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

Abstract

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 F0F1ATPase operon (containing the eight genesatpBEFHAGDC) 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 theatpBgene. RT-PCR studies confirmed the alkaline pH induction of the F0F1operon and the existence of theatpIgene. TheatpIgene, located upstream of the F0F1operon, 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 F0F1operon, 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 inC. glutamicumandEscherichia 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 vegetativeσfactor ofC. glutamicum, whereas the −35 and −10 boxes of P-atp2 fitted the consensus sequence forσH-recognizedMycobacterium tuberculosispromoters CC/GGGA/GAC 17–22 ntC/GGTTC/G, known to be involved in expression of heat-shock and other stress-response genes. These results suggest that the F0F1operon is highly expressed at alkaline pH, probably using aσHRNA polymerase.