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

Marco Fischer,a Dörte Falke,a Carolin Naujoks,a* R. Gary Sawersa

aInstitute of Microbiology, Martin Luther University Halle-Wittenberg, Halle (Saale), Germany

ABSTRACT Streptomyces coelicolor A3(2) is a filamentously growing, spore-forming,obligately aerobic actinobacterium that uses both a copper aa3-type cytochrome coxidase and a cytochrome bd oxidase to respire oxygen. Using defined knockoutmutants, we demonstrated that either of these terminal oxidases was capable of al-lowing the bacterium to grow and complete its developmental cycle. The genes en-coding the bcc complex and the aa3 oxidase are clustered at a single locus. UsingWestern blot analyses, we showed that the bcc-aa3 oxidase branch is more prevalentin spores than the bd oxidase. The level of the catalytic subunit, CydA, of the bd oxi-dase was low in spore extracts derived from the wild type, but it was upregulated ina mutant lacking the bcc-aa3 supercomplex. This indicates that cytochrome bd oxi-dase can compensate for the lack of the other respiratory branch. Components ofboth oxidases were abundant in growing mycelium. Growth studies in liquid me-dium revealed that a mutant lacking the bcc-aa3 oxidase branch grew approximatelyhalf as fast as the wild type, while the oxygen reduction rate of the mutant remainedclose to that of the wild type, indicating that the bd oxidase was mainly functioning incontrolling electron flux. Developmental defects were observed for a mutant lacking thecytochrome bd oxidase during growth on buffered rich medium plates with glucose asthe energy substrate. Evidence based on using the redox-cycling dye methylene bluesuggested that cytochrome bd oxidase is essential for the bacterium to grow and com-plete its developmental cycle under oxygen limitation.

IMPORTANCE Respiring with oxygen is an efficient means of conserving energy inbiological systems. The spore-forming, filamentous actinobacterium Streptomycescoelicolor grows only aerobically, synthesizing two enzyme complexes for O2 reduc-tion, the cytochrome bcc-aa3 cytochrome oxidase supercomplex and the cytochromebd oxidase. We show in this study that the bacterium can survive with either ofthese respiratory pathways to oxygen. Immunological studies indicate that the bcc-aa3 oxidase is the main oxidase present in spores, but the bd oxidase compensatesif the bcc-aa3 oxidase is inactivated. Both oxidases are active in mycelia. Growth con-ditions were identified, revealing that cytochrome bd oxidase is essential for aerialhypha formation and sporulation, and this was linked to an important role of theenzyme under oxygen-limiting conditions.

KEYWORDS actinobacteria, cytochrome bd oxidase, cytochrome oxidases,menaquinol, mycelium, respiration, spores

Streptomyces coelicolor A3(2) is an actinobacterium with a complex developmentalcycle and, like other streptomycetes, it produces a complex spectrum of secondary

metabolites. The bacterium grows as a substrate mycelium and, upon nutrient limita-tion, produces hydrophobic aerial mycelia that develop into chains of spores (1, 2).Growth and development of S. coelicolor depend on oxygen respiration, and strepto-

Received 23 April 2018 Accepted 14 May2018

Accepted manuscript posted online 21 May2018

Citation Fischer M, Falke D, Naujoks C, SawersRG. 2018. Cytochrome bd oxidase has animportant role in sustaining growth anddevelopment of Streptomyces coelicolor A3(2)under oxygen-limiting conditions. J Bacteriol200:e00239-18. https://doi.org/10.1128/JB.00239-18.

Editor Conrad W. Mullineaux, Queen MaryUniversity of London

Copyright © 2018 American Society forMicrobiology. All Rights Reserved.

Address correspondence to R. Gary Sawers,gary.sawers@mikrobiologie.uni-halle.de.

* Present address: Carolin Naujoks, IDTBiologika, Am Pharmapark, Dessau-Roßlau,Germany.

