the cioab genes from pseudomonas aeruginosa code for a novel cyanide-insensitive terminal oxidase...

The cioAB genes from Pseudomonas aeruginosa codefor a novel cyanide-insensitive terminal oxidase relatedto the cytochrome bd quinol oxidases

Louise Cunningham, † Melinda Pitt and Huw D.Williams *

Department of Biology, Imperial College of Science,Technology and Medicine, Prince Consort Road, London,SW7 2BB, UK.

Summary

The structural genes for the cyanide-insensitive term-inal oxidase (CIO) of Pseudomonas aeruginosa weresequenced. The locus comprised two open readingframes, cioA and cioB , coding for gene products of488 and 335 amino acid residues with predicted mole-cular masses of 54 241 and 37 016 Da respectively.These genes were encoded by a 2.7 kb transcript andprobably comprise an operon. Upstream of a majortranscriptional start site is a ¹10 promoter regionand, approximately at nucleotides ¹50 and þ13, thereare sequences homologous to the binding site of thetranscriptional regulator Anr. The deduced aminoacid sequences of CioA and CioB are homologousto the cytochrome bd quinol oxidases of Escherichiacoli and Azotobacter vinelandii . However, no cyto-chrome d -like signals were found in wild-type P. aeru-ginosa strains. An atypical cytochrome d -like signalwas seen under low-aeration growth conditions butonly in strains in which the cioAB genes were presenton a high-copy-number plasmid. The appearance ofthese cytochrome d -like signals was not paralleledby a concomitant increase in CIO activity. These datasupport the hypothesis that the CIO of P. aeruginosadoes not contain haem d. This raises the possibilitythat there is a family of bacterial quinol oxidasesrelated to the cytochrome bd of E. coli that can differin their haem composition from the E. coli paradigm.

Introduction

Current knowledge of the nature of prokaryotic andeukaryotic terminal oxidases indicates that they fall into

two categories. The large majority, including the mito-chondrial aa3-type cytochrome c oxidases, are membersof the haem–copper oxidase superfamily (Garcıa-Hors-man et al., 1994; Calhoun et al., 1994). This family is char-acterized by the homology of the major subunit, whichcontains an iron–copper bimetallic centre where the reduc-tion of molecular oxygen to water takes place. Mostmembers of the superfamily also contain homologues ofsubunits II and III of the mitochondrial enzyme (Garcıa-Horsman et al., 1994; Calhoun et al., 1994). They oxidizea quinol or a cytochrome c and the site of electron entryinto the oxidase is a second haem which in cytochromec oxidases is usually associated with two atoms of copper.

The only well-characterized prokaryotic oxidase that isclearly not a member of this superfamily is the cytochromebd quinol oxidase (Poole, 1988; Trumpower and Gennis,1994). The best studied cytochrome bd is from Escheri-chia coli. This oxidase contains three haem components:a low-spin haem b558, a high-spin haem b595 and a high-spin haem d. The three haems are present in the ratio1:1:1 (Miller and Gennis, 1983; Hata et al., 1985; Hata-Tanaka et al., 1987; Meinhardt et al., 1989). This oxidasedoes not contain copper and therefore it does not contain ahaem–copper bimetallic centre. The enzyme is a hetero-dimer of two integral membrane polypeptides, subunit I(CydA, 58 kDa) and subunit II (CydB, 43 kDa, Miller etal., 1988). Subunit I contains the haem b558 and is prob-ably the site of quinol oxidation (Lorence et al., 1987).Both haem b595 and haem d bind exogenous ligands.The site of oxygen reduction is thought to be a haem d –haem b595 binuclear centre analogous to the haem–CuB

bimetallic centre of the haem–copper oxidases (Poole etal., 1983; Rothery and Ingledew, 1989; D’Mello et al.,1996; Hill et al., 1993). Cytochrome bd has a very highaffinity for O2 (Km = 3–5 nM) and its expression increasesat low O2 tension (D’Mello et al., 1996; Rice and Hemp-fling, 1978; Cotter et al., 1990; Iuchi et al., 1990). Thiscytochrome is relatively insensitive to inhibition by theclassical cytochrome oxidase inhibitor KCN and it is res-ponsible for cyanide-insensitive respiration in E. coli (Riceand Hempfling, 1978; Pudek and Bragg, 1974). The aero-bic diazotroph Azotobacter vinelandii contains a spectrallysimilar cytochrome bd oxidase whose protein subunitsshow homology to E. coli CydA and CydB (Green et al.,1988; Moshiri et al., 1991a). However, the A. vinelandii

Molecular Microbiology (1997) 24(3), 579–591

Q 1997 Blackwell Science Ltd

Received 22 October, 1996; revised 17 February, 1997; accepted 21February, 1997. †Present address: Department of Molecular Biologyand Biotechnology, University of Sheffield, Western Bank, Sheffield,S10 2TN, UK. *For correspondence. E-mail [email protected];Tel. (0171) 5945383; Fax (0171) 5842056.

