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 gamma-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 degrees 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 x 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 x 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.

FIG. 1

Construction and genotypic characterization of M. smegmatis cyd mutants. (A) Restriction map of the cyd locus of M. smegmatis showing the insertion sites of the aph cassette in the cyd knockout mutants. The 2.4-kb KpnI fragment from pBK1, used as the probe for Southern blot analysis (see panel B), is denoted by a dashed, double-headed arrow. Restriction sites: Bg, BglII; H, HindIII; K, KpnI; P, PstI; S, SphI. The sequence extending from the PstI site upstream of cydA to a SalI site downstream of cydC has been deposited in GenBank under accession no. AF196488. A hypothetical open reading frame that showed high homology to Rv1624c from M. tuberculosis was found upstream of cydA, confirming that the genetic organization at this locus is conserved between M. smegmatis and M. tuberculosis. (B) Southern blot analysis of cyd mutants. Genomic DNA from the various strains was digested with the indicated enzyme(s) and probed with the 2.4-kb KpnI fragment from pBK1. In all cases, the sizes of the hybridizing fragments derived from the mutant strains were consistent with site-specific, allelic exchange.

FIG. 2

Sodium dithionite-reduced minus potassium ferricyanide-oxidized difference spectra on wild-type and cyd mutant membranes. (A) mc²155, (B) mc²155(pOTBCYD), (C) cydA::aph, (D) cydA::aph(pOTBCYD), (E) cydB::aph, or (F) cydB::aph(pOTBCYD). In all cases, samples were suspended in 1 ml of TC buffer (pH 7.4) at a protein concentration of 2.6 mg/ml.

FIG. 3

Growth of cocultures of wild-type and cyd mutant strains at various air saturation levels. The growth curves of mixed cultures of mc²155::pHINT (●) and cydA::aph (○) are shown for 21% (A), 5% (B), 1% (C), or 0.5% (D) air saturation for 24 h (see Materials and Methods for more details). The data are shown as the averages and standard deviations from three independent experiments. Statistical analysis of the data by using an analysis of variance test revealed that the cell counts of the mutant after 24 h of growth were significantly lower than for wild type at the lower aerations (0.5 and 1% air saturation; P < 0.05).

FIG. 4

Expression analysis of the M. smegmatis cyd gene cluster. (A) Genetic organization at the cyd locus of the reporter strain, mc²155::pBK4. The hatched arrow denotes the putative cyd promoter, P_cyd. The genotype of this site-specific single recombinant was confirmed by Southern blotting (not shown). (B) Dependence of β-galactosidase reporter activity on pO₂. The specific activity of a reporter gene product (β-galactosidase) was studied at 21, 5, 1, and 0.5% air saturation as described in Materials and Methods. The data shown are an average of three independent experiments for each air saturation level. A statistical pairwise comparison showed that the data followed a significant second-order relationship, as represented in the figure (P < 0.05).

FIG. 5

Spectral characterization of membrane fractions isolated from M. smegmatis grown in an oxystat. The spectral profiles of purified membrane fractions from M. smegmatis grown at 21% (A) and 1% (B) air saturation are shown. Reduction was accomplished by the addition of sodium dithionite, and oxidation was accomplished by the addition of potassium ferricyanide.

FIG. 6

Cyanide inhibition of wild-type and cydA::aph strains under oxystatic conditions. The growth of mixed cultures of mc²155 (●) and cydA::aph (○) in the presence of 1 mM KCN are shown. KCN was added 30 min postinoculation, and the growth of each strain was monitored by differential plating as described in Materials and Methods. The data are shown as the averages and standard errors from two independent experiments.

FIG. 7

Proposed aerobic respiratory pathways in M. smegmatis and related organisms. The pathways in B. subtilis were adapted from references and . The M. tuberculosis pathways were deduced from its genome sequence (7), and those of M. smegmatis are based on this study and analysis of the preliminary genome sequence data obtained from The Institute for Genomic Research website (http://www.tigr.org). Solid arrows denote confirmed pathways, and dashed arrows denote putative pathways. The mycobacterial cytochrome reductase complex (QcrCAB) is denoted as “bcc” in accordance with the designation of QcrC as cytochrome cc (54).