Involvement of the LuxR-type transcriptional regulator RamA in regulation of expression of the gapA gene, encoding glyceraldehyde-3-phosphate dehydrogenase of Corynebacterium glutamicum

Abstract

SugR, RamA, GlxR, GntR1, and a MarR-type transcriptional regulator bind to the promoter region of the gapA gene encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH), essential for glycolysis in Corynebacterium glutamicum. We previously showed that SugR, a transcriptional repressor of phosphotransferase system genes for the sugar transport system, is involved in the downregulation of gapA expression in the absence of sugar. In this study, the role of RamA in the expression of the gapA gene was examined. Comparing the gapA expression and GAPDH activity of a ramA mutant with those of the wild type revealed that RamA is involved in upregulation of gapA expression in glucose-grown cells. DNase I footprint analyses and electrophoretic mobility shift assays revealed that RamA binds with different affinities to three sites in the gapA promoter. lacZ reporter assays with mutated RamA binding sites in the gapA promoter showed that the middle binding site is the most important for RamA to activate gapA expression and that binding of RamA to the gapA promoter activates the gene expression not only in glucose-grown cells but also in acetate-grown cells. Furthermore, RamA also directly activates sugR expression, indicating that two global regulators, RamA and SugR, are coordinately involved in the complex regulation of gapA expression in C. glutamicum.

FIG. 1.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins binding to the gapA promoter region. Cell extracts of C. glutamicum R grown in minimal medium containing 1% acetate (lane 1) or glucose (lane 2) were incubated with magnetic beads linked to biotin-labeled gapA promoter DNA fragment, and bound proteins were eluted with 1 M NaCl. The N-terminal amino acid sequence of the proteins in bands a to g was analyzed: band a, termination factor Rho (CgR1278); band b, RamA (CgR2464); band c, SugR (CgR1761); band d, GntR1 (CgR2434); bands e and f, GlxR; band g, MarR-type transcriptional regulator (CgR2877). A molecular mass standard is shown to the left.

FIG. 2.

Effect of deletion of the ramA gene on growth (A, B), gapA expression (C), and GAPDH activity (D). (A, B) Growth of the wild-type R (white circles) and the ramA mutant KT6 (black circles) in nutrient-rich A medium containing 1% glucose (A) or acetate (B). Mean values from at least four independent cultures are shown, with standard deviations. OD610, optical density at 610 nm. (C) The level of gapA mRNA in the wild-type R (white) and ramA mutant KT6 (black) during growth in A medium with 1% glucose or acetate at the exponential (3 h) and the stationary (6 h) phase was analyzed by qRT-PCR, and the levels relative to that in the wild type grown in the presence of glucose at 3 h were determined. Mean values from three independent cultures are shown, with standard deviations. (D) Activities of GAPDH in the wild-type R (white) and the ramA mutant KT6 (black) grown in A medium with 1% glucose or acetate. The activity was determined at the stationary (6 h) phase. Mean values from at least three independent cultures are shown, with standard deviations.

FIG. 3.

Binding of RamA to the gapA promoter region. (A) DNA fragments (P1 to P6) used to determine the location of the RamA binding site in the gapA promoter are shown. The numbers above the diagram indicate the position relative to the TSP (+1) of the gapA gene. The regions included in the DNA fragments are indicated, with positions with respect to the TSP at the right side. The gapA gene is indicated with a black arrow. The whiA gene, which encodes a WhiA-like protein, is located upstream of gapA with the same direction and is indicated with a white arrow. Three RamA binding sites (P, M, and D sites) are indicated with white boxes. (B) Results of EMSAs using the gapA promoter region and His-tagged RamA. The DNA fragments (P1 to P6) used are indicated at the top of the gels. Each well contained 10 nM of DNA fragment. Lanes 1 to 5 show EMSA results using 0, 200, 400, 800, and 1,600 nM of RamA protein, respectively. Probe DNA and DNA-RamA complexes are indicated with white and black arrowheads, respectively. Nonspecific bands are indicated with asterisks. (C) DNA sequence of the gapA promoter showing the putative RamA binding sites. The TSP (+1) is indicated with a bold letter and an arrow. The numbers indicate the position relative to the TSP (+1). The coding region of gapA is indicated with capital letters and the amino acid sequence. Three RamA binding sites (P, M, and D sites) and the mutated sequences shown under the sites are boxed. The mutated sequences are indicated with bold letters. The binding sites of SugR and GlxR are underlined and shaded in gray, respectively. (D) The results of EMSAs using DNA fragments without (WT) or with (Mut) the mutations in the RamA binding site are shown. The DNA fragments (P1, P3, and P4) used are indicated at the top of the gels. Each well contained 10 nM of DNA fragment. Lanes 1 to 5 show results of EMSAs using 0, 200, 400, 800, and 1,600 nM of RamA protein, respectively. The mutated P3 fragment contains the mutated P binding site. The mutated P4 fragment contains the mutated middle and native distal binding sites. The mutated P1 fragment contains mutations in all the binding sites. A nonspecific band is indicated with an asterisk.