RESEARCH ARTICLE

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mycetes are predicted to have a branched, menaquinone-based respiratory chain, ashas been demonstrated for other members of the actinobacteria, such as Corynebac-terium, Mycobacterium, and Rhodococcus species (3–8). Moreover, Streptomyces speciesalso lack a soluble cytochrome c and instead have a diheme QcrC protein as part oftheir menaquinol:cytochrome bcc oxidoreductase (bcc complex) (9). The bcc complexand a cytochrome c oxidase of the aa3 copper family (aa3-type oxidase) form oneoxygen-reducing respiratory branch, while a menaquinol-oxidizing cytochrome bdoxidase forms the other. The bcc-aa3 branch is bioenergetically more favorable for ATPgeneration and proton motive force (PMF)-driven substrate transport processes, be-cause the combined action of the bcc complex-driven Q cycle and the proton-pumpingaa3-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 neverthelessis electrogenic, releasing 2 H� for each 2 e� transferred, owing to its menaquinoloxidation site being on the outer face of the cytoplasmic membrane (11, 12). Thus, thebd oxidase completes a redox loop when coupled with quinone dehydrogenases thatreceive electrons from NADH, pyruvate, D-lactate, or acyl coenzyme A (acyl-CoA) (9). Itis likely that the bd oxidase is functional under microaerobic conditions, as in otherbacteria (13, 14), and the induction of expression of the cydAB genes in response to anincreased NADH/NAD� ratio would support this (15).

The multiprotein enzyme complexes of the cytochrome bcc-aa3 oxidase branch ofthe aerobic respiratory chain in actinobacteria have a number of unusual features,which have been summarized previously (3, 16–19). These features are also conservedin the bcc complex and the aa3 oxidase of S. coelicolor, and together they suggest thatboth enzymes form a supercomplex, as has been demonstrated for Corynebacteriumglutamicum and for Mycobacterium species (5, 17, 18, 20). This is underlined by the factsthat 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 terminaloxidase, is located immediately upstream of the qcrCAB operon (Fig. 1A).

While the phenotypic consequence of a deletion in the genes encoding the cyto-chrome 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 mutationsin 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 respiratorychain. 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 growthmedia (26). However, no studies have been undertaken to examine the consequencesto the physiology of Streptomyces coelicolor caused by deleting the genes encoding thecytochrome bd oxidase. Because Streptomyces species are important model systems forstudying both bacterial development and secondary metabolism, it is important tounderstand the impact of each respiratory branch on these processes. We show in thisstudy that, in contrast to other characterized actinobacteria, both respiratory branchesare individually capable of sustaining growth and development of the bacterium. Wealso identify growth conditions under which a functional cytochrome bd oxidase isessential for development beyond the formation of substrate mycelium.

RESULTSStrains with either the cytochrome bd oxidase or the bcc-aa3 oxidase complex

complete their developmental cycle on solid medium. Mutant COE190 lacks thegenes SCO3945 to SCO3946, encoding cytochrome bd oxidase, and mutant COE192lacks the complete locus encompassing genes SCO2148 to SCO2156, encoding thecytochrome bcc complex and the aa3 oxidase (Fig. 1). Growth of strain COE192 (lackingthe bcc-aa3 supercomplex), and that of the wild-type strain M145, was compared onsoya flour and mannitol (SFM) medium (Fig. 2A). Despite this medium normallyallowing rapid colony development, only small colonies of COE192 appeared after 2 to3 days, and by the 5th day they were roughly 50% of the size of M145 colonies. In

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contrast, colonies of M145 were visible after 1 day of growth (Fig. 2A). Notably, despiteits markedly slower growth, morphological development of strain COE192 was unim-paired 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 thecomplete locus into COE192 on the integrative plasmid derivative, pMS2148-56(Fig. 2A). Strain COE192 also completed its developmental cycle on buffered yeastextract-peptone (YP) medium, with or without sugar supplementation (see Fig. S1 inthe supplemental material), and this was also observed for several other solid richmedia tested (see Fig. S2 in the supplemental material for yeast extract [YE] plusglucose medium; also data not shown). Moreover, no reproducible developmentalphenotype 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 wasessentially indistinguishable from the wild-type strain M145 (Fig. 2B). Together, thesefindings indicate that the bcc-aa3 oxidase complex is important for rapid colony

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 isdepicted above the gene. The genes encoding the cytochrome bcc complex and the aa3 oxidase arecolored blue and orange, respectively (A), while those encoding the cytochrome bd oxidase are shownin red (B). The functions of the gene products of the gray genes are unknown. The extent of the DNAfragments cloned in the respective complementation plasmids is shown below the loci.

