the PPi loop (23). Dimerization of B. subtilis PyrR involves the α3 helix, the β6 strand, and the dimer loop (20). Amino acid substitutions in L. plantarum PyrR1 (*) and B. subtilis (diamonds [8] and triangles [20]) that lead to defects in pyrimidine regulation are marked. Black triangles indicate B. subtilis residues that were mutated and impaired mRNA binding without changing protein folding. The names of the L. plantarum mutants are indicated above the mutated residues.