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

Marco Fischer; Dörte Falke; Carolin Naujoks; R Gary Sawers

doi:10.1128/JB.00239-18

. 2018 Jul 25;200(16):e00239-18. doi: 10.1128/JB.00239-18

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

Marco Fischer ^a, Dörte Falke ^a, Carolin Naujoks ^a,^*, R Gary Sawers ^a,^✉

Editor: Conrad W Mullineaux^b

PMCID: PMC6060352 PMID: 29784883

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

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.

INTRODUCTION

Streptomyces coelicolor A3(2) is an actinobacterium with a complex developmental cycle and, like other streptomycetes, it produces a complex spectrum of secondary metabolites. The bacterium grows as a substrate mycelium and, upon nutrient limitation, produces hydrophobic aerial mycelia that develop into chains of spores (1, 2). Growth and development of S. coelicolor depend on oxygen respiration, and streptomycetes are predicted to have a branched, menaquinone-based respiratory chain, as has been demonstrated for other members of the actinobacteria, such as Corynebacterium, Mycobacterium, and Rhodococcus species (3 –8). Moreover, Streptomyces species also lack a soluble cytochrome c and instead have a diheme QcrC protein as part of their menaquinol:cytochrome bcc oxidoreductase (bcc complex) (9). The bcc complex and a cytochrome c oxidase of the aa₃ copper family (aa₃-type oxidase) form one oxygen-reducing respiratory branch, while a menaquinol-oxidizing cytochrome bd oxidase forms the other. The bcc-aa₃ branch is bioenergetically more favorable for ATP generation and proton motive force (PMF)-driven substrate transport processes, because the combined action of the bcc complex-driven Q cycle and the proton-pumping aa₃-type oxidase results in translocation of 6 H⁺ for each 2 electrons (e⁻) transferred (10). In contrast, the cytochrome bd oxidase does not pump protons but nevertheless is electrogenic, releasing 2 H⁺ for each 2 e⁻ transferred, owing to its menaquinol oxidation site being on the outer face of the cytoplasmic membrane (11, 12). Thus, the bd oxidase completes a redox loop when coupled with quinone dehydrogenases that receive electrons from NADH, pyruvate, d-lactate, or acyl coenzyme A (acyl-CoA) (9). It is likely that the bd oxidase is functional under microaerobic conditions, as in other bacteria (13, 14), and the induction of expression of the cydAB genes in response to an increased NADH/NAD⁺ ratio would support this (15).

The multiprotein enzyme complexes of the cytochrome bcc-aa₃ oxidase branch of the aerobic respiratory chain in actinobacteria have a number of unusual features, which have been summarized previously (3, 16 –19). These features are also conserved in the bcc complex and the aa₃ oxidase of S. coelicolor, and together they suggest that both enzymes form a supercomplex, as has been demonstrated for Corynebacterium glutamicum and for Mycobacterium species (5, 17, 18, 20). This is underlined by the facts that the operons encoding both complexes are adjacent to each other in the S. coelicolor genome and that the ctaE gene, which encodes subunit III of the terminal oxidase, is located immediately upstream of the qcrCAB operon (Fig. 1A).

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.

While the phenotypic consequence of a deletion in the genes encoding the cytochrome bd oxidase on the physiology of C. glutamicum (21) or Mycobacterium species (16, 22) is minimal under laboratory conditions, this is not the case for similar mutations in the genes encoding the bcc-aa₃ supercomplex (16, 23 –25). The loss of the bcc-aa₃ respiratory branch leads to moderate to severe growth phenotypes in C. glutamicum, while Mycobacterium tuberculosis cannot grow without this branch of the respiratory chain. A recent study (26) has shown that deletions in the genes encoding the bcc-aa₃ supercomplex led to a developmental phenotype in S. coelicolor on certain growth media (26). However, no studies have been undertaken to examine the consequences to the physiology of Streptomyces coelicolor caused by deleting the genes encoding the cytochrome bd oxidase. Because Streptomyces species are important model systems for studying both bacterial development and secondary metabolism, it is important to understand the impact of each respiratory branch on these processes. We show in this study that, in contrast to other characterized actinobacteria, both respiratory branches are individually capable of sustaining growth and development of the bacterium. We also identify growth conditions under which a functional cytochrome bd oxidase is essential for development beyond the formation of substrate mycelium.

RESULTS

Strains with either the cytochrome bd oxidase or the bcc-aa₃ oxidase complex complete their developmental cycle on solid medium.

Mutant COE190 lacks the genes SCO3945 to SCO3946, encoding cytochrome bd oxidase, and mutant COE192 lacks the complete locus encompassing genes SCO2148 to SCO2156, encoding the cytochrome bcc complex and the aa₃ oxidase (Fig. 1). Growth of strain COE192 (lacking the bcc-aa₃ supercomplex), and that of the wild-type strain M145, was compared on soya flour and mannitol (SFM) medium (Fig. 2A). Despite this medium normally allowing rapid colony development, only small colonies of COE192 appeared after 2 to 3 days, and by the 5th day they were roughly 50% of the size of M145 colonies. In contrast, colonies of M145 were visible after 1 day of growth (Fig. 2A). Notably, despite its markedly slower growth, morphological development of strain COE192 was unimpaired on solid SFM medium, and the strain completed its life cycle after 4 to 5 days (Fig. 2A). Complementation of the growth defect was achieved by introducing the complete locus into COE192 on the integrative plasmid derivative, pMS2148-56 (Fig. 2A). Strain COE192 also completed its developmental cycle on buffered yeast extract-peptone (YP) medium, with or without sugar supplementation (see Fig. S1 in the supplemental material), and this was also observed for several other solid rich media tested (see Fig. S2 in the supplemental material for yeast extract [YE] plus glucose medium; also data not shown). Moreover, no reproducible developmental phenotype could be observed after growth of COE192 on Bennett's/glucose medium, as was previously described (26; data not shown).