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cytochrome bd has a much lower O2 affinity than the E.coli cytochrome bd (Km = 4.5 mM; D’Mello et al., 1994),its expression increases at high O2 tensions and it has arole in respiratory protection of nitrogenase (Kelly et al.,1990). A survey of bacteria that can perform cyanide-insensitive respiration (defined as respiration in the pre-sence of 1 mM KCN) found that many bacteria containedspectrally detectable cytochrome bd (Akimenko and Trutko,1984). However, a significant number, including Pseudo-monas aeruginosa, did not.

Early studies indicated that the opportunistic pathogenP. aeruginosa had a branched aerobic respiratory chain,composed of b- and c-type cytochromes, containing atleast two terminal oxidases (Matsushita et al., 1980;1983). Cytochrome co is a cytochrome c oxidase thatmay be related to the cytochrome cbb3-type oxidase ofthe haem–copper superfamily (Matsushita et al., 1982;Thony-Meyer et al., 1994). The second is a cyanide-insen-sitive oxidase (CIO) of unknown molecular composition.Cyanide-insensitive respiration is of particular interest inP. aeruginosa. Under low oxygen tensions this bacteriumproduces HCN as a metabolic product at concentrationsthat would inhibit haem–copper oxidases. Therefore aCIO might have a role in allowing aerobic respirationunder cyanogenic growth conditions (Castric, 1983). Cya-nide has been detected in tissue samples infected with P.aeruginosa where it may be a potential virulence factor(Goldfarb and Margraf, 1967). We are interested in themolecular structure and function of the CIO of P. aerugi-nosa. We have previously isolated and characterized amutant defective in the CIO and cloned the complementinggenes. The mutant did not show any clear change in thecomposition of its membrane-bound cytochromes, lendingsome support to the earlier suggestion that the CIO mightbe a non-haem oxidase (Matsushita et al., 1983; Cunning-ham and Williams, 1995).

In this study we report the sequence analysis of thestructural genes for the CIO of P. aeruginosa and provideevidence for the CIO being a novel enzyme related to thecytochrome bd quinol oxidases.

Results

Nucleotide sequence of the cioAB genes ofP. aeruginosa

We determined the nucleotide sequence of the genesencoding the CIO of P. aeruginosa. The nucleotide sequ-ence data will appear in the EMBL, GenBank and DDBJNucleotide Sequence Databases under the Accessionno. Y10528. The recombinant plasmid pLC2, containinga 5.2 kb HindIII fragment cloned into the broad-host-range vector pUCP18, is able to complement the P. aeru-ginosa cio mutant PAO7701 (Cunningham and Williams,

1995). Nucleotide sequencing of 3 kb of this fragmentrevealed two long open reading frames (ORFs) of1467 bp (OrfA) and 1008 bp (OrfB), separated by 3 bp.We wished to confirm that these two ORFs were sufficientto complement the cio mutation in PAO7701. Therefore,DNA containing these two ORFs was amplified by poly-merase chain reaction (PCR) and cloned into the vectorpGEM-T to give the plasmid pT3. An NdeI site locateddownstream of OrfB and a unique NdeI site contained inthe multiple-cloning site of pT3 allowed a 2.7 kb NdeI frag-ment, containing both ORFs, to be cloned into the uniqueNdeI site of pUCP18. The resulting plasmid pLC8 wastransformed into PAO7701. It was able to fully comple-ment the mutant for growth on Luria–Bertani (LB) mediumcontaining 1 mM sodium azide and it restored the CIOactivity of cytoplasmic membrane preparations (data notshown; Cunningham and Williams, 1995). Upstream ofthe ATG start codon of OrfA there is a putative ribo-some-binding site (see Fig. 3C later). Downstream of theTGA stop codon following OrfB no inverted repeat couldbe found. The GþC content of the two ORFs is 65%,similar to the P. aeruginosa average of 67% (Rothmel etal., 1991). However, it was noticeable that the regionupstream from OrfA was more AT rich (43%) than thetwo ORFs and the expected value for P. aeruginosa.The codon usage of the two ORFs (not shown) was char-acteristic of that expected for P. aeruginosa (West andIglewski, 1988). The two ORFs will henceforth be referredto as cioA and cioB.