FIG. 4.

DNase I footprint analysis of the interaction between RamA and the gapA promoter regions examined on the coding and the noncoding strands. A DNA fragment (4 nM) was incubated with different concentrations of RamA: lanes 1 and 7, no protein; lane 2, 200 nM; lane 3, 400 nM; lane 4, 800 nM; lane 5, 1,600 nM; lane 6, 3,200 nM. The three RamA binding sites are indicated with brackets on the left side of each panel. Protected regions are indicated by bars, and hypersensitive sites are indicated by arrows. The DNA sequencing reactions were set up using the same labeled primer and plasmid as for generating labeled footprinting probes.

FIG. 5.

RamA has different affinities for the three binding sites in the gapA promoter region. The gapA promoter fragment without (WT) or with mutated RamA binding sites was incubated with different concentrations of RamA: lane 1, no protein; lane 2, 100 nM; lane 3, 200 nM; lane 4, 400 nM. The binding sites mutated are indicated at the top of the gels. Probe DNA and DNA-RamA complexes are indicated with white and black arrowheads, respectively.

FIG. 6.

Effects of mutation of the RamA binding sites and deletion of the ramA and sugR genes on expression of the gapA promoter-lacZ translational fusion. (A) The gapA promoter-lacZ fusion constructs are indicated. The strains carrying the fusion constructs in their genome are indicated at the left side. The TSP of the gapA gene is indicated with +1. The three RamA binding sites (P, M, and D) without or with mutations are indicated with white or black boxes, respectively. The mutated gapA promoter-lacZ fusions, containing RamA binding sites mutated as shown in Fig. 3C, were introduced into the wild type, yielding KT13 derivatives (KT13P, KT13M, KT13D, KT13PM, KT13MD, KT13PD, and KT13PMD). The gapA promoter-lacZ fusion was also introduced into the wild-type strain (KT2), the ramA mutant (KT11), the sugR mutant (KT3), and the ramA-sugR double mutant (KT12). (B, C) β-Galactosidase activity in each strain grown in nutrient-rich A medium containing 1% glucose (B) or acetate (C) at the onset of the stationary phase. Mean values from at least three independent cultures are shown, with standard deviations.

FIG. 7.

RamA directly regulates sugR expression. (A) The levels of sugR mRNA in the wild-type R (white) and the ramA mutant KT6 (black) during growth in nutrient-rich A medium with 1% glucose or acetate at the exponential (3 h) and the stationary (6 h) phases were analyzed by qRT-PCR, and the levels relative to that in the wild type grown in the presence of glucose at 3 h were determined. Mean values from three independent cultures are shown, with standard deviations. (B) DNA sequence of the upstream region of sugR of C. glutamicum. The proposed RamA binding sites are boxed. The start codon of sugR is indicated by a bold arrow and bold letters. Thin arrows indicate the primers used to prepare probes for EMSAs. (C) The sugR promoter fragments prepared by PCR using primers indicated at the top of the gels were incubated with different concentrations of RamA: lane 1, no protein; lane 2, 200 nM; lane 3, 400 nM; lane 4, 800 nM; lane 5, 1,600 nM. Probe DNA and DNA-RamA complexes are indicated with white and black arrowheads, respectively.