Growth and sporulation of strain COE190 (no bd oxidase) on SFM medium was essentially indistinguishable from the wild-type strain M145 (Fig. 2B). Together, these findings indicate that the bcc-aa₃ oxidase complex is important for rapid colony growth, while a strain with only a functional bd oxidase, despite growing more slowly, nevertheless completes its developmental cycle.

Growth rate of the bcc-aa₃ oxidase-negative mutant is reduced in liquid culture.

S. coelicolor grows in liquid culture as mycelium, but it fails to sporulate under these conditions (2). We determined the doubling times of M145 (both terminal oxidases present), COE190 (only bcc-aa₃ oxidase present), COE192 (only bd oxidase present), and COE192 complemented with plasmid pMS2148-56 during cultivation in buffered, half-strength liquid tryptic soy broth (TSB) medium (Fig. 3). From the same initial amounts of spore suspension, mycelium of M145 and COE190 increased in cell density at a similar rate (doubling of mycelium “cell” density every 2.5 h), while strain COE192 grew approximately 55% more slowly compared to the wild type. Note that streptomycetes grow by tip growth (2), and therefore “growth” cannot be quantified in terms of “growth rate” and “doubling time” as for standard bacteria, and this is why the representation of growth rate is linear and not semilogarithmic (Fig. 3). Introduction of the complete genetic locus, including all genes necessary for synthesis of the bcc-aa₃ complex (Fig. 1A), on the integrative plasmid pMS82 into COE192 restored growth of the mutant to one similar to that of the wild-type M145 (Fig. 3). We ruled out the possibility that the genes SCO2152, which encodes a putative response regulator, and SCO2153, which encodes a predicted secreted protein (Fig. 1A; strepdb.streptomyces.org.uk), were responsible for the growth phenotype of mutant COE192, because strains carrying in-frame deletion mutations in either gene failed to exhibit either a growth or a developmental phenotype under all conditions tested in this study (data not shown). Together with the plate growth study (Fig. 2A), these data indicate that, while the presence of the bcc-aa₃ complex affords more efficient energy conservation and hence growth, a strain lacking this complex and that is reliant only on the bd oxidase for growth can nevertheless grow and sporulate.

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 (Δ*cydAB*), COE192 (Δ*qcr-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.

A strain lacking the diheme c-type cytochrome, QcrC, is also devoid of aa₃ oxidase activity.

To demonstrate that strain COE192 indeed lacks cytochrome aa₃ oxidase, we initially qualitatively assessed this by in-gel activity staining (27). A single activity band could be detected in extracts of both spores (Fig. 4A) and mycelium (Fig. 4B) derived from wild type M145 after nondenaturing PAGE. As anticipated, no enzyme activity could be detected in extracts derived from strain COE192 in either spores or mycelium (Fig. 4A and B), confirming that the activity was due to the aa₃-type oxidase. As an additional control, we constructed a mutant carrying an in-frame deletion in gene SCO2150 (qcrC in Fig. 1A), encoding the diheme cytochrome c subunit of the bcc complex and an extract derived from this mutant also lacked aa₃ oxidase enzyme activity (Fig. 4A and B). Introduction of pMS2148-56 into strains COE192 and COE502 restored aa₃ oxidase enzyme activity to both mutants in spores (Fig. 4A). Strain COE502 had a growth phenotype that was indistinguishable from that of COE192 on all media tested (data not shown).

An extract derived from mycelium of strain COE190 exhibited aa₃-type oxidase enzyme activity (Fig. 4B), clearly demonstrating that this oxidase activity was independent of the cytochrome bd oxidase.

Quantitative determination of enzyme activity showed a barely detectable oxidase activity in mycelial extracts derived from COE192 of 0.8 mU · mg⁻¹, while extracts of M145 had a specific activity of 16.2 mU · mg⁻¹, and extracts of COE190 had an activity of 38.5 mU · mg⁻¹. The higher oxidase activity measured in the quantitative assay in extracts from COE190 was not observed in the qualitative in-gel activity assay (Fig. 4B). It is conceivable that aa₃ oxidase enzyme activity was increased in mycelium of strain COE190 to compensate for the lack of the cytochrome bd oxidase.

Lower levels of the CydA polypeptide are present in wild-type spores compared with mycelium.