FIG 2 Growth and developmental phenotypes of cytochrome oxidase-negative mutants. (A) Spores ofM145 (wild type), COE192 (Δqcr-cta) carrying a deletion of the genes SCO2148 through SCO2156, whichencode the bcc complex and cytochrome aa3 oxidase, and COE192 (comp.) transformed with plasmidpMS2148-56 (Fig. 1A), were grown on SFM solid medium for the days indicated. (B) The indicated strainswere grown for 3 days on SFM medium. Spores were streaked out in the three sectors (I, streaked as athick flat layer; II, streaked as a layer of colonies; III, streaked to single colonies) to analyze the influenceof culture density on sporulation.

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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 liquidculture. S. coelicolor grows in liquid culture as mycelium, but it fails to sporulate underthese conditions (2). We determined the doubling times of M145 (both terminaloxidases present), COE190 (only bcc-aa3 oxidase present), COE192 (only bd oxidasepresent), and COE192 complemented with plasmid pMS2148-56 during cultivation inbuffered, half-strength liquid tryptic soy broth (TSB) medium (Fig. 3). From the sameinitial amounts of spore suspension, mycelium of M145 and COE190 increased in celldensity at a similar rate (doubling of mycelium “cell” density every 2.5 h), while strainCOE192 grew approximately 55% more slowly compared to the wild type. Note thatstreptomycetes grow by tip growth (2), and therefore “growth” cannot be quantified interms of “growth rate” and “doubling time” as for standard bacteria, and this is why therepresentation of growth rate is linear and not semilogarithmic (Fig. 3). Introduction ofthe 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 ofthe mutant to one similar to that of the wild-type M145 (Fig. 3). We ruled out thepossibility that the genes SCO2152, which encodes a putative response regulator, andSCO2153, which encodes a predicted secreted protein (Fig. 1A; strepdb.streptomyces.org.uk), were responsible for the growth phenotype of mutant COE192, because strainscarrying in-frame deletion mutations in either gene failed to exhibit either a growth ora 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 thepresence of the bcc-aa3 complex affords more efficient energy conservation and hencegrowth, a strain lacking this complex and that is reliant only on the bd oxidase forgrowth can nevertheless grow and sporulate.

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 singleactivity 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 enzymeactivity could be detected in extracts derived from strain COE192 in either spores ormycelium (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 geneSCO2150 (qcrC in Fig. 1A), encoding the diheme cytochrome c subunit of the bcccomplex and an extract derived from this mutant also lacked aa3 oxidase enzymeactivity (Fig. 4A and B). Introduction of pMS2148-56 into strains COE192 and COE502

FIG 3 Growth analysis of oxidase-negative mutants grown in liquid culture. Strains were grown asdescribed in Materials and Methods, and cell density was measured as described previously (28). StrainsM145, COE190 (ΔcydAB), COE192 (Δqcr-cta), and COE192 (complemented [comp.] with pMS2148-56) areshown. The experiment was performed four times (i.e., from four independent spore harvests), each timein triplicate. In the interest of clarity, error bars are shown only for strain COE192.

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restored aa3 oxidase enzyme activity to both mutants in spores (Fig. 4A). Strain COE502had a growth phenotype that was indistinguishable from that of COE192 on all mediatested (data not shown).

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

Quantitative determination of enzyme activity showed a barely detectable oxidaseactivity in mycelial extracts derived from COE192 of 0.8 mU · mg�1, while extracts ofM145 had a specific activity of 16.2 mU · mg�1, and extracts of COE190 had an activityof 38.5 mU · mg�1. The higher oxidase activity measured in the quantitative assay inextracts 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 strainCOE190 to compensate for the lack of the cytochrome bd oxidase.

