Function of the cytochrome bc1-aa3 branch of the respiratory network in mycobacteria and network adaptation occurring in response to its disruption

Abstract

The aerobic electron transport chain in Mycobacterium smegmatis can terminate in one of three possible terminal oxidase complexes. The structure and function of the electron transport pathway leading from the menaquinol-menaquinone pool to the cytochrome bc1 complex and terminating in the aa3-type cytochrome c oxidase was characterized. M. smegmatis strains with mutations in the bc1 complex and in subunit II of cyctochome c oxidase were found to be profoundly growth impaired, confirming the importance of this respiratory pathway for mycobacterial growth under aerobic conditions. Disruption of this pathway resulted in an adaptation of the respiratory network that is characterized by a marked up-regulation of cydAB, which encodes the bioenergetically less efficient and microaerobically induced cytochrome bd-type menaquinol oxidase that is required for the growth of M. smegmatis under O2-limiting conditions. Further insights into the adaptation of this organism to rerouting of the electron flux through the branch terminating in the bd-type oxidase were revealed by expression profiling of the bc1-deficient mutant strain using a partial-genome microarray of M. smegmatis that is enriched in essential genes. Although the expression profile was indicative of an increase in the reduced state of the respiratory chain, blockage of the bc1-aa3 pathway did not induce the sentinel genes of M. smegmatis that are induced by oxygen starvation and are regulated by the DosR two-component regulator.

FIG. 1.

Genomic organization of bc₁-aa₃ respiratory pathway genes in mycobacteria. The arrows denote the respiratory pathway (black) and neighboring genes (gray). The gene annotation for M. tuberculosis H37Rv is taken from Tuberculist (http://genolist.pasteur.fr/TubercuList/), and the annotation for M. smegmatis mc²155 is from the TIGR Comprehensive Microbial Resource (http://www.tigr.org/tigr-scripts/CMR2/CMRHomePage.spl).

FIG. 2.

Targeted knockout of bc₁-aa₃ respiratory pathway genes in M. smegmatis mc²155. Each panel shows a schematic representation of the mutant allele. The gene(s) in which internal deletions were made is denoted by a black arrow, the hyg gene is denoted by a white block, and neighboring genes are shown as gray arrows. The positions of the restriction enzymes used for Southern blot analysis shown on the right of each panel are indicated by vertical arrows, and the hybridization probes used in the analysis are shown as hatched boxes above each map. Chromosomal DNA from up- and downstream single-crossover (sco) recombinants, double crossovers (dco), and the parental wild type (mc²155) was digested with ApaI (qcrCAB), BamHI (ctaDI), or SalI (ctaC) and hybridized with the corresponding probe shown above the restriction map (hatched box).

FIG. 3.

Growth of the ΔqcrCAB::hyg and ΔctaC::hyg mutants of M. smegmatis under aerobic conditions. (A) Growth in liquid medium. Strains were grown at 37°C in shaking flasks (350 rpm) in MADC-Tw medium, and growth was monitored by the absorbance at 600 nm (OD₆₀₀). ▴, ΔqcrCAB::hyg; ▪, ΔctaC::hyg; ⧫, wild-type mc²155. (B) Growth on solid medium. Strains were grown oxystatically (21% air saturation) in MADC-Tw in a bioreactor, and aliquots were withdrawn and plated on LA. Plates were incubated at 37°C for 3 days (wild type) or 6 to 8 days (mutant).

FIG. 4.

Effect of cytochrome bc₁ disruption on expression of the cyd operon in M. smegmatis. Expression of the cyd operon was assessed using a previously described lacZ reporter under control of the cyd promoter (16) that was integrated at the cyd locus in the wild-type (open bars) and ΔqcrCAB::hyg (black bars) strains. The specific activity of the reporter gene product (β-galactosidase) was assessed at 21, 5, and 1% air saturation, as described under Materials and Methods. All assays were performed in duplicate and the data shown represent the averages and standard deviation of at least two independent experiments.