Because strain COE192 completes its developmental cycle when growing on buffered rich medium (Fig. 2A), this indicates that the cytochrome bd oxidase activity must be sufficient to allow spores to form and for them to retain viability. To assess the distribution of this oxidase in spores and mycelium, we used peptide antibodies to detect the catalytic CydA (encoded by SCO3945 in Fig. 1B) polypeptide of the bd oxidase in Western blots (Fig. 4C). A signal corresponding to the CydA polypeptide was barely detectable in extracts derived from spores of M145 (Fig. 4C), while a polypeptide of significantly stronger intensity, migrating at approximately 50 kDa (deduced molecular mass of CydA = 55.8 kDa), was visible in an extract derived from mycelium (Fig. 4D). As a negative control, no CydA polypeptide could be detected in extracts derived from spores (Fig. 4C) or mycelium (Fig. 4D) of strain COE190 (ΔcydAB). Notably, while the level of CydA was essentially unaffected by the absence of the bcc-aa₃ complex in extracts of mycelium derived from COE192 (Fig. 4D), amounts of the polypeptide were increased to detectable levels in spores of the mutant (Fig. 4C). Complementation of the mutation in COE192 with the genes SCO2148 to SCO2156 resulted in reduced, barely detectable levels of CydA in spores of the complemented strain.

Finally, we analyzed the levels of CydA polypeptide in spore extracts derived from strain COE502 (ΔqcrC), and again, increased levels of the CydA polypeptide were detected, which correlated with the absence of activity of the aa₃ oxidase in the mutant (Fig. 4C). A Coomassie-stained SDS-PAGE gel of the same samples of spore extracts as those used for the Western blots (Fig. 4E) confirmed equal protein loading. Together, these data suggest that synthesis of the CydA component of the cytochrome bd oxidase, which is otherwise synthesized at very low levels, is increased in spores when the alternative aa₃-type oxidase is nonfunctional.

Mycelium of a strain reliant on bd oxidase exhibits wild-type rates of O₂ respiration but reduced biomass production.

Next, we wished to determine the contribution of each oxidase in the individual oxidase-negative mutants to oxygen respiration rates in exponentially growing mycelium cultivated in buffered, half-strength liquid TSB medium. To do this, cell material was removed after 20 h of growth, and the rate of O₂ consumption was measured in the wild type and in the oxidase mutants COE190 and COE192 (see Materials and Methods for details). Strain M145 exhibited a rate of O₂ reduction that was approximately equal to 50 nmol O₂ reduced per min per 1,000 cell amount equivalents (CAE) (Fig. 5A) (28), while COE190 consumed O₂ at a rate that was only 27% of this value. Complementation of this mutant with pMS3945-46 restored the rate of O₂ reduction to a level that was approximately 90% of that observed for the wild type (Fig. 5A). The rate of O₂ consumption by mycelia of strain COE192 (only bd oxidase present) was approximately 80% of the rate of that of M145 (wild type), and this difference was not considered statistically significant. These data indicate that mutants lacking the bcc-aa₃ branch of the respiratory pathway reduced O₂ at a rate similar to that of the wild type, while the absence of the bd-type oxidase caused a much more significant reduction in the O₂ consumption rate.

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.

To determine how the mutations in the oxidase genes affected growth and biomass production, the cell density was determined and compared for the same samples used to measure O₂ reduction rates (Fig. 5B). The results show that the cell density of M145, COE190 (lacking bd oxidase), and strain COE190 complemented with pMS3945-46 were similar, around 1,200 CAE · ml⁻¹ (see Materials and Methods) (28), while the cell density attained by strain COE192 (lacking the bcc complex and aa₃ oxidase) was reduced by approximately 30% compared to that of M145. Together, these data are in accord with those shown in Fig. 3, and they indicate that cytochrome bd oxidase is sufficient to support growth of S. coelicolor mycelium in the absence of a bcc-aa₃ pathway.

Cytochrome bd oxidase is required to allow sporulation under oxygen-limiting conditions.

While testing growth of the wild type M145 and the bd oxidase mutant, COE190, for their developmental phenotypes on different solid growth media (see Table S1 in the supplemental material), it was noted that strain COE190, which lacks the bd oxidase, reproducibly failed to form aerial hyphae or spores on buffered yeast extract-peptone (YP)–glucose medium within 6 days (Fig. 6A). Complementation of the phenotype could be achieved by introducing the cydAB genes on integrative plasmid pMS3945-46 (Table 1), resulting in restoration of the sporulating phenotype within 4 days and confirming that this phenotype was due solely to the absence of cytochrome bd oxidase (Fig. 6A). The developmental phenotype was not reproducibly observed for strain COE190 if the solid medium was not supplemented with a sugar, whereas M145 and the complemented COE190 strain (COE437) sporulated without sugar supplementation (Table S1). Supplementation of YP medium with ribose, mannose, fructose, cellobiose, or glycerol led to a reproducible nonsporulating phenotype for COE190, while supplementation with sucrose, trehalose, lactose, or raffinose led to a sporulation phenotype that was not reproducible (see Table S1 and Fig. S1 in the supplemental material).

FIG 6 — Phenotypes of strain COE190 (Δ*cydAB*). (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.

TABLE 1.