Lower levels of the CydA polypeptide are present in wild-type spores com-pared with mycelium. Because strain COE192 completes its developmental cyclewhen growing on buffered rich medium (Fig. 2A), this indicates that the cytochrome bdoxidase activity must be sufficient to allow spores to form and for them to retainviability. To assess the distribution of this oxidase in spores and mycelium, we usedpeptide 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 theCydA polypeptide was barely detectable in extracts derived from spores of M145 (Fig.4C), while a polypeptide of significantly stronger intensity, migrating at approximately50 kDa (deduced molecular mass of CydA � 55.8 kDa), was visible in an extract derivedfrom mycelium (Fig. 4D). As a negative control, no CydA polypeptide could be detectedin extracts derived from spores (Fig. 4C) or mycelium (Fig. 4D) of strain COE190

FIG 4 Characterization of cytochrome oxidases in spores and mycelium. In-gel cytochrome c oxidase activity was determinedin crude extracts of spores. (A and B) Crude extracts (80 �g of protein) derived from spores (A) and from the mycelium (40 �gof protein) (B) were separated by nondenaturing 10% (wt/vol) native PAGE, and gels were subsequently stained forcytochrome c oxidase activity (see Materials and Methods). The arrow indicates the cytochrome c oxidase activity band. (C andD) Western blot analysis for the presence of CydA polypeptide in crude extracts of spores and mycelium. Aliquots of crudeextracts (45 �g) derived from spores (C) and mycelium (D) were separated by 10% (wt/vol) denaturing SDS-PAGE andtransferred to nitrocellulose membranes (see Materials and Methods). To detect the CydA polypeptide, the membrane wasprobed with anti-SCO3945 (1:10,000) antibodies. An arrow identifies the migration position of the CydA polypeptide. (E) Thesame 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 theleft. COE192 (comp.) and COE502 (comp.) signify the respective mutants complemented with pMS2148-56.

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(ΔcydAB). Notably, while the level of CydA was essentially unaffected by the absence ofthe bcc-aa3 complex in extracts of mycelium derived from COE192 (Fig. 4D), amountsof 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 SCO2156resulted in reduced, barely detectable levels of CydA in spores of the complementedstrain.

Finally, we analyzed the levels of CydA polypeptide in spore extracts derived fromstrain COE502 (ΔqcrC), and again, increased levels of the CydA polypeptide weredetected, 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 asthose used for the Western blots (Fig. 4E) confirmed equal protein loading. Together,these data suggest that synthesis of the CydA component of the cytochrome bdoxidase, which is otherwise synthesized at very low levels, is increased in spores whenthe 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 thecontribution of each oxidase in the individual oxidase-negative mutants to oxygenrespiration 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 oxidasemutants COE190 and COE192 (see Materials and Methods for details). Strain M145exhibited a rate of O2 reduction that was approximately equal to 50 nmol O2 reducedper min per 1,000 cell amount equivalents (CAE) (Fig. 5A) (28), while COE190 consumedO2 at a rate that was only 27% of this value. Complementation of this mutant withpMS3945-46 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 ofstrain COE192 (only bd oxidase present) was approximately 80% of the rate of that ofM145 (wild type), and this difference was not considered statistically significant. Thesedata indicate that mutants lacking the bcc-aa3 branch of the respiratory pathwayreduced O2 at a rate similar to that of the wild type, while the absence of the bd-typeoxidase caused a much more significant reduction in the O2 consumption rate.

To determine how the mutations in the oxidase genes affected growth and biomassproduction, the cell density was determined and compared for the same samples usedto 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 weresimilar, around 1,200 CAE · ml�1 (see Materials and Methods) (28), while the cell densityattained by strain COE192 (lacking the bcc complex and aa3 oxidase) was reduced byapproximately 30% compared to that of M145. Together, these data are in accord withthose shown in Fig. 3, and they indicate that cytochrome bd oxidase is sufficient tosupport growth of S. coelicolor mycelium in the absence of a bcc-aa3 pathway.

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 foraliquots of mycelium from the indicated strains after 20 h of growth in buffered, half-strength TSBmedium (see Materials and Methods). The experiment was performed three times, each time in triplicate.

