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. 2004 Apr;70(4):2146-53.
doi: 10.1128/AEM.70.4.2146-2153.2004.

Cloning, deletion, and characterization of PadR, the transcriptional repressor of the phenolic acid decarboxylase-encoding padA gene of Lactobacillus plantarum

Jérôme Gury  1 , Lise Barthelmebs, Ngoc Phuong Tran, Charles Diviès, Jean-François Cavin
Affiliations

Cloning, deletion, and characterization of PadR, the transcriptional repressor of the phenolic acid decarboxylase-encoding padA gene of Lactobacillus plantarum

Jérôme Gury et al. Appl Environ Microbiol. 2004 Apr.

Abstract

Lactobacillus plantarum displays a substrate-inducible padA gene encoding a phenolic acid decarboxylase enzyme (PadA) that is considered a specific chemical stress response to the inducing substrate. The putative regulator of padA was located in the padA locus based on its 52% identity with PadR, the padA gene transcriptional regulator of Pediococcus pentosaceus (L. Barthelmebs, B. Lecomte, C. Diviès, and J.-F. Cavin, J. Bacteriol. 182:6724-6731, 2000). Deletion of the L. plantarum padR gene clearly demonstrates that the protein it encodes is the transcriptional repressor of divergently oriented padA. The padR gene is cotranscribed with a downstream open reading frame (ORF1), the product of which may belong to a group of universal stress proteins (Usp). The padR deletion mutant overexpressed padA constitutively, and the padA promoter appears to be tightly regulated in this bacterium. Gel mobility shift assays using the padA gene promoter region and purified PadR expressed in Escherichia coli indicated that operator DNA binding by PadR was not eliminated by addition of p-coumarate. Gel mobility shift assays using partially purified extracts of native PadR protein from both phenolic acid-induced and noninduced L. plantarum cells demonstrate that inactivation of PadR by phenolic acids requires the integrity of L. plantarum and mediation by a specific protein absent in E. coli.