Strains and vectors used in this study

Species, strain, plasmid, or cosmid	Genotype and characterics	Reference or source
Species and strains
Streptomyces coelicolor A3(2)
M145 (wild type)	SCP1⁻ SCP2⁻	37
COE190	M145 Δ(SCO3945–SCO3946) (deletion of 2,525 bp, removing cydAB)	J. Alderson (John Innes Center)
COE192	M145 Δ(SCO2148–SCO2156) (deletion of 9,398 bp, removing qcrCAB-ctaE-SCO2152-SCO2153-ctaCDF)	J. Alderson (John Innes Center)
COE502	M145 SCO2150::Tn5062	This study
COE437	COE190 complemented with pMS3945-46	This study
COE634	COE192 complemented with pMS2148-56	This study
COE639	COE502 complemented with pMS2148-56	This study
Escherichia coli
DH5α	F⁻ ϕ80lacZΔM15 endA recA hsdR(r_K⁻ m_K⁻) supE thi gyrA relA Δ(lacZYA-argF)U169	Laboratory stock
ET12567/pUZ8002	Dam⁻ Dam⁻; with trans-mobilizing plasmid pUZ8002	40
Plasmids and cosmids
6G10A.1.F02	Cosmid St6G10A disrupted in SCO2150 with Tn5062	42
pIJ773	aac(3)IV (Apra^r) + oriT	41
pIJ778	aadA from Ω-fragment (Spec^r, Strep^r) + oriT	41
pMS82	ΦBT1 attP-int-derived integration vector for the conjugal transfer of DNA from E. coli to Streptomyces (Hyg^r)	43
pMS2148-56	pMS82 SCO2148–SCO2156 (with 200-bp upstream and downstream sequences)	This study
pMS3945-46	pMS82 SCO3945–SCO3946 (with 150-bp upstream and 9-bp downstream sequences)	This study

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Comparing growth and development of strain COE190 with that of M145 on different solid media revealed that, apart from soya flour and mannitol (SFM) medium, both strains grew and sporulated on meat extract medium (but only when the concentration was minimally 3 g · liter⁻¹), on yeast extract medium supplemented with 10 g · liter⁻¹ sugar, and on buffered peptone medium (summarized in Table S1). Moreover, neither strain completed the developmental cycle consistently and reproducibly within 4 to 5 days when grown on yeast extract-peptone-malt extract-glucose (YEME), on buffered meat extract containing glucose or mannitol, or on Difco nutrient broth (DNB) (peptone, meat extract with glucose or mannitol buffered to pH 7) medium (also summarized in Table S1).

The intriguing phenotype on YP medium of the S. coelicolor mutant lacking the cytochrome bd oxidase, whereby growth and sporulation were restricted (Fig. 6A), suggested a link to either energy or oxygen limitation. To test this, we analyzed whether adding the redox-cycling dye methylene blue (MB) (29) also affected growth or development of the three strains, M145, COE190, and COE192. Addition of 25 μM MB to MOPS (morpholinepropanesulfonic acid)-buffered yeast extract agar plates supplemented with glucose severely restricted growth and development only of strain COE190 (ΔcydAB), while M145 (wild type) and COE192 (Δqcr-cta) cells grew and sporulated after 3 days of aerobic growth (Fig. 6B). Growth inhibition of COE190 by inclusion of MB occurred regardless of whether the plates were incubated in the light or the dark. MB-induced oxidative stress is caused by UV light (21) and thus the inhibitory effect on growth of strain COE190 in the dark indicates this was not caused by oxidative stress, but rather by oxygen restriction due to the redox-cycling action of the dye (21). Sporulation of COE190 was already affected at a MB concentration of 15 μM, and growth inhibition was already noted at 20 μM MB (see Fig. S2 in the supplemental material). Specific inhibition of sporulation of COE190 on tryptic soy agar (TSA) and SFM agar plates supplemented with 25 μM MB could also be demonstrated (see Fig. S3 in the supplemental material).

DISCUSSION

Coupling of the Q cycle to the proton-pumping capacity in the bcc-aa₃ oxidase branch of the respiratory chain generates the bulk of the PMF per O₂ reduced and results in a high H⁺/O ratio (10). The efficient PMF generation by this complex explains why strain COE190, which has only the bcc-aa₃ oxidase branch, grows essentially like the wild type, despite oxygen consumption being lower than that of the wild type. In comparison, the cytochrome bd oxidase does not pump protons, but is electrogenic and mainly functions to carry the electron flux to reduce O₂. Consequently, the PMF generated by this branch of the respiratory chain is lower (30). This explains why the rate of oxygen consumption by COE192 (lacking the bcc-aa₃ complex) was only marginally reduced compared to that of the wild type, yet the cell density was considerably lower. Synthesizing both respiratory branches to oxygen therefore offers the bacterium considerable flexibility in balancing electron flux and PMF generation in response to oxygen availability and gives S. coelicolor the capability of maintaining ATP generation and growth at both high and low O₂ concentrations.

Nevertheless, we demonstrate here that S. coelicolor can grow and complete its complex developmental cycle without the bcc-aa₃ complex. This indicates that cytochrome bd oxidase is sufficient to allow S. coelicolor spores to survive and germinate and to allow outgrowth of the substrate mycelium. Indeed, the energy conserved by this means of O₂ respiration is adequate to allow the bacterium to sporulate and to produce at least some of its secondary metabolites, namely, the colored antibiotics actinorhodin and undecylprodigiosin (see Fig. S1 and S2 in the supplemental material). Notably, high levels of glucose were used in the solid growth medium used to grow strain COE192, and this aided sporulation. A recent study revealed that omission of buffer in the growth medium caused a strain lacking the bcc-aa₃ complex to grow more slowly, and it resulted in a significant reduction of the pH of the extracellular growth medium (26). This suggests that these strains reduce glucose to lactate and use substrate-level phosphorylation as the main means of ATP generation, and that they use the cytochrome bd oxidase to off-load the excess redox equivalents onto oxygen. This is reminiscent of the Warburg-Crabtree effect observed in yeast and cancer cells when they are exposed to high glucose concentrations (31). Under these conditions, they shut down respiration and essentially ferment. It should be stressed, however, that oxygen respiration is essential for growth of S. coelicolor, because it has not been possible to generate a viable strain lacking both terminal oxidases (D. Falke, M. Fischer, and R. G. Sawers, unpublished data). This also indicates that, although S. coelicolor is able to synthesize three respiratory nitrate reductases, these reductases only contribute to anaerobic survival, presumably through maintenance of a PMF, and are incapable of allowing growth of the bacterium by nitrate respiration (32, 33).