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Cytochrome bd oxidase is required to allow sporulation under oxygen-limitingconditions. While testing growth of the wild type M145 and the bd oxidase mutant,COE190, for their developmental phenotypes on different solid growth media (seeTable S1 in the supplemental material), it was noted that strain COE190, which lacks thebd oxidase, reproducibly failed to form aerial hyphae or spores on buffered yeastextract-peptone (YP)– glucose medium within 6 days (Fig. 6A). Complementation of thephenotype could be achieved by introducing the cydAB genes on integrative plasmidpMS3945-46 (Table 1), resulting in restoration of the sporulating phenotype within 4days and confirming that this phenotype was due solely to the absence of cytochromebd oxidase (Fig. 6A). The developmental phenotype was not reproducibly observed forstrain COE190 if the solid medium was not supplemented with a sugar, whereas M145and the complemented COE190 strain (COE437) sporulated without sugar supplemen-tation (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 sporulationphenotype that was not reproducible (see Table S1 and Fig. S1 in the supplementalmaterial).

Comparing growth and development of strain COE190 with that of M145 ondifferent solid media revealed that, apart from soya flour and mannitol (SFM) medium,both strains grew and sporulated on meat extract medium (but only when theconcentration was minimally 3 g · liter�1), on yeast extract medium supplemented with10 g · liter�1 sugar, and on buffered peptone medium (summarized in Table S1).Moreover, neither strain completed the developmental cycle consistently and repro-ducibly 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 nutrientbroth (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 thecytochrome bd oxidase, whereby growth and sporulation were restricted (Fig. 6A),suggested a link to either energy or oxygen limitation. To test this, we analyzedwhether adding the redox-cycling dye methylene blue (MB) (29) also affected growth

FIG 6 Phenotypes of strain COE190 (ΔcydAB). (A) Strains were grown on buffered YP medium platessupplemented with glucose for the time period indicated below each plate. The key to the right of the agarplates 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 platesalso included 50 mM MOPS buffer (pH 7) and 25 �M methylene blue.

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or development of the three strains, M145, COE190, and COE192. Addition of 25 �M MBto MOPS (morpholinepropanesulfonic acid)-buffered yeast extract agar plates supple-mented with glucose severely restricted growth and development only of strainCOE190 (ΔcydAB), while M145 (wild type) and COE192 (Δqcr-cta) cells grew andsporulated after 3 days of aerobic growth (Fig. 6B). Growth inhibition of COE190 byinclusion of MB occurred regardless of whether the plates were incubated in the lightor the dark. MB-induced oxidative stress is caused by UV light (21) and thus theinhibitory effect on growth of strain COE190 in the dark indicates this was not causedby oxidative stress, but rather by oxygen restriction due to the redox-cycling action ofthe dye (21). Sporulation of COE190 was already affected at a MB concentration of 15�M, and growth inhibition was already noted at 20 �M MB (see Fig. S2 in thesupplemental material). Specific inhibition of sporulation of COE190 on tryptic soy agar(TSA) and SFM agar plates supplemented with 25 �M 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 oxidasebranch of the respiratory chain generates the bulk of the PMF per O2 reduced andresults in a high H�/O ratio (10). The efficient PMF generation by this complex explainswhy strain COE190, which has only the bcc-aa3 oxidase branch, grows essentially likethe wild type, despite oxygen consumption being lower than that of the wild type. Incomparison, the cytochrome bd oxidase does not pump protons, but is electrogenicand mainly functions to carry the electron flux to reduce O2. Consequently, the PMFgenerated by this branch of the respiratory chain is lower (30). This explains why therate of oxygen consumption by COE192 (lacking the bcc-aa3 complex) was onlymarginally reduced compared to that of the wild type, yet the cell density wasconsiderably lower. Synthesizing both respiratory branches to oxygen therefore offersthe bacterium considerable flexibility in balancing electron flux and PMF generation inresponse to oxygen availability and gives S. coelicolor the capability of maintaining ATPgeneration and growth at both high and low O2 concentrations.

