Characterization of the cydAB -Encoded Cytochrome bd Oxidase from Mycobacterium smegmatis

Abstract

The cydAB genes from Mycobacterium smegmatis have been cloned and characterized. The cydA and cydB genes encode the two subunits of a cytochrome bd oxidase belonging to the widely distributed family of quinol oxidases found in prokaryotes. The cydD and cydC genes located immediately downstream of cydB encode a putative ATP-binding cassette-type transporter. At room temperature, reduced minus oxidized difference spectra of membranes purified from wild-type M. smegmatis displayed spectral features that are characteristic of the ␥-proteobacterial type cytochrome bd oxidase. Inactivation of cydA or cydB by insertion of a kanamycin resistance marker resulted in loss of d-heme absorbance at 631 nm. The d-heme could be restored by transformation of the M. smegmatis cyd mutants with a replicating plasmid carrying the highly homologous cydABDC gene cluster from Mycobacterium tuberculosis. Inactivation of cydA had no effect on the ability of M. smegmatis to exit from stationary phase at 37 or 42°C. The growth rate of the cydA mutant was tested under oxystatic conditions. Although no discernible growth defect was observed under moderately aerobic conditions (9.2 to 37.5 ؋ 10 2 Pa of pO 2 or 5 to 21% air saturation), the mutant displayed a significant growth disadvantage when cocultured with the wild type under extreme microaerophilia (0.8 to 1.7 ؋ 10 2 Pa of pO 2 or 0.5 to 1% air saturation). These observations were in accordance with the two-to threefold increase in cydAB gene expression observed upon reduction of the pO 2 of the growth medium 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. smegmatis cultured at 1% air saturation. Finally, the cydA mutant displayed a competitive growth disadvantage in the presence of the terminal oxidase inhibitor, cyanide, when cocultured with wild type at 21% air saturation in an oxystat. In conjunction with these findings, our results suggest that cytochrome bd is an important terminal oxidase in M. smegmatis.

Profiling the Proteome of Mycobacterium tuberculosis during Dormancy and Reactivation

“…In addition, up-regulation of Rv1623c, a subunit of cytochrome D terminal oxidase complex, during NRP2 is indicative of ATP synthesis through electron transport chain. The homolog of this protein in E. coli is a component of the aerobic respiratory chain that is predominant when cells are grown under low aeration (37,38). Our study suggests that cytochrome D terminal oxidase of MTB also might perform a similar function when the bacterium gets ready to enter dormancy.…”

Host cell-free growth of the Q fever bacterium Coxiella burnetii

“…burnetii replication in ACCM is optimal in a 2.5% oxygen environment and the presence of genes encoding cytochrome bd (i.e., cydAB) with high affinity for oxygen provides a biochemical/ physiological explanation for the observed growth phenotype. Interestingly, the intracellular bacteria Mycobacterium tuberculosis (19), Chlamydia trachomatis (20), and Rickettsia rickettsii all encode cydAB, suggesting adaptation to microaerobic metabolism in intracellular bacteria may be underappreciated. Indeed, transcriptional analysis of M. tuberculosis during infection of macrophages indicates the organism adapts to a reduced oxygen environment (21), and improved growth of Chlamydia pneumoniae is observed under low oxygen conditions (22).…”

Role of Cytochrome bd Oxidase from Corynebacterium glutamicum in Growth and Lysine Production

“…The behavior of the 13032⌬cydAB mutant could be due to low dissolved oxygen concentrations at the end of the exponential growth phase, which strongly restrict the activity of the low-affinity cytochrome aa 3 oxidase but not that of the high-affinity cytochrome bd oxidase. In Mycobacterium smegmatis, like C. glutamicum a member of the suborder Corynebacterineae (36), cytochrome bd oxidase was recently shown to be important for growth under microaerobic conditions and to be induced two-to threefold upon reduction of the O 2 partial pressure in the growth medium from 21% to 0.5% air saturation (14).…”