An important role for the cytochrome bd oxidase in the stationary-phase mycelium and development of aerial hyphae is suggested by the developmental phenotype observed in strain COE190 (ΔcydAB) when it is grown on buffered rich medium with either glucose or ribose as the carbon source. Not only does this strain fail to sporulate, but it also does not produce either of the colored antibiotics undecylprodigiosin and actinorhodin (1, 2, 34) when grown on this medium. This suggests that under these conditions, the bd oxidase branch is important for induction of aerial hypha development. It is conceivable that this requirement is due to oxygen limitation occurring in the densely growing substrate mycelium. Support for this hypothesis was provided using the redox-cycling dye MB, which inhibited growth specifically of the strain lacking cytochrome bd oxidase (Fig. 6B). Although MB can generate reactive oxygen species, this requires UV light (35). MB is also capable of cycling between MBH₂ and MB, due to cytochrome c-dependent reoxidation, whereby MB is rereduced, for example, by flavin-dependent enzymes, using NAD(P)H as an electron source (29). Electrons are thus diverted out of the respiratory chain by MB (standard redox potential at pH 7 [E°′] = +71 mV) and do not reach the proton-pumping aa₃ oxidase. Consequently, the PMF is reduced and growth is reduced. The inhibition of growth of strain COE190 caused by MB was also observed in the dark, indicating that MB is not acting via production of reactive oxygen species. Thus, when cytochrome bd oxidase is not present, MB causes the bcc-aa₃ complex to be bypassed, and the cells are effectively starved of the electrons required to reduce oxygen, despite oxygen being present in the immediate environment of the cells. Reintroduction of the genes encoding cytochrome bd oxidase restores growth to the mutant. Thus, cytochrome bd oxidase, which receives its electrons from menaquinol and which is unaffected by MB, allows oxygen reduction to occur. This is because electrons flow along a different route and do not reach the diheme c-type cytochrome QcrC.

Taken together, our data indicate that the cytochrome bd oxidase plays a significant role in maintaining electron flow through the respiratory chain under oxygen-limiting conditions, which can occur in the soil habitats in which streptomycetes are frequently found (2). The bd oxidase is therefore crucial to the physiology of streptomycetes, and particularly for completion of the developmental cycle, when oxygen levels drop below a concentration that is below the K_m (50 μM for C. glutamicum [23]) for oxygen of the aa₃ oxidase, compared with the K_m for the generally high-affinity of cytochrome bd oxidases, which lies in the nM range (13, 14). This might also explain why the cytochrome bd oxidase has a lesser role in other actinobacteria, such as corynebacteria or the pathogen M. tuberculosis, which are more reliant on the bcc-aa₃ oxidase respiratory branch (16, 24, 25, 36) and which have neither such a complex life cycle nor an extensive secondary metabolism.

MATERIALS AND METHODS

Bacterial strains and culture conditions.

Media and culture conditions for S. coelicolor and E. coli were the same as those described previously (37, 38). Strains are listed in Table 1. S. coelicolor A3(2) wild-type strain M145 and mutant derivatives (Table 1) were grown on SFM (soya flour and mannitol), on LB (Luria broth), or on Difco nutrient broth (DNB) agar medium, as indicated (37). Details of other growth media used for plate growth are listed in Table S1 in the supplemental material. To observe the developmental phenotype of mutant COE190, YP agar medium (3 g/liter yeast extract, 5 g/liter peptone, 50 mM glucose or ribose, 20 g/liter agar, and 40 mM MOPS-NaOH [pH 7.0]) was used. Fresh spores were streaked on buffered YP agar, and plates were incubated for up to 6 days at 30°C. To observe the methylene blue-dependent phenotype of mutant COE190, yeast extract medium with sugar (4 g/liter yeast extract, 10 g/liter glucose, 20 g/liter agar, 50 mM MOPS-NaOH [pH 7.0]) was used. Filter-sterilized methylene blue (MB) was added to the indicated final concentrations after autoclaving the medium.

For cultivation in liquid medium, Streptomyces strains were grown in tryptic soy broth (TSB; Oxoid) or DNB supplemented with appropriate antibiotics to maintain selection. S. coelicolor A3(2) strains were grown as highly dispersed liquid cultures in Duran F tubes with MOPS-buffered half-strength TSB, as described previously (39). Standardized 15 h exponential cultures (20 ml) were inoculated with 2 ml of a standard mycelium suspension. This suspension was prepared from a highly dispersed preculture by the determination of the cell pellet size after centrifugation (2,000 × g, 10 min, 6°C). A pellet volume of 200 μl was diluted to 10 ml. Afterwards the cultures were incubated in Duran F vials for 15 h, as described previously (39).