Nevertheless, we demonstrate here that S. coelicolor can grow and complete its

TABLE 1 Strains and vectors used in this study

Species, strain, plasmid, or cosmid Genotype and characterics Reference or source

Species and strainsStreptomyces coelicolor A3(2)

M145 (wild type) SCP1� SCP2� 37COE190 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 studyCOE437 COE190 complemented with pMS3945-46 This studyCOE634 COE192 complemented with pMS2148-56 This studyCOE639 COE502 complemented with pMS2148-56 This study

Escherichia coliDH5� F� �80lacZΔM15 endA recA hsdR(rK

� mK�) supE thi gyrA relA

Δ(lacZYA-argF)U169Laboratory stock

ET12567/pUZ8002 Dam� Dam�; with trans-mobilizing plasmid pUZ8002 40

Plasmids and cosmids6G10A.1.F02 Cosmid St6G10A disrupted in SCO2150 with Tn5062 42pIJ773 aac(3)IV (Aprar) � oriT 41pIJ778 aadA from �-fragment (Specr, Strepr) � oriT 41pMS82 �BT1 attP-int-derived integration vector for the conjugal transfer of DNA

from E. coli to Streptomyces (Hygr)43

pMS2148-56 pMS82 SCO2148–SCO2156 (with 200-bp upstream and downstreamsequences)

This study

pMS3945-46 pMS82 SCO3945–SCO3946 (with 150-bp upstream and 9-bp downstreamsequences)

This study

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complex developmental cycle without the bcc-aa3 complex. This indicates that cyto-chrome bd oxidase is sufficient to allow S. coelicolor spores to survive and germinateand to allow outgrowth of the substrate mycelium. Indeed, the energy conserved bythis means of O2 respiration is adequate to allow the bacterium to sporulate and toproduce at least some of its secondary metabolites, namely, the colored antibioticsactinorhodin 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 growstrain COE192, and this aided sporulation. A recent study revealed that omission ofbuffer in the growth medium caused a strain lacking the bcc-aa3 complex to grow moreslowly, and it resulted in a significant reduction of the pH of the extracellular growthmedium (26). This suggests that these strains reduce glucose to lactate and usesubstrate-level phosphorylation as the main means of ATP generation, and that theyuse 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 cellswhen they are exposed to high glucose concentrations (31). Under these conditions,they shut down respiration and essentially ferment. It should be stressed, however, thatoxygen respiration is essential for growth of S. coelicolor, because it has not beenpossible 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 isable to synthesize three respiratory nitrate reductases, these reductases only contributeto anaerobic survival, presumably through maintenance of a PMF, and are incapable ofallowing growth of the bacterium by nitrate respiration (32, 33).

An important role for the cytochrome bd oxidase in the stationary-phase myceliumand development of aerial hyphae is suggested by the developmental phenotypeobserved in strain COE190 (ΔcydAB) when it is grown on buffered rich medium witheither 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 andactinorhodin (1, 2, 34) when grown on this medium. This suggests that under theseconditions, the bd oxidase branch is important for induction of aerial hypha develop-ment. It is conceivable that this requirement is due to oxygen limitation occurring in thedensely growing substrate mycelium. Support for this hypothesis was provided usingthe redox-cycling dye MB, which inhibited growth specifically of the strain lackingcytochrome 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 tocytochrome c-dependent reoxidation, whereby MB is rereduced, for example, byflavin-dependent enzymes, using NAD(P)H as an electron source (29). Electrons are thusdiverted 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 isreduced and growth is reduced. The inhibition of growth of strain COE190 caused byMB was also observed in the dark, indicating that MB is not acting via production ofreactive oxygen species. Thus, when cytochrome bd oxidase is not present, MB causesthe bcc-aa3 complex to be bypassed, and the cells are effectively starved of theelectrons required to reduce oxygen, despite oxygen being present in the immediateenvironment of the cells. Reintroduction of the genes encoding cytochrome bd oxidaserestores growth to the mutant. Thus, cytochrome bd oxidase, which receives itselectrons from menaquinol and which is unaffected by MB, allows oxygen reduction tooccur. This is because electrons flow along a different route and do not reach thediheme c-type cytochrome QcrC.

