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 F0 F1 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 F0 F1 operon and the existence of the atpI gene. The atpI gene, located upstream of the F0 F1 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 F0 F1 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 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 F0 F1 operon is highly expressed at alkaline pH, probably using a sigma (H) RNA polymerase.