Involvement of the global regulator GlxR in 3-hydroxybenzoate and gentisate utilization by Corynebacterium glutamicum

Abstract

Corynebacterium glutamicum is an industrially important producer of amino acids and organic acids, as well as an emerging model system for aromatic assimilation. An IclR-type regulator GenR has been characterized to activate the transcription of genDFM and genKH operons for 3-hydroxybenzoate and gentisate catabolism and represses its own expression. On the other hand, GlxR, a global regulator of the cyclic AMP (cAMP) receptor protein-fumarate nitrate reductase regulator (CRP-FNR) type, was also predicted to be involved in this pathway. In this study, electrophoretic mobility shift assays and footprinting analyses demonstrated that GlxR bound to three sites in the promoter regions of three gen operons. A combination of site-directed mutagenesis of the biding sites, promoter activity assay, and GlxR overexpression demonstrated that GlxR repressed their expression by binding these sites. One GlxR binding site (DFMx) was found to be located -13 to +8 bp upstream of the genDFM promoter, which was involved in negative regulation of genDFM transcription. The GlxR binding site R-KHx01 (located between positions -11 to +5) was upstream of the genKH promoter sequence and involved in negative regulation of its transcription. The binding site R-KHx02, at which GlxR binds to genR promoter to repress its expression, was found within a footprint extending from positions -71 to -91 bp. These results reveal that GlxR represses the transcription of all three gen operons and then contributes to the synchronization of their expression for 3-hydroxybenzoate and gentisate catabolism in collaboration with the specific regulator GenR.

FIG 1

Organization of the gen cluster, physical locations of promoters for activity detection, and probes for EMSAs. The region of the DNA fragment in the genome of Corynebacterium glutamicum ATCC 13032 is indicated (54). The promoter or probes below the gen cluster contained the following mutated GlxR binding sites: mutated site DFMx (from GTGN⁸TTC to CACN⁸GTG), indicated with an asterisk; mutated site R-KHx01 (from TGTN⁹CAC to CACN⁹GTG), indicated with a rhombus (◆); mutated site R-KHx02 (from GTGN⁸AAC to CACN⁸GTG), indicated with a filled circle (•).

FIG 2

Electrophoretic mobility shift assays for determination of GlxR binding sites upstream of genDFM, genR, and genKH operons. (A and C) Effects of cAMP on the binding affinity of GlxR for the upstream regions of genDFM, genR, and genKH operons. The probes for genDFM (DFMan in panel A), genR, and genKH operons (R-KHan in panel C) (25) were incubated with increasing amounts of His₆-GlxR, while the amounts of cAMP were constant. The concentrations (nM) of purified His₆-GlxR and the presence of 500 nM cAMP (+) or absence of cAMP (−) are indicated above the lanes. Glucose served as a control. (B and D) Competition assays using unlabeled specific competitor (SCR) and nonspecific competitor (NSCR) DNA. Labeled probe and unlabeled competitor of 25-fold, 50-fold, and 100-fold more in amount were incubated with His₆-GlxR for 30 min at 25°C. Each lane contained 0.5 ng (0.3 to 0.4 nM) of ³²P-labeled DFMan and R-KHan probes. The free probes are indicated by open arrows and the retarded DNA fragments by solid arrows.

FIG 3

Identification of the GlxR binding sites upstream of genDFM (A), genR (B), and genKH (C) operons by DNase I footprinting analysis. The reaction mixtures contained approximately 200 ng of end-labeled PCR products. These were amplified with prnagRD02 primer and ³²P-labeled pegenDFM01 primer (A), pgenR01 and ³²P-labeled pegenR01 (B), or pgenKH02 and ³²P-labeled pegenKH04 (C) (25). Before DNase I treatment, labeled DNA was preincubated with His₆-GlxR for 30 min at 25°C. Standards were generated by sequencing with ³²P-labeled primers pegenDFM01(A), pegenR01 (B), and pegenKH04 (C). The concentrations (μM) of purified His₆-GlxR and the GlxR-protected sequences are indicated. The nucleotide sequence around the TSS (+1) (25) is shown by solid arrows, and the GlxR binding sites are represented by brackets. Arrows indicate the direction of transcription. (D and E) Organization of the upstream region of the genDFM operon (D) and the genR-genKH promoter region (E) is shown in detail. The first codon of each gene is given in italics and boldface. The sequences in the dotted boxes are site R-KHxu as proposed previously (30), and the regulator GlxR binding sites are showed in the box. The TSSs are shown by +1 (boldface) and arrows and the putative −10 and −35 promoter sequences are underlined. The regulator GenR binding sites are highlighted in gray.

FIG 4

Sequence logos of motifs identified in GlxR binding sites by MEME (multiple Em for motif elicitation). (A) Motifs identified in GlxR binding sites for 3-HBA/GEN catabolic genes by MEME. (B) Data modified from references , , , and .

FIG 5

Mutational analyses of the four GlxR binding sites. EMSAs using the wild-type and mutated DNA fragments. Probes DFMan, R-KHax01, and R-KHax02 contained the intact GlxR binding sites DFMx, R-KHx01, and R-KHx02, respectively. Probes DFMaxm, R-KHax01m, and R-KHax02m contained the mutated GlxR binding sites described in Materials and Methods. The amounts (nM) of His₆-GlxR used in lanes 1 to 5 are indicated. The free probes are indicated by open arrows, and the retarded DNA fragments are indicated by solid arrows.

FIG 6

GlxR attenuates upregulation of the transcription of genDFM, genR, and genKH operons. (A) qRT-PCR analyses examining the transcription of genDFM, genR, and genKH in strains RES167 and RES167/pZWHJ033. RNA samples were isolated from strains RES167 and RES167/pZWHJ033 grown on MM with 2 mM 3-HBA overnight. The levels of gene expression in each sample were calculated as the fold expression ratio after normalization to the 16S rRNA gene transcript. The values are averages of two independent RT-qPCR experiments. Error bars indicate standard deviations. These data are derived from at least three independent measurements. There was a significant difference in the transcription of genDFM, genR, and genKH between strains RES167 and RES167/pZWHJ033 (P < 0.001, paired-samples test). (B) β-Galactosidase activity driven by genDFM, genR, and genKH promoters with their corresponding GlxR binding sites in strain RES167. DFMa, Ra, and KHa containing GlxR binding sites are the GenR binding sites reported as previously (25). DFMax01m, Rax01m, Rax02m, Rax12m, KHax01m, KHax02m, and KHax12m are the constructs containing the mutated GlxR binding sites described in Materials and Methods and shown in Fig. 1. The β-galactosidase activity analyses were performed as given in the text. The data are derived from at least three independent measurements, and error bars indicate standard deviations. There was a significant difference in promoter activity between GlxR binding sites and its mutated sites from strains RES167 grown on MM with GEN or 3-HBA (P < 0.001, one-way ANOVA).