Taken together, our data indicate that the cytochrome bd oxidase plays a significantrole in maintaining electron flow through the respiratory chain under oxygen-limitingconditions, which can occur in the soil habitats in which streptomycetes are frequentlyfound (2). The bd oxidase is therefore crucial to the physiology of streptomycetes, andparticularly for completion of the developmental cycle, when oxygen levels drop belowa concentration that is below the Km (50 �M for C. glutamicum [23]) for oxygen of theaa3 oxidase, compared with the Km for the generally high-affinity of cytochrome bdoxidases, which lies in the nM range (13, 14). This might also explain why the

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cytochrome bd oxidase has a lesser role in other actinobacteria, such as corynebacteriaor the pathogen M. tuberculosis, which are more reliant on the bcc-aa3 oxidaserespiratory branch (16, 24, 25, 36) and which have neither such a complex life cycle noran extensive secondary metabolism.

MATERIALS AND METHODSBacterial 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), onLB (Luria broth), or on Difco nutrient broth (DNB) agar medium, as indicated (37). Details of other growthmedia used for plate growth are listed in Table S1 in the supplemental material. To observe thedevelopmental 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 werestreaked on buffered YP agar, and plates were incubated for up to 6 days at 30°C. To observe themethylene blue-dependent phenotype of mutant COE190, yeast extract medium with sugar (4 g/literyeast extract, 10 g/liter glucose, 20 g/liter agar, 50 mM MOPS-NaOH [pH 7.0]) was used. Filter-sterilizedmethylene 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 DNBsupplemented with appropriate antibiotics to maintain selection. S. coelicolor A3(2) strains were grown ashighly dispersed liquid cultures in Duran F tubes with MOPS-buffered half-strength TSB, as describedpreviously (39). Standardized 15 h exponential cultures (20 ml) were inoculated with 2 ml of a standardmycelium suspension. This suspension was prepared from a highly dispersed preculture by the determinationof the cell pellet size after centrifugation (2,000 � g, 10 min, 6°C). A pellet volume of 200 �l was diluted to10 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 cellculture 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 �l of a fresh spore suspension (or spores from aglycerol-frozen spore stock) with an optical density at 450 nm (OD450) of 10 was added to each well. The cellculture plates were incubated at 30°C with continuous shaking at 200 rpm and an amplitude of 25 mm. Forgrowth curve measurements, three wells were used as a triplicate for each time point, and probes of between0.25 to 1.25 ml were taken. Cell density was measured in these samples, using the MB method as describedpreviously (28). Due to the small volume in the wells (1.5 ml of medium and up to 1.25 ml of sample), thesamples for the different time points were taken from different wells.

E. coli DH5� (Stratagene) was used as a host for cosmids and for plasmid constructions. E. coliET12567/pUZ8002 (40) is the nonmethylating plasmid donor strain used for intergeneric conjugationwith S. coelicolor strain M145 and its derivatives (37). Apramycin (Apra, 25 �g · ml�1), carbenicillin (Carb,100 �g · ml�1), chloramphenicol (Cm, 25 �g · ml�1), kanamycin (Kan, 25 �g · ml�1), spectinomycin (Spc,25 �g · ml�1) or hygromycin (Hyg, 25 �g · ml�1), all from Sigma, was added to growth media whenrequired.

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 encompassingthe genes SCO2148 to SCO2156 (Fig. 1A) (using the oligonucleotides BCF and BCR), while the aadA (Specr

Strepr) resistance cassette from pIJ778 (41) was used to create a deletion in the SCO3945 and SCO3946genes, encoding cytochrome bd oxidase (using oligonucleotides BDF and BDR). The oligonucleotidesused for these disruptions are given in Table S2 in the supplemental material, and the extent of therespective deletions is indicated in Table 1. Subsequent to mutant construction, the antibiotic resistancecassettes were removed by FLP-mediated recombination leaving an in-frame 81-bp “scar” sequence asdescribed previously (41). The deletions in COE190 and COE192 were checked by PCR, using theoligonucleotides Sco2156con plus Sco2148con and Sco3945con plus Sco3946con (Table S2), respec-tively, which hybridize with sequences flanking the deletions.

