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 F 0 F 1 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 F 0 F 1 operon and the existence of the atpI gene. The atpI gene, located upstream of the F 0 F 1 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 F 0 F 1 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 s factor of C. glutamicum, whereas the "35 and "10 boxes of P-atp2 fitted the consensus sequence for s 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 F 0 F 1 operon is highly expressed at alkaline pH, probably using a s H RNA polymerase.

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