Identification and characterization of glxR, a gene involved in regulation of glyoxylate bypass in Corynebacterium glutamicum

Abstract

A corynebacterial clone, previously isolated by scoring repression of lacZYA fused to the aceB promoter of Corynebacterium glutamicum, was analyzed further. In the clone, an open reading frame designated glxR, consisting of 681 nucleotides and encoding a 24,957-Da protein, was found. The molecular mass of a native GlxR protein was estimated by gel filtration column chromatography to be 44,000 Da, suggesting that the protein formed dimers. The predicted amino acid sequence contained both cyclic AMP (cAMP)- and DNA-binding motifs and was homologous with the cAMP receptor protein family of proteins. The aceB-repressing activity of the glxR clone was markedly relieved in an Escherichia coli cya mutant, but the activity was restored in growth medium containing cAMP. In glucose medium, the intracellular cAMP concentration of C. glutamicum reached 22 nmol/mg of protein in the early exponential phase and then decreased further; but in acetate medium, the intracellular cAMP concentration was only 5 nmol/mg of protein and remained low throughout the growth phase. The expression of glxR was not affected by the carbon source. Binding of purified GlxR to the promoter region of aceB could be demonstrated only in the presence of cAMP. These data suggest that GlxR may form dimers which bind to the aceB promoter region in the presence of cAMP and repress the glyoxylate bypass genes.

FIG. 1.

Glyoxylate bypass and associated pathways of C. glutamicum. The glyoxylate bypass is carried out by isocitrate lyase and malate synthase, which are encoded by the aceA and aceB genes, respectively. Abbreviations: ACK, acetate kinase; ICDH, isocitrate dehydrogenase; ICL, isocitrate lyase; MS, malate synthase; PTA, phosphotransacetylase; CoA, coenzyme A. Dashed arrows indicate multiple steps.

FIG. 2.

Schematic diagram of clones and subclones. The colony color (blue or white) of E. coli DH5αF′-145 cells carrying each clone was tested on LB plates containing X-Gal, tetracycline, and ampicillin. The arrow indicates the glxR gene. The cloning vector pMT1 is not shown. Abbreviations: B, BamHI; E, EcoRI; K, KpnI; Sa, SalI; Sc, ScaI; Sp, SphI; X, XhoI; Xb, XbaI.

FIG. 3.

Multiple sequence alignment of the GlxR protein of C. glutamicum with homologous sequences. Conserved and functionally similar amino acids are marked with black and shaded boxes, respectively. The cyclic nucleotide monophosphate-binding motif and helix-turn-helix DNA-binding motif are marked with solid bars. Abbreviations: CG, GlxR of C. glutamicum (accession no. AF220150); MT, putative transcriptional regulator Rv3676 of Mycobacterium tuberculosis H37RV (E70790); SC, putative transcriptional regulator of Streptomyces coelicolor (T36556); VC, CRP of Vibrio cholerae (NP232242); EC, CRP of Escherichia coli (J01598).

FIG. 4.

Measurement of intracellular cAMP in C. glutamicum. Cells were grown in MCGC containing glucose (A) or acetate (B) as a carbon source. Intracellular cAMP (shaded bars) was measured as described in Materials and Methods. The data represent three independent experiments. Symbols: •, growth; ○, glucose. OD, optical density at 600 nm.

FIG. 5.

Effect of glxR on the expression of malate synthase (MS). Cells were grown on MB containing acetate. Crude extracts were prepared as described in Materials and Methods. For each lane, a total of 5 μg of protein was loaded. Lanes: M, molecular mass markers (sizes are shown in daltons); 1, C. glutamicum/pMT1; 2, C. glutamicum/pSL08; 3, C. glutamicum/pSL08-glxR.

FIG. 6.

Expression and purification of GlxR in E. coli. (A) The GlxR protein was expressed from the pKK-glxR vector as described in Materials and Methods. Lanes: 1, E. coli JM105/pKK223-3; 2, E. coli JM105/pKK-glxR (uninduced); 3, E. coli JM105/pKK-glxR (induced). (B) Purification of GlxR. The protein was expressed from pMAL-glxR. Lanes: 4, total protein; 5, soluble fraction; 6, purified MBP-GlxR fusion protein; 7, after factor X treatment; 8, purified GlxR protein. Lanes M, molecular mass standards (sizes are shown in daltons). Arrows indicate GlxR protein.

FIG. 7.

Gel shift assay with purified GlxR. Purified GlxR proteins were incubated with the probe, and the mixtures were analyzed by 6% PAGE. (A) Schematic diagram of the aceB promoter region. (B) Gel shift in the absence of cAMP. (C) Gel shift in the presence of cAMP. cAMP was added (to 0.2 mM) to the assay mixture, gels, and PAGE buffer. Lane 1 contained no protein; lanes 2 through 8 contained 30 ng, 60 ng, 130 ng, 250 ng, 500 ng, 1.0 μg, and 2.0 μg of purified GlxR protein, respectively. Arrows indicate shifted bands.