Strain COE502 (ΔSCO2150) was constructed by first introducing cosmid 6G10A.1.F02 (Table 1) witha transposon insertion (Tn5062 [42]) in the gene SCO2150 (qcrC) into E. coli ET12567/pUZ8002 byelectroporation, with subsequent transfer to S. coelicolor by conjugation (37). Exconjugants with doublecrossovers were selected for Kans and Aprar. The authenticity of the S. coelicolor mutant strain wasconfirmed by PCR (data not shown).

Complementation of gene disruptions. The sequences of the oligonucleotides used are shown inTable S2. For complementation studies involving the large SCO2148 to SCO2156 gene disruption in strainCOE192, we constructed plasmid pMS2148-56. The 9,804-bp DNA fragment corresponding to the codingregion from SCO2148 to SCO2156 (including 200 bp upstream and 200 bp downstream as flankingsequences) 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 wereamplified 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 seriesbetween the HindIII and NsiI restriction sites of plasmid pMS82 (43) using the NEBuilder kit, exactly asdescribed by the manufacturer (New England BioLabs). For construction of pMS3945-46 for the complemen-tation of the gene disruption in strain COE190 (ΔSCO3945 to SCO3946), the corresponding DNA fragment was

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amplified using oligonucleotides SCO3945_HindIII_fw and SCO3946_KpnI-rv and was cloned between theHindIII and KpnI restriction sites of plasmid pMS82 to deliver pMS3945-46.

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

Measurement and calculation of oxygen respiration. A 1-ml aliquot of a 20-h, aerobically grownmycelium in shaking cell culture plates (representing 700 to 1,000 CAE; reference28) was transferreddirectly from flushed cultures (described above) to a vial containing 9 ml of air-saturated and bufferedhalf-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 opticaloxygen meter (FirestingO2; PyroScience, Aachen, Germany). Vials were stoppered (air tight) and stirredwith a magnetic bar at 1,200 rpm. The linear rate of oxygen consumption was analyzed using FirestingLogger Software (PyroScience, Aachen, Germany) over a period of 5 to 10 min. A control in which theprotein synthesis inhibitor chloramphenicol was added at 100 �g · ml�1 showed no difference in the rateof 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 wereperformed minimally three times and in triplicate.

Determination of cytochrome aa3 oxidase activity. Cytochrome aa3 oxidase enzyme activity wasdetermined spectrophotometrically by measuring the absorbance change at 550 nm, using membranefractions 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 mMTris-HCl buffer (pH 7) containing 20 �M of reduced cytochrome c. Enzyme activity was measured threetimes from two biological replicates. The assay was performed at room temperature (RT), and one mUrepresents 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 wereseparated by native polyacrylamide gel electrophoresis (PAGE). Typically, gels were prepared usingnondenaturing 10% (wt/vol) polyacrylamide. Cytochrome aa3 oxidase activity was visualized byincubating the gel in 10 ml of 50 mM potassium phosphate buffer (pH 7.2) containing 1 ml ofcytochrome c as the substrate (stock solution 10 mg · ml�1) and 0.5 ml of DAB reagent (diamino-benzidine stock solution of 10 mg · ml�1). For activity staining, gels were incubated at RT and forbetween 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 acidsequence, NPPTKIGGDLRDADK). Antibodies were affinity purified against the respective synthesizedpeptide. Aliquots (typically 25 to 60 �g of protein) from the crude extracts (typically 45 �g of protein)were separated by SDS-PAGE using 10% (wt/vol) polyacrylamide gels (46) and transferred to nitrocellu-lose membranes, as described previously (47). Affinity-purified antibodies were generally used at adilution of 1:8,000 (1:10,000 for anti-CydA) unless otherwise specified. Secondary antibody conjugated tohorseradish peroxidase was obtained from Bio-Rad. Visualization was done by enhanced chemilumines-cent reaction (Stratagene).

SUPPLEMENTAL MATERIAL

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

SUPPLEMENTAL FILE 1, PDF file, 6.7 MB.

ACKNOWLEDGMENTSWe 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, andClaudia Hammerschmidt for expert technical assistance. We are also grateful to RobertPoole 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 therespiration experiments along with M.F. R.G.S. conceived the study and wrote themanuscript.

We declare no conflict of interest.

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