MOLECULAR GENETICS OF BACILLUS SUBTILIS CYTOCHROME AA3

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Cytochrome aa3 is a multi-subunit enzyme complex that functions as a terminal oxidase in the respiratory chains of prokaryotic and eukaryotic cells. Although cytochrome aa3 has been well-studied biochemically, its genetic regulation is poorly understood. A number of hereditary human myopathies and encephalomyopathies have been traced to genes regulating formation of the mature complex. Molecular studies of cytochrome aa3 regulation are facilitated in bacteria because of its biochemical simplicity in these organisms. In addition, some bacteria contain variants of cytochrome aa3, for example caa3 or ba3, which because of unique characteristics can offer new insights into mechanism and regulation of the cytochrome aa3 family. This project addresses the regulation of cytochrome caa3 formation in Bacillus subtilis. The system has several attributes that make it particularly suitable: i) B. subtilis endospore formation is presently being described in molecular terms, ii) proper regulation of the respiratory chain is known to be critical to initiation of sporulation, iii) classical and molecular genetic technologies are well-developed; iv) B. subtilis is the only organism thus far shown to possess both cytochromes aa3 and caa3. Of particular importance are several regulatory genes identified in B. subtilis that affect cytochrome aa3 and caa3 formation. The structural genes for B. subtilis cytochrome caa3 (the ctaBCDEF cluster) recently have been cloned and sequenced. Physical, genetic, and transcriptional analyses are to be made of this cluster. Approaches will include integrational disruption, low- and high-resolution transcript mapping, and formation of gene fusions, a particular focus will be on the ctaA and strC gene products as possible transcriptional factors. The strC gene will be cloned for further study. Possibilities for participation of the ctaA and ctaB gene products in post-transcriptional regulation of cytochrome caa3 will be tested by immunochemical measurement of its subunits in ctaA and ctaB mutant backgrounds. The sca and scs suppressors of ctaA are to be studied genetically to gain insight into the functioning of these genes in the regulatory network that encompasses regulation of cytochrome aa3 and cytochrome caa3, as well as early events in the sporulation process.