Cultivation of mycelium for growth curves and respiration measurements was performed in 24-well cell culture plates. Each well was filled with 5 glass beads (4 mm) and 1.5 ml of buffered half-strength TSB medium (100 mM MOPS-NaOH [pH 7.0]). For inoculation, 15 μl of a fresh spore suspension (or spores from a glycerol-frozen spore stock) with an optical density at 450 nm (OD₄₅₀) of 10 was added to each well. The cell culture plates were incubated at 30°C with continuous shaking at 200 rpm and an amplitude of 25 mm. For growth curve measurements, three wells were used as a triplicate for each time point, and probes of between 0.25 to 1.25 ml were taken. Cell density was measured in these samples, using the MB method as described previously (28). Due to the small volume in the wells (1.5 ml of medium and up to 1.25 ml of sample), the samples for the different time points were taken from different wells.

E. coli DH5α (Stratagene) was used as a host for cosmids and for plasmid constructions. E. coli ET12567/pUZ8002 (40) is the nonmethylating plasmid donor strain used for intergeneric conjugation with S. coelicolor strain M145 and its derivatives (37). Apramycin (Apra, 25 μg · ml⁻¹), carbenicillin (Carb, 100 μg · ml⁻¹), chloramphenicol (Cm, 25 μg · ml⁻¹), kanamycin (Kan, 25 μg · ml⁻¹), spectinomycin (Spc, 25 μg · ml⁻¹) or hygromycin (Hyg, 25 μg · ml⁻¹), all from Sigma, was added to growth media when required.

Construction of gene disruption mutants.

The large deletion mutations in strains COE190 (ΔSCO3945 and ΔSCO3946) and COE192 (Δ[SCO2148 to SCO2156]) were constructed using a PCR-targeted gene replacement technique, as described previously (41). Initially, the aac(3)IV (apramycin) resistance cassette from pIJ773 (41) was used to create a deletion of the complete region encompassing the genes SCO2148 to SCO2156 (Fig. 1A) (using the oligonucleotides BCF and BCR), while the aadA (Spec^r Strep^r) resistance cassette from pIJ778 (41) was used to create a deletion in the SCO3945 and SCO3946 genes, encoding cytochrome bd oxidase (using oligonucleotides BDF and BDR). The oligonucleotides used for these disruptions are given in Table S2 in the supplemental material, and the extent of the respective deletions is indicated in Table 1. Subsequent to mutant construction, the antibiotic resistance cassettes were removed by FLP-mediated recombination leaving an in-frame 81-bp “scar” sequence as described previously (41). The deletions in COE190 and COE192 were checked by PCR, using the oligonucleotides Sco2156con plus Sco2148con and Sco3945con plus Sco3946con (Table S2), respectively, which hybridize with sequences flanking the deletions.

Strain COE502 (ΔSCO2150) was constructed by first introducing cosmid 6G10A.1.F02 (Table 1) with a transposon insertion (Tn5062 [42]) in the gene SCO2150 (qcrC) into E. coli ET12567/pUZ8002 by electroporation, with subsequent transfer to S. coelicolor by conjugation (37). Exconjugants with double crossovers were selected for Kan^s and Apra^r. The authenticity of the S. coelicolor mutant strain was confirmed by PCR (data not shown).

Complementation of gene disruptions.

The sequences of the oligonucleotides used are shown in Table S2. For complementation studies involving the large SCO2148 to SCO2156 gene disruption in strain COE192, we constructed plasmid pMS2148-56. The 9,804-bp DNA fragment corresponding to the coding region from SCO2148 to SCO2156 (including 200 bp upstream and 200 bp downstream as flanking sequences) was generated from four distinct but partially overlapping DNA fragments (fragment 1, 2,680 bp; fragment 2, 2,582 bp; fragment 3, 2,591 bp; and fragment 4, 1,951 bp). The individual fragments were amplified by PCR using the oligonucleotides pMS_Fragment1_fw and AnnealFrag1_rv for fragment 1, AnnealFrag2_fw and AnnealFrag2_rv for fragment 2, AnnealFrag3_fw and AnnealFrag3_rv for fragment 3, and AnnealFrag4_fw and pMS_Fragment4_rv for fragment 4. The four DNA fragments were cloned in series between the HindIII and NsiI restriction sites of plasmid pMS82 (43) using the NEBuilder kit, exactly as described by the manufacturer (New England BioLabs). For construction of pMS3945-46 for the complementation of the gene disruption in strain COE190 (ΔSCO3945 to SCO3946), the corresponding DNA fragment was amplified using oligonucleotides SCO3945_HindIII_fw and SCO3946_KpnI-rv and was cloned between the HindIII and KpnI restriction sites of plasmid pMS82 to deliver pMS3945-46.

The authenticity of the cloned fragments in all complementation plasmids was verified by DNA sequencing, and plasmids were introduced into strains COE190 or COE192 via conjugation, using the plasmid-containing E. coli strain ET12567/pUZ8002 (Table 1).

Measurement and calculation of oxygen respiration.

