Characterization of the cydAB-encoded cytochrome bd oxidase from Mycobacterium smegmatis

The cydAB genes from Mycobacterium smegmatis have been cloned and character ized. The cyd4 and cydB genes encode the two subunits of a cytochrome bd ox idase belonging to the widely distributed family of quinol oxidases found i n prokaryotes. The cydD and cydC genes located immediately downstream of cy dB encode a putative ATP-binding cassette-type transporter. At room tempera ture, reduced minus oxidized difference spectra of membranes purified from wild-type M. smegmatis displayed spectral features that are characteristic of the gamma -proteobacterial type cytochrome bd oxidase. Inactivation of c yd-4 or cydB by insertion of a kanamycin resistance marker resulted in loss of d-heme absorbance at 631 run. The d-heme could be restored by transform ation of the M. smegmatis cyd mutants with a replicating plasmid carrying t he highly homologous cydABDC gene cluster from Mycobacterium tuberculosis. Inactivation of cyd4 had no effect on the ability of M. smegmatis to exit f rom stationary phase at 37 or 42 degreesC. The growth. rate of the cydA mut ant was tested under oxystatic conditions. Although no discernible growth d efect was observed under moderately aerobic conditions (9.2 to 37.5 x 10(2) Pa of pO(2) or 5 to 21% air saturation), the mutant displayed a significan t growth disadvantage hen cocultured with the wild type under extreme micro aerophilia (0.8 to 1.7 x 10(2) Pa of pO(2) or 0.5 to 1% air saturation). Th ese observations were in accordance with the two- to threefold increase in cydAB gene expression observed upon reduction of the pO(2), of the growth m edium from 21 to 0.5% air saturation and with the concomitant increase in d -heme absorbance in spectra of membranes isolated from wild-type M. smegmat is cultured at 1% air saturation. Finally, the cydA mutant displayed a comp etitive growth disadvantage in the presence of the terminal oxidase inhibit or, cyanide, when cocultured with wild type at 21% air saturation in an oxy stat. In conjunction with these findings, our results suggest that cytochro me bd is an important terminal oxidase in M. smegmatis.