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Figures

FIG. 1.
FIG. 1.
Alignment of amino acid sequences of PadR. LPPADR, PadR of L. plantarum (accession no. AJ289188); BSPADR, PadR of B. subtilis (accession no. P94443) (unpublished data); PPPADR, PadR of P. pentosaceus (accession no. AJ276891). Asterisks designate identical residues, colons designate conserved substitutions, and periods designate semiconserved substitutions. Predicted coiled-coil domains, obtained by using the COILS computer program with a 21-amino-acid window (32-34), are boxed. Gaps in the alignment (dashes) are indicated.
FIG. 2.
FIG. 2.
(A) Physical map of the padA locus in a wild-type strain of L. plantarum and characterization of physical deletion in an LPNC8ΔR strain. Large open arrows represent the different genes and ORF1 and their orientation. Small bars indicate the restriction sites. Small horizontal arrows indicate primers for PCR. (B) PCR of the padR region with two primer pairs on chromosomal DNAs of a mutant strain and a wild-type strain, respectively. Lanes 1 and 2, LPMINV2-LPD8 amplification. Lanes 3 and 4, LPREP2-LPD8 amplification. Smart ladder (Eurogentec). (C) Nucleotide sequence of the overlapping diverging promoter region of the padA gene and the padR-ORF1 operonic structure (accession no. AJ289188). Putative promoters are indicated with their putative −10 and −35 boxes. ATG start codons of padA and padR genes are boldfaced. The two putative ribosome binding site (RBS) regions are underlined. The transcription starting points (+1) of padA (6) and padR (see Fig. 4A) are underlined. The inverted-repeat (IR) sequence of the PadR putative DNA binding site is boldfaced and underlined.
FIG. 3.
FIG. 3.
(A) SDS-PAGE of crude protein extracts from noninduced (lane 3) and 2.4 mM p-coumaric acid-induced (lane 2) LPNC8 cells and from the noninduced LPNC8ΔR mutant (lane 1). M, SDS-PAGE molecular mass standards (Invitrogen). Arrow indicates the protein band corresponding to the constitutively overexpressed PadA enzyme in the LPNC8ΔR mutant. (B) Identical to panel A, with a simple increase in brightness and contrast to enable evaluation of the relative concentration of PadA in the LPNC8ΔR mutant compared to the concentrations of the other two main protein bands (indicated by arrowheads) that are constitutively expressed in the wild-type and mutant strains.
FIG. 4.
FIG. 4.
Transcriptional analysis of padR gene. (A) Mapping of the 5′ end of padR mRNA by extension analysis using primer LPD9 with total L. plantarum RNA from noninduced (NI) and 2.4 mM p-coumaric acid-induced (I) cells. The products of the reverse transcriptase reactions were analyzed by 6% sequencing gel reactions (ACGT) with the same primer. Arrow indicates the 5′ end of padR gene mRNA (C for the coding sequence). (B) Northern blot analysis of total RNAs purified from NI and I cultures of L. plantarum. A padR-specific probe was used. (C) RT-PCR of the padR gene and the 5′-most 180 bp of putative ORF1 by using total RNAs purified from NI and I cells of L. plantarum as the matrix. This region of interest was amplified by PCR using LPYFMUT1 and LPD6. Lane 1, positive control from chromosomal DNA; lanes 2 and 3, RT-PCR with total RNAs from NI and I cells, respectively; lanes 4 and 5, negative controls with no RT step; M, DNA Smart ladder (Eurogentec).
FIG. 5.
FIG. 5.
Overexpression, purification, and mobility shift assays with purified PadR. (A) SDS-PAGE analysis of crude extracts and purified His-tagged PadR from E. coli BL21(DE3) carrying the pER plasmid. Lane 1, molecular mass standards; lane 2, crude protein extract from noninduced E. coli; lane 3, crude protein extract from 1 mM IPTG-induced E. coli; lane 4, PadR purified by Ni-NTA affinity chromatography; lane 5, purified PadR treated with 10 mM glutaraldehyde. Dimer PadR, protein band corresponding to the putative dimerized purified PadR with a molecular mass of about 45 kDa. (B) Mobility shift assays of the DNA probe corresponding to the padA promoter region, generated and labeled by PCR amplification with [α-32P]dATP, with or without purified PadR. Lane 1, probe (3 × 10−7 M pad promoter DNA) without protein; lanes 2 to 7, probes with increasing concentrations of purified PadR (lane 2, 10−6 M; lane 3, 2 × 10−6 M; lane 4, 3 × 10−6 M; lane 5, 4 × 10−6 M; lane 6, 5 × 10−6 M; lane 7, 6 × 10−6 M).
FIG. 6.
FIG. 6.
Mobility shift assay of a DNA probe corresponding to the padA promoter region (2 × 10−8 M), generated and labeled by PCR with [α-32P]dATP, with crude protein extracts, partially purified with (NH4)2SO4 and by gel filtration, from noninduced (NI) or p-coumaric acid-induced (I) L. plantarum cells. (A) Binding assay with (NH4)2SO4 fractions from NI or I cells. P, DNA probe without protein extract; lane 1, crude extract from NI cells; lanes 2 and 5, 30% (NH4)2SO4 fraction; lanes 3 and 6, 40% (NH4)2SO4 fraction; lanes 4 and 7, 50% (NH4)2SO4 fraction. (B) Binding assays with 50% (NH4)2SO4 fractions from NI and I cells preincubated with (+) or without (−) 3 × 10−7 M unlabeled ldhL promoter DNA as a competitor (see Materials and Methods). P, DNA probe without protein extract; lanes 1 and 3, 0.025 μg of protein/μl; lanes 2 and 4, 0.075 μg of protein/μl. (C) Binding assays with the four pools of protein obtained by gel filtration (GF) of the 50% (NH4)2SO4 protein fraction from the NI protein extract with (+) or without (−) ldhL promoter DNA as a competitor. P, probe without protein extract; lanes 1, 2, 3, and 4, respectively, the first, second, third, and fourth pools of proteins collected by gel filtration elution. The protein concentration of the pools was the same as that in the binding assays (0.025 μg/μl). C1, band corresponding to a specific binding of the probe with a protein in extracts or protein fractions exclusively from NI cells; C2, band corresponding to the binding of the probe with a high-molecular-mass protein.
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