A 1-ml aliquot of a 20-h, aerobically grown mycelium in shaking cell culture plates (representing 700 to 1,000 CAE; reference28) was transferred directly from flushed cultures (described above) to a vial containing 9 ml of air-saturated and buffered half-strength TSB. The vial had an oxygen-dependent luminescence sensor spot (PyroScience, Aachen, Germany) affixed to the inner side (glass wall), and the vial was noninvasively connected to an optical oxygen meter (FirestingO2; PyroScience, Aachen, Germany). Vials were stoppered (air tight) and stirred with a magnetic bar at 1,200 rpm. The linear rate of oxygen consumption was analyzed using Firesting Logger Software (PyroScience, Aachen, Germany) over a period of 5 to 10 min. A control in which the protein synthesis inhibitor chloramphenicol was added at 100 μg · ml⁻¹ showed no difference in the rate of oxygen consumption compared with a that of a sample without inhibitor. The specific respiration rate (nmol O₂ × min⁻¹ × [1,000 CAE]⁻¹) was calculated according to reference 39. Experiments were performed minimally three times and in triplicate.

Determination of cytochrome aa₃ oxidase activity.

Cytochrome aa₃ oxidase enzyme activity was determined spectrophotometrically by measuring the absorbance change at 550 nm, using membrane fractions derived from the mycelium (39, 44) and the oxidation of reduced horse heart cytochrome c (45). Cytochrome c (0.22 mM) was prereduced by adding freshly prepared solution of dithiothreitol (0.5 mM) and measuring the A₅₅₅/A₅₆₅ ratio. Activity was determined in a total assay volume of 0.2 ml in 10 mM Tris-HCl buffer (pH 7) containing 20 μM of reduced cytochrome c. Enzyme activity was measured three times from two biological replicates. The assay was performed at room temperature (RT), and one mU represents the oxidation of 1 nmol of ferrocytochrome c per min at pH 7.

In-gel staining for cytochrome aa₃ oxidase enzyme activity was determined according to Sabar et al. (27). Briefly, protein complexes in extracts or membrane fractions derived from spores or mycelium were separated by native polyacrylamide gel electrophoresis (PAGE). Typically, gels were prepared using nondenaturing 10% (wt/vol) polyacrylamide. Cytochrome aa₃ oxidase activity was visualized by incubating the gel in 10 ml of 50 mM potassium phosphate buffer (pH 7.2) containing 1 ml of cytochrome c as the substrate (stock solution 10 mg · ml⁻¹) and 0.5 ml of DAB reagent (diaminobenzidine stock solution of 10 mg · ml⁻¹). For activity staining, gels were incubated at RT and for between 1 and 2 h.

Antibody preparation, SDS-PAGE, and Western blotting.

Antibodies were prepared commercially (Eurogentec, Seraing, Belgium) against a 15-amino-acid peptide specific for SCO3945 (amino acid sequence, NPPTKIGGDLRDADK). Antibodies were affinity purified against the respective synthesized peptide. Aliquots (typically 25 to 60 μg of protein) from the crude extracts (typically 45 μg of protein) were separated by SDS-PAGE using 10% (wt/vol) polyacrylamide gels (46) and transferred to nitrocellulose membranes, as described previously (47). Affinity-purified antibodies were generally used at a dilution of 1:8,000 (1:10,000 for anti-CydA) unless otherwise specified. Secondary antibody conjugated to horseradish peroxidase was obtained from Bio-Rad. Visualization was done by enhanced chemiluminescent reaction (Stratagene).

Supplementary Material

Supplemental material

supp_200_16_e00239-18__index.html^{(1.1KB, html)}

ACKNOWLEDGMENTS

We are grateful to Jesse Alderson (formerly of the John Innes Center) for help with constructing mutants, Marcell Barth for help in performing the respiration studies, and Claudia Hammerschmidt for expert technical assistance. We are also grateful to Robert Poole for discussion.

This work was supported by the Deutsche Forschungsgemeinschaft (grant Sa-494/4-2).

All authors read the final version of the manuscript. M.F. and D.F. helped conceive the study and were involved in performing all of the experiments. C.N. performed the respiration experiments along with M.F. R.G.S. conceived the study and wrote the manuscript.

We declare no conflict of interest.

Footnotes

Supplemental material for this article may be found at https://doi.org/10.1128/JB.00239-18.

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Supplementary Materials

Supplemental material

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[B1] 1.Hodgson DA. 2000. Primary metabolism and its control in streptomycetes: a most unusual group of bacteria. Adv Microb Physiol 42:47–238. doi: 10.1016/S0065-2911(00)42003-5. [DOI] [PubMed] [Google Scholar]

[B2] 2.Hopwood DA. 2006. Soil to genomics: the Streptomyces chromosome. Annu Rev Genet 40:1–23. doi: 10.1146/annurev.genet.40.110405.090639. [DOI] [PubMed] [Google Scholar]

[B3] 3.Bott M, Niebisch A. 2003. The respiratory chain of Corynebacterium glutamicum. J Biotechnol 104:129–153. doi: 10.1016/S0168-1656(03)00144-5. [DOI] [PubMed] [Google Scholar]

