, 2005; Cha et al., 2010). One of the CRP homologues, glxR (cg0350), has been reported to regulate the gene expression of glyoxylate bypass enzymes involved in acetate

metabolism, aceB [encoding malate synthase (MS)] (Kim et al., 2004). Letek check details et al. (2006) showed the possibility that GlxR acts as a transcriptional regulator of the catabolite repression of two genes, gntP (encoding gluconate permease) and gntK (encoding gluconate kinase), involved in gluconate catabolism. Recently, GlxR has been reported to bind to the upstream regions of several genes involved in central carbon metabolism, including glycolysis, gluconeogenesis and the tricarboxylic acid cycle (Han et al., 2007, 2008). In addition, Kohl et al. (2008) identified 51 binding sites in vitro using electrophoretic mobility shift assays, where the sites were selected from 201 potential GlxR-binding R428 solubility dmso sites based on in silico analysis of the C. glutamicum genome. Thus, GlxR has been suggested to be an important transcriptional regulator involved in the regulation of several metabolic genes. However, a C.

glutamicum mutant deficient in the glxR gene has not yet been characterized, due to the difficulties involved in constructing such a mutant. Accordingly, in this study, a glxR deletion mutant was constructed and characterized to analyse its role in C. glutamicum. The resulting data revealed that GlxR acts as a transcriptional repressor of the aceA [encoding isocitrate lyase (ICL)] and aceB genes involved in acetate metabolism. In addition, the derepression of the gluA gene of the glutamate uptake system in the glxR mutant on glucose medium suggests that GlxR plays a role as a global regulator controlling both carbon catabolite repression (CCR) and acetate metabolism. The bacterial strains, plasmids and oligonucleotides used in this study are listed in Table 1. The E. coli strain was grown in Luria–Bertani medium (10 g L−1

tryptone, 5 g L−1 yeast extract, 10 g L−1 NaCl) at 37 °C, and the C. glutamicum ATCC 13032 and glxR mutant Y-27632 2HCl strains were grown at 30 °C in MB medium (15 g L−1 tryptone, 5 g L−1 yeast extract, 5 g L−1 soytone, 5 g L−1 NaCl) (Follettie et al., 1993) or brain–heart infusion (BHI) medium (Eggeling & Reyes, 2004). As the carbon source, glucose, fructose, acetate, pyruvate or glutamate was added to the media at 1% (w/v). When appropriate, ampicillin, kanamycin and chloramphenicol were added at concentrations of 50, 20 and 10 μg mL−1, respectively. The oligonucleotides used in this study were purchased from Genotech (Korea). Standard molecular cloning procedures were followed in this study (Sambrook et al., 1989). The chromosomal DNA from the C. glutamicum cells was isolated using a genomic DNA purification kit (SolGent, Korea), and the DNA fragments from the agarose gel were eluted using the Qiagen Gel Extraction Kit (Qiagen, Germany). The plasmids were introduced into C. glutamicum by electroporation (Tauch et al., 2002).