Cytochrome bd Oxidase Has an Important Role in Sustaining Growth and Development of Streptomyces coelicolor A3(2) under Oxygen-Limiting Conditions

Abstract

Streptomyces coelicolor A3(2) is a filamentously growing, spore-forming, obligately aerobic actinobacterium that uses both a copper aa₃ -type cytochrome c oxidase and a cytochrome bd oxidase to respire oxygen. Using defined knockout mutants, we demonstrated that either of these terminal oxidases was capable of allowing the bacterium to grow and complete its developmental cycle. The genes encoding the bcc complex and the aa₃ oxidase are clustered at a single locus. Using Western blot analyses, we showed that the bcc-aa₃ oxidase branch is more prevalent in spores than the bd oxidase. The level of the catalytic subunit, CydA, of the bd oxidase was low in spore extracts derived from the wild type, but it was upregulated in a mutant lacking the bcc-aa₃ supercomplex. This indicates that cytochrome bd oxidase can compensate for the lack of the other respiratory branch. Components of both oxidases were abundant in growing mycelium. Growth studies in liquid medium revealed that a mutant lacking the bcc-aa₃ oxidase branch grew approximately half as fast as the wild type, while the oxygen reduction rate of the mutant remained close to that of the wild type, indicating that the bd oxidase was mainly functioning in controlling electron flux. Developmental defects were observed for a mutant lacking the cytochrome bd oxidase during growth on buffered rich medium plates with glucose as the energy substrate. Evidence based on using the redox-cycling dye methylene blue suggested that cytochrome bd oxidase is essential for the bacterium to grow and complete its developmental cycle under oxygen limitation.IMPORTANCE Respiring with oxygen is an efficient means of conserving energy in biological systems. The spore-forming, filamentous actinobacterium Streptomyces coelicolor grows only aerobically, synthesizing two enzyme complexes for O₂ reduction, the cytochrome bcc-aa₃ cytochrome oxidase supercomplex and the cytochrome bd oxidase. We show in this study that the bacterium can survive with either of these respiratory pathways to oxygen. Immunological studies indicate that the bcc-aa₃ oxidase is the main oxidase present in spores, but the bd oxidase compensates if the bcc-aa₃ oxidase is inactivated. Both oxidases are active in mycelia. Growth conditions were identified, revealing that cytochrome bd oxidase is essential for aerial hypha formation and sporulation, and this was linked to an important role of the enzyme under oxygen-limiting conditions.

Keywords: actinobacteria; cytochrome bd oxidase; cytochrome oxidases; menaquinol; mycelium; respiration; spores.

FIG 1

Schematic representation of the genes encoding the terminal oxidases of Streptomyces coelicolor. The SCO number is placed under the respective gene and the gene product of the respective gene is depicted above the gene. The genes encoding the cytochrome bcc complex and the aa₃ oxidase are colored blue and orange, respectively (A), while those encoding the cytochrome bd oxidase are shown in red (B). The functions of the gene products of the gray genes are unknown. The extent of the DNA fragments cloned in the respective complementation plasmids is shown below the loci.

FIG 2

Growth and developmental phenotypes of cytochrome oxidase-negative mutants. (A) Spores of M145 (wild type), COE192 (Δqcr-cta) carrying a deletion of the genes SCO2148 through SCO2156, which encode the bcc complex and cytochrome aa₃ oxidase, and COE192 (comp.) transformed with plasmid pMS2148-56 (Fig. 1A), were grown on SFM solid medium for the days indicated. (B) The indicated strains were grown for 3 days on SFM medium. Spores were streaked out in the three sectors (I, streaked as a thick flat layer; II, streaked as a layer of colonies; III, streaked to single colonies) to analyze the influence of culture density on sporulation.

FIG 3

Growth analysis of oxidase-negative mutants grown in liquid culture. Strains were grown as described in Materials and Methods, and cell density was measured as described previously (28). Strains M145, COE190 (ΔcydAB), COE192 (Δqcr-cta), and COE192 (complemented [comp.] with pMS2148-56) are shown. The experiment was performed four times (i.e., from four independent spore harvests), each time in triplicate. In the interest of clarity, error bars are shown only for strain COE192.

FIG 4

Characterization of cytochrome oxidases in spores and mycelium. In-gel cytochrome c oxidase activity was determined in crude extracts of spores. (A and B) Crude extracts (80 μg of protein) derived from spores (A) and from the mycelium (40 μg of protein) (B) were separated by nondenaturing 10% (wt/vol) native PAGE, and gels were subsequently stained for cytochrome c oxidase activity (see Materials and Methods). The arrow indicates the cytochrome c oxidase activity band. (C and D) Western blot analysis for the presence of CydA polypeptide in crude extracts of spores and mycelium. Aliquots of crude extracts (45 μg) derived from spores (C) and mycelium (D) were separated by 10% (wt/vol) denaturing SDS-PAGE and transferred to nitrocellulose membranes (see Materials and Methods). To detect the CydA polypeptide, the membrane was probed with anti-SCO3945 (1:10,000) antibodies. An arrow identifies the migration position of the CydA polypeptide. (E) The same protein samples of crude extracts derived from spores as used in the gel shown in panel C were separated by 10% SDS-PAGE, and polypeptides were stained with Coomassie brilliant blue. The molecular mass in kilodaltons is indicated on the left. COE192 (comp.) and COE502 (comp.) signify the respective mutants complemented with pMS2148-56.

FIG 5

Strain COE192 lacking the bcc complex-aa₃ oxidase branch shows a reduced rate of O₂ reduction. Oxygen consumption (A) and biomass production, measured as cell density (B), were determined for aliquots of mycelium from the indicated strains after 20 h of growth in buffered, half-strength TSB medium (see Materials and Methods). The experiment was performed three times, each time in triplicate.

FIG 6

Phenotypes of strain COE190 (ΔcydAB). (A) Strains were grown on buffered YP medium plates supplemented with glucose for the time period indicated below each plate. The key to the right of the agar plates indicates the location of each strain. (B) The same strains as in panel A were grown on agar plates, including yeast extract (4 g · liter⁻¹) and glucose (10 g · liter⁻¹) for 3 d at 30°C. Where indicated, the plates also included 50 mM MOPS buffer (pH 7) and 25 μM methylene blue.