Expression of the pyr operon of Lactobacillus plantarum is regulated by inorganic carbon availability through a second regulator, PyrR2, homologous to the pyrimidine-dependent regulator PyrR1

Abstract

Inorganic carbon (IC), such as bicarbonate or carbon dioxide, stimulates the growth of Lactobacillus plantarum. At low IC levels, one-third of natural isolated L. plantarum strains are nutritionally dependent on exogenous arginine and pyrimidine, a phenotype previously defined as high-CO2-requiring (HCR) prototrophy. IC enrichment significantly decreased the amounts of the enzymes in the pyrimidine biosynthetic pathway encoded by the pyrR1BCAa1Ab1DFE operon, as demonstrated by proteomic analysis. Northern blot and reverse transcription-PCR experiments demonstrated that IC levels regulated pyr genes mainly at the level of transcription or RNA stability. Two putative PyrR regulators with 62% amino acid identity are present in the L. plantarum genome. PyrR1 is an RNA-binding protein that regulates the pyr genes in response to pyrimidine availability by a mechanism of transcriptional attenuation. In this work, the role of PyrR2 was investigated by allelic gene replacement. Unlike the pyrR1 mutant, the DeltapyrR2 strain acquired a demand for both pyrimidines and arginine unless bicarbonate or CO2 was present at high concentrations, which is known as an HCR phenotype. Analysis of the IC- and pyrimidine-mediated regulation in pyrR1 and pyrR2 mutants suggested that only PyrR2 positively regulates the expression levels of the pyr genes in response to IC levels but had no effect on pyrimidine-mediated repression. A model is proposed for the respective roles of PyrR1 and PyrR2 in the pyr regulon expression.

FIG. 1.

UMP and arginine biosynthetic pathways in L. plantarum. (A) The pyrBCAa₁Ab₁DFE operon codes for the enzymes involved in UMP synthesis. For each step, the corresponding enzyme and gene are indicated. CPS-P stands for carbamoyl phosphate synthase pyrimidine-regulated protein. The carAB operon codes for CPS-A, the carbamoyl phosphate synthase arginine-regulated protein (20). (B) Genetic organization of pyr genes. Flag, transcriptional start; t, transcriptional terminator; circle, transcriptional attenuators as defined previously (19).

FIG. 2.

SDS-PAGE protein patterns in response to inorganic carbon, arginine, and uracil. Proteins were extracted from L. plantarum wild-type strain CCM 1904 under different growth conditions: in ordinary air (Low IC), with KHCO₃ added at 2 g/liter, in a 4% CO₂-enriched atmosphere, or with arginine and uracil (+). M, protein size marker (BenchMark protein ladder; Invitrogen). The 90-kDa constitutive band of an unknown protein served as the internal standard for quantification of the signal corresponding to PyrAb₁ (band shown at 130 kDa). Numbers indicate the relative amounts of the PyrAb₁ polypeptide expressed as percentages of the value obtained for cells cultivated at low IC levels, in the absence of arginine and uracil. The values shown are averages for at least four independent experiments.

FIG. 3.

Comparison of proteomic profiles at low and high inorganic carbon levels. L. plantarum wild-type strain CCM 1904 was cultivated in defined DLA medium (in the absence of arginine or uracil), with agitation in CO₂-enriched or ordinary air. Spots corresponding to the proteins encoded by the pyr genes are circled and identified by numbers referenced in Table 3. Sizes of protein markers (BenchMark protein ladder; Invitrogen) are shown in 10-kDa increments from 10 to 220 kDa.

FIG. 4.

Transcription of pyr genes in response to IC and uracil. Slot blot hybridizations detecting pyr mRNA were performed with probes specific to pyr genes (described in Table 2) and standardized with probe rrn, specific to 16S RNA encoding genes. The relative amounts (arbitrary units) were calculated by dividing the signal for each pyr probe by the signal obtained with probe rrn. RNAs were extracted from cells cultivated in DLA under different conditions, in normal air (1 and 2), in a 4% CO₂-enriched atmosphere (3 and 4), with KHCO₃ added at 2 g/liter (5 and 6), without uracil (1, 3, and 5), or with uracil (2, 3, and 6). (A) Quantification test with probe Ab₁#1, with different amounts of total RNA prepared from strain CCM1904. (B) Transcription of different pyr genes in the wild-type strain CCM 1904. (C) Gene pyrAb₁ transcription efficiencies in strains harboring wild-type or mutated pyrR₁ or pyrR₂ genes. Relative amounts calculated for different growth conditions were compared to relative amounts obtained for the corresponding wild-type isogenic strain grown at low IC levels without uracil added (condition marked 1). Each “% of the relative mRNA amount” is therefore expressed as a percentage of the amount obtained for condition 1, defined as 100%. Names of strains are indicated in parentheses.

FIG. 5.

Model for PyrR₁ and PyrR₂ regulation of L. plantarum pyr regulon in response to pyrimidine availability and inorganic carbon. The regulated pyr genes studied include the pyrR₁BCAa₁Ab₁DFE operon and pyrP. The gray circle schematizes pyr gene cis transcription regulatory elements that are involved in response to pyrimidine availability and IC at the DNA or the mRNA level. The activity of the RNA-binding PyrR₁ protein is regulated by binding to antagonist effectors such as 5-phospho-d-ribosyl-1-pyrophosphate (PRPP) and UMP. The UMP-PyrR₁ complex binds to an attenuation site of the pyr mRNA, leading to terminated transcription (19). Another unknown mechanism independent of PyrR₁ and PyrR₂ activity operates under conditions of elevated intracellular pyrimidine nucleotide levels. IC regulation may occur at the level of pyrR₂ expression or the gene product activity. PyrR₂* represents the functional PyrR₂ regulator.