[B4] 4.Lu P, Heineke MH, Koul A, Andries K, Cook GM, Lill H, van Spanning R, Bald D. 2015. The cytochrome bd-type quinol oxidase is important for survival of Mycobacterium smegmatis under peroxide and antibiotic-induced stress. Sci Rep 5:10333. doi: 10.1038/srep10333. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B5] 5.Kim MS, Jang J, Ab Rahman NB, Pethe K, Berry EA, Huang LS. 2015. Isolation and characterization of a hybrid respiratory supercomplex consisting of Mycobacterium tuberculosis cytochrome bcc and Mycobacterium smegmatis cytochrome aa3. J Biol Chem 290:14350–14360. doi: 10.1074/jbc.M114.624312. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B6] 6.Kalia NP, Hasenoehr EJ, Ab Rahman NB, Koh VH, Ang MLT, Sajord DR, Hards K, Grüber G, Alonso S, Cook GM, Berney M, Pethe K. 2017. Exploiting the synthetic lethality between terminal respiratory oxidases to kill Mycobacterium tuberculosis and clear host infection. Proc Natl Acad Sci U S A 114:7426–7431. doi: 10.1073/pnas.1706139114. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B7] 7.Collins MD, Jones D. 1981. Distribution of isoprenoid quinone structural types in bacteria and their taxonomic implication. Microbiol Rev 45:316–354. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B8] 8.Dairi T. 2009. An alternative menaquinone biosynthetic pathway operating in microorganisms: an attractive target for drug discovery to pathogenic Helicobacter and Chlamydia strains. J Antibiot (Tokyo) 62:347–352. doi: 10.1038/ja.2009.46. [DOI] [PubMed] [Google Scholar]

[B9] 9.Sawers RG, Falke D, Fischer M. 2016. Oxygen and nitrate respiration in Streptomyces coelicolor A3(2). Adv Microb Physiol 68:1–40. doi: 10.1016/bs.ampbs.2016.02.004. [DOI] [PubMed] [Google Scholar]

[B10] 10.Nicholls DG, Ferguson SJ. 2013. Bioenergetics, 4th ed Academic Press, Waltham, MA. [Google Scholar]

[B11] 11.Cook GM, Poole RK. 2016. A bacterial oxidase like no other? Science 352:518–519. doi: 10.1126/science.aaf5514. [DOI] [PubMed] [Google Scholar]

[B12] 12.Safarian S, Rajendran C, Müller H, Preu J, Langer JD, Ovchinnikov S, Hirose T, Kusumoto T, Sakamoto J, Michel H. 2016. Structure of a bd oxidase indicates similar mechanisms for membrane-integrated oxygen reductases. Science 352:583–586. doi: 10.1126/science.aaf2477. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B13] 13.D'Mello R, Hill S, Poole RK. 1996. The cytochrome bd quinol oxidase in Escherichia coli has an extremely high oxygen affinity and two oxygen-binding haems: implications for regulation of activity in vivo by oxygen inhibition. Microbiology 142:755–763. doi: 10.1099/00221287-142-4-755. [DOI] [PubMed] [Google Scholar]

[B14] 14.Borisov VB, Gennis RB, Hemp J, Verkhovsky MI. 2011. The cytochrome bd respiratory oxygen reductases. Biochim Biophys Acta 1807:1398–1413. doi: 10.1016/j.bbabio.2011.06.016. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B15] 15.Brekasis D, Paget MS. 2003. A novel sensor of NADH/NAD⁺ redox poise in Streptomyces coelicolor A3(2). EMBO J 22:4856–4865. doi: 10.1093/emboj/cdg453. [DOI] [PMC free article] [PubMed] [Google Scholar]

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PERMALINK

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

Marco Fischer

Dörte Falke

Carolin Naujoks

R Gary Sawers

Roles

ABSTRACT

INTRODUCTION

FIG 1.

RESULTS

Strains with either the cytochrome bd oxidase or the bcc-aa3 oxidase complex complete their developmental cycle on solid medium.

FIG 2.

Growth rate of the bcc-aa3 oxidase-negative mutant is reduced in liquid culture.

FIG 3.

A strain lacking the diheme c-type cytochrome, QcrC, is also devoid of aa3 oxidase activity.

FIG 4.

Lower levels of the CydA polypeptide are present in wild-type spores compared with mycelium.

Mycelium of a strain reliant on bd oxidase exhibits wild-type rates of O2 respiration but reduced biomass production.

FIG 5.

Cytochrome bd oxidase is required to allow sporulation under oxygen-limiting conditions.

FIG 6.

TABLE 1.

DISCUSSION

MATERIALS AND METHODS

Bacterial strains and culture conditions.

Construction of gene disruption mutants.

Complementation of gene disruptions.

Measurement and calculation of oxygen respiration.

Determination of cytochrome aa3 oxidase activity.

Antibody preparation, SDS-PAGE, and Western blotting.

Supplementary Material

ACKNOWLEDGMENTS

Footnotes

REFERENCES

Associated Data

Supplementary Materials

ACTIONS

PERMALINK

RESOURCES

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Links to NCBI Databases

Strains with either the cytochrome bd oxidase or the bcc-aa₃ oxidase complex complete their developmental cycle on solid medium.

Growth rate of the bcc-aa₃ oxidase-negative mutant is reduced in liquid culture.

A strain lacking the diheme c-type cytochrome, QcrC, is also devoid of aa₃ oxidase activity.

Mycelium of a strain reliant on bd oxidase exhibits wild-type rates of O₂ respiration but reduced biomass production.

Determination of cytochrome aa₃ oxidase activity.