The alcohol dehydrogenase gene adhA in Corynebacterium glutamicum is subject to carbon catabolite repression

Abstract

Corynebacterium glutamicum has recently been shown to grow on ethanol as a carbon and energy source and to possess high alcohol dehydrogenase (ADH) activity when growing on this substrate and low ADH activity when growing on ethanol plus glucose or glucose alone. Here we identify the C. glutamicum ADH gene (adhA), analyze its transcriptional organization, and investigate the relevance of the transcriptional regulators of acetate metabolism RamA and RamB for adhA expression. Sequence analysis of adhA predicts a polypeptide of 345 amino acids showing up to 57% identity with zinc-dependent ADH enzymes of group I. Inactivation of the chromosomal adhA gene led to the inability to grow on ethanol and to the absence of ADH activity, indicating that only a single ethanol-oxidizing ADH enzyme is present in C. glutamicum. Transcriptional analysis revealed that the C. glutamicum adhA gene is monocistronic and that its expression is repressed in the presence of glucose and of acetate in the growth medium, i.e., that adhA expression is subject to catabolite repression. Further analyses revealed that RamA and RamB directly bind to the adhA promoter region, that RamA is essential for the expression of adhA, and that RamB exerts a negative control on adhA expression in the presence of glucose or acetate in the growth medium. However, since the glucose- and acetate-dependent down-regulation of adhA expression was only partially released in a RamB-deficient mutant, there might be an additional regulator involved in the catabolite repression of adhA.

FIG. 1.

Transcriptional characterization of the adhA gene. (A) Northern blot analysis after growth of C. glutamicum in minimal medium containing ethanol (lanes 1 to 3) or glucose (lanes 4 to 6) as carbon sources. Total RNA (5 μg in lanes 1 and 4; 10 μg in lanes 2 and 5; 15 μg in lanes 3 and 6) was electrophoresed and probed with a radioactively labeled adhA-specific DNA probe. Sizes are shown on the right. (B) Primer extension analysis of the transcriptional start site in front of the adhA gene. The primer extension product is shown in lane 5. Lanes A, C, G, and T represent the products of sequencing reactions with the same primer (CM4) used for the primer extension reaction. The relevant DNA sequence (coding strand) is shown on the right, and the transcriptional start site is indicated by an asterisk.

FIG. 2.

Genomic locus of the adhA promoter region and DNA fragments used for mapping the relevant RamA and RamB binding sites (A) and representative EMSAs using hexahistidyl-tagged RamA (B) and RamB (C) proteins. cg3108 codes for a putatively secreted, unknown protein. The transcriptional start site is denoted as TS. The black boxes represent the putative RamA binding sites and the grey boxes the RamB binding sites. Their respective localizations relative to the transcriptional start site are given. The fragments used for the binding assays are given as bars and designated as indicated to the left, whereas the respective sizes are indicated to the right. The fragments used in the EMSAs with hexahistidyl-tagged RamA (B) and RamB (C) are indicated below the different parts of the gels. Lanes 1 to 4 show EMSAs using 0, 0.25, 0.5, and 1 μg of RamA or RamB, respectively. Lane 5 shows a negative control using 1 μg of bovine serum albumin protein with the respective probe.

FIG. 3.

adhA promoter activity during growth of WT C. glutamicum(pET2-adhAP2) on 1% glucose (A), 0.125% glucose and 1% ethanol (B), 1% ethanol (C), and 1% glucose (D). Black circles, growth; black bars, specific CAT activity.