Uracil salvage pathway in Lactobacillus plantarum: Transcription and genetic studies

Abstract

The uracil salvage pathway in Lactobacillus plantarum was demonstrated to be dependent on the upp-pyrP gene cluster. PyrP was the only high-affinity uracil transporter since a pyrP mutant no longer incorporated low concentrations of radioactively labeled uracil and had increased resistance to the toxic uracil analogue 5-fluorouracil. The upp gene encoded a uracil phosphoribosyltransferase (UPRT) enzyme catalyzing the conversion of uracil and 5-phosphoribosyl-alpha-1-pyrophosphate to UMP and pyrophosphate. Analysis of mutants revealed that UPRT is a major cell supplier of UMP synthesized from uracil provided by preformed nucleic acid degradation. In a mutant selection study, seven independent upp mutants were isolated and all were found to excrete low amounts of pyrimidines to the growth medium. Pyrimidine-dependent transcription regulation of the biosynthetic pyrimidine pyrR1-B-C-Aa1-Ab1-D-F-E operon was impaired in the upp mutants. Despite the fact that upp and pyrP are positioned next to each other on the chromosome, they are not cotranscribed. Whereas pyrP is expressed as a monocistronic message, the upp gene is part of the lp_2376-glyA-upp operon. The lp_2376 gene encodes a putative protein that belongs to the conserved protein family of translation modulators such as Sua5, YciO, and YrdC. The glyA gene encodes a putative hydroxymethyltransferase involved in C1 unit charging of tetrahydrofolate, which is required in the biosynthesis of thymidylate, pantothenate, and purines. Unlike upp transcription, pyrP transcription is regulated by exogenous pyrimidine availability, most likely by the same mechanism of transcription attenuation as that of the pyr operon.

FIG. 1.

Uracil salvage and pyrimidine biosynthetic pathways. Enzymes are identified by their corresponding gene designation for the uracil salvage pathway (thin arrows), de novo pyrimidine synthesis (thick arrows), and the IMP biosynthetic pathway (arrowheads), including steps involved in the incorporation of C₁ units from serine into THF. The enzymes catalyzing UMP synthesis are encoded by the pyrR1-B-C-Aa1-Ab1-D-F-E operon (9). Abbreviations for genes encoding enzymes are as follows: glyA, serine hydroxymethyltransferase (EC 2.1.2.1); ndk, nucleoside-diphospho kinase (EC 2.7.4.14); udk, uridine kinase (EC 2.7.1.48); udp, uridine phosphorylase (EC 2.4.2.3); upp, uracil phosphoribosyltransferase (EC 2.4.2.9); pdp, pyrimidine-nucleoside phosphorylase (EC 2.4.2.2); pyrP, uracil permease; tdk, thymidine kinase (EC:2.7.1.21); and thyA, thymidylate synthase (EC 2.1.1.45).

FIG. 2.

Analysis of the upp and pyrP mutations found in spontaneous uracil-resistant clones. Genes are schematized as white rectangles when the resulting protein is wild type (wt) or as gray rectangles when mutations led to chimeric proteins. Strain names are indicated on the left. PyrP was inactivated only in strains U30 and U2. Insertion and deletion events delineated by brackets are oriented outwards and inwards, respectively. In strain HN38, the mobile insertion sequence ISLpl1 inactivated upp (25). In mutant U27, the sequence 5′-TATGCCGTTGAAGGACATTGAG-3′ was inserted at nucleotide 448. Numbers refer to nucleotide positions in the EMBL database sequence accession no. AJ012720. The PRPP- and uracil-binding sites were determined using UPRT sequence alignments as determined in reference .

FIG. 3.

Uracil transport in the pyrP strain. Radioactively labeled uracil was added at 0.5 μM to L. plantarum U2 (upp, pyrP) (diamonds), 0.5 μM to U27 (upp) (squares), and 1.0 μM to U27 (triangles). Samples were filtered at 15-second intervals, and the radioactivity retained by the cells was measured and plotted against the incubation time.

FIG. 4.

Results of transcriptional studies of the upp-pyrP locus in L. plantarum. (A) Gene organization and transcription terminator localization. The orientation and the name of the genes are indicated in rectangles. Sequences involved in RNA hairpin structures are schematized with facing arrows and labeled t (terminator), at (antiterminator), or aat (anti-antiterminator). In circles, primer sets are named from a to g. Filled diamonds represent forward primers, and filled arrowheads represent reverse primers. Thick black lines delineate DNA probes used in Northern slot blot analysis. (B) Results of reverse transcriptase coupled with PCR amplification. The sizes of the expected bands were controlled by PCR amplification in the presence of CCM 1904 genomic DNA (lanes marked with an asterisk), and band sizes were estimated using DNA molecular marker bands (lane M). RNAs were extracted from cells grown with (+) or without (−) 50 μg/ml of uracil in defined DLA medium (for the presented experiment, cells were grown in aerobiosis but similar amplification patterns were obtained with cells grown in air enriched with 4% CO₂). RT-PCR was performed with different RNA amounts: 20 ng for the 16S rrn gene (lanes 1 and 2) and the pyrAb1 gene (lanes 3 and 4), ng for the pyrP gene (lanes 6 and 7), the upp gene (lane 15), and the lp_2376-glyA-upp gene cluster (lanes 17, 19, and 20), and 200 ng for the intergenic upp-pyrP region (lanes 9 to 10 and 12 to 13). (C) Northern slot blots with probes specific to the rrn gene, the pyrP gene, the upp gene, and the pyrAb1 gene in the parental strain harboring a wild-type upp gene (lanes 1 to 10) and in its upp derivative strain U27 (lanes 11 to 14). The percentage of expression was calculated with two independent experiments. The relative amount of mRNA (signal measured with the test probe divided by the signal detected with the rrn probe) of the mutant was compared to the relative amount obtained with the wild type grown without uracil (100%).

FIG. 5.

Model of pyrimidine-dependent transcription attenuation. The transcription attenuation site sequence of the pyrP gene is shown (nucleotides 1924 to 2033 in EMBL database sequence accession no. AJ012720). (A) When the PyrR regulator remains unbound, the formation of the RNA loop antiterminator allows for the transcription of the downstream gene. (B) When the PyrR-UMP complex is bound to the hexaloop CAGAGA (in bold) at the tip of the anti-antiterminator hairpin, the terminator RNA loop can form and transcription stops before the downstream pyr gene. The ΔG values of the proposed RNA loops are indicated in parentheses.

FIG. 6.

Gene linkage of lp_2376-glyA-upp genes in gram-positive bacteria. Black, gray, and light-gray circles refer to the distributions of the upp, glyA, and lp_2376 genes, respectively. Names of bacteria in which a genetic linkage of two genes was observed (cluster) are found in the circle intersection. In the intersection of the three circles are bacteria names with linked lp_2376-glyA-upp genes.