Cloning of the cta operon from alkaliphilic Bacillus firmus OF4 and characterization of the pH-regulated cytochrome caa3 oxidase it encodes

Abstract

We have cloned and sequenced the DNA of alkaliphilic Bacillus firmus OF4 encompassing the cta operon that encodes a pH-regulated cytochrome caa3 oxidase. The gene organization is identical with that of the homologous Bacillus subtilis caa3 oxidase locus (van der Oost, J., von Wachenfeld, C., Hederstedt, L. & Saraste, M. (1991) Mol. Microbiol. 5, 2063-2072). The deduced amino acid sequences of the four putative structural subunits (CtaC-F) indicate substantial similarity to caa3-type oxidases from other Bacillus species and to other members of the family of mitochondrial-type aa3 oxidases. A marked paucity of basic residues was noted in the cytochrome c-containing domain of CtaC, which faces the highly alkaline external milieu. We have also purified the enzyme as a three-subunit complex, with possible trace amounts of a fourth subunit. N-terminal sequence analysis of the two largest subunits confirmed them to be encoded by the cloned cta genes. An additional, minor caa3 component with distinctive chromatographic properties was noted during purification. Analysis of mRNA with a ctaD probe revealed an abundant 4-kilobase message of the right size to encode CtaC-F. The cellular content of this message varied with growth pH. Cells grown at pH 10.5 contained 2 to 2.5 times more message than those grown at pH 7.5, in good correspondence with the relative amounts of caa3 oxidase found in the cells. The ctaB gene, immediately upstream from the ctaC-F genes, was found to be transcribed onto a low abundance 5-kilobase message, which is likely also to encode CtaC-F. Levels of this message were not affected by growth pH.