Transcriptional analysis of the F0F1 ATPase operon of Corynebacterium glutamicum ATCC 13032 reveals strong induction by alkaline pH

Corynebacterium glutamicum, a soil Gram-positive bacterium used for industrial amino acid production, was found to grow optimally at pH 7.0-9.0 when incubated in 5 litre fermenters under pH-controlled conditions. The highest biomass was accumulated at pH 9.0. Growth still occurred at pH 9.5 but at a reduced rate. The expression of the pH-regulated F0F1 ATPase operon (containing the eight genes atpBEFHAGDC) was induced at alkaline pH. A 7.5 kb transcript, corresponding to the eight-gene operon, was optimally expressed at pH 9.0. The same occurred with a 1.2 kb transcript corresponding to the atpB gene. RT-PCR studies confirmed the alkaline pH induction of the F0F1 operon and the existence of the atpl gene. The atpl gene, located upstream of the F0F1 operon, was expressed at a lower level than the polycistronic 7.5 kb mRNA, from a separate promoter (P-atp1). Expression of the major promoter of the F0F1 operon, designated P-atp2, and the P-atp1 promoter was quantified by coupling them to the pET2 promoter-probe vector. Both P-atp1 and P-atp2 were functional in C. glutamicum and Escherichia coli. Primer extension analysis identified one transcription start point inside each of the two promoter regions. The P-atp1 promoter fitted the consensus sequence of promoters recognized by the vegetative sigma factor of C. glutamicum, whereas the -35 and -10 boxes of P-atp2 fitted the consensus sequence for a sigma(H)-recognized Mycobacterium tuberculosis promoters C-C/(G)GG(A)/(G)AC 17-22 nt (C)/(G)GTT(C)/G, known to be involved in expression of heat-shock and other stress-response genes. These results suggest that the F0F1 operon is highly expressed at alkaline pH, probably using a sigma(H) RNA polymerase.