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

¹ Instituto de Biotecnología de León (INBIOTEC), Parque Científico de León, Av. Real 1, 24006 León, Spain
² University of León, Facultad de Ciencias Biológicas y Ambientales, Campus de Vegazana s/n, 24071 León, Spain

Correspondence
Juan F. Martín
degjmm{at}unileon.es

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 atpI gene. The atpI 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 factor of C. glutamicum, whereas the –35 and –10 boxes of P-atp2 fitted the consensus sequence for ^H-recognized Mycobacterium tuberculosis promoters C^C/_GGG^A/_GAC 17–22 nt ^C/_GGTT^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 ^H RNA polymerase.

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