The cio locus encodes the subunits of a terminaloxidase homologous to the cytochrome bd quinoloxidases

cioA encodes a hydrophobic protein of 488 amino acidresidues with a predicted molecular mass of 54 241 Da.cioB encodes a protein, which also has the characteristicsof a membrane protein, containing 335 amino acids witha predicted molecular mass of 37 016 Da. Homologysearches of the cioA and cioB gene products indicatedthat they were homologous to the products of the E. colicydA and cydB genes, respectively (27.5% and 24.4%positional identity respectively), which encode the twosubunits of the cytochrome bd quinol oxidase of E. coli.Taking into account conservative amino acid substitutions,the similarities rise to 62.5% for CioA versus CydA, and67% for CioB versus CydB. CioA and CioB are also homo-logous to the products of the cydAB genes of the diazo-trophic bacterium A. vinelandii and to the appCB genesof E. coli which also encode for cytochrome bd quinol oxi-dases (Moshiri et al., 1991a; Dassa et al., 1991; Sturr etal., 1996). CioA and CioB show 29.5% and 28.3% identityto A. vinelandii CydA and CydB respectively, and 31.1%and 26.2% identity to AppC and AppB, respectively, in

Q 1997 Blackwell Science Ltd, Molecular Microbiology, 24, 579–591

580 L. Cunningham, M. Pitt and H. D. Williams


Fig. 1. The alignment of the amino acid sequences of P. aeruginosa CioA and CioB with corresponding sequences from E. coli and A.vinelandii.A. P. aeruginosa CioA (PSCIOA.PRO) compared with E. coli CydA (ECCYDA.PRO) and AppC (ECAPPC.PRO) and A. vinelandii CydA(AVCYDA.PRO).B. P. aeruginosa CioB (PSCIOB.PRO) compared with E. coli CydB (ECCYDB.PRO) and AppB (ECAPPB.PRO) and A. vinelandii CydB(AVCYDB.PRO).Identical residues are shaded in black while boxed residues all match the residue group in the P. aeruginosa sequence. Residue groupingsare: (DE), (HKR), (AFILMPVW), (CGNQSTY). Gaps are indicated by -. The proposed haem-binding ligands in CydA, His-19, His-186 andMet-393 are marked with solid circles. The 11 amino acid sequence that forms the epitope to the monoclonal antibody A14–5 against theE. coli CydA is boxed in all four sequences.

Pseudomonas aeruginosa terminal oxidase 581

pairwise alignments. The multiple alignment of CioA andCioB with these proteins is shown in Fig. 1. Further evi-dence for the overall similarity of CioA and CioB to thecytochrome bd quinol oxidase is provided by comparisonof the hydropathy profiles (Fig. 2). Figure 2A comparesthe hydropathy plots of CioA and E. coli CydA and thecomparison shows a strong similarity between the distri-bution of the various hydrophobic and hydrophilic domains.This suggests that the membrane topology of CioA isvery similar to the model proposed for CydA, based onimmunological and gene-fusion data, in which there areseven transmembrane helices (Georgiou et al., 1988;Newton et al., 1991). The hydropathy plots emphasizethat CioA is shorter than CydA, for two main reasons.First, CioA has a shorter hydrophilic C-terminus thanCydA. Second, the large periplasmic loop in E. coli CydA(amino acids 238–392 in Fig. 1A) is more than 60 aminoacids shorter in CioA, mainly as a result of fewer aminoacids in the C-terminal end of the loop. The presence ofthis periplasmic loop has been experimentally verified inCydA and shown to be the probable site of quinol binding(Dueweke and Gennis, 1990). However, the portion of theloop that is present in CioA does contain the proposed qui-nol-binding site of CydA (Newton et al., 1991; Duewekeand Gennis, 1990; 1991). Gennis and co-workers havecharacterized two different monoclonal antibodies thatbind to cytochrome bd and specifically inhibit the quinoloxidase activity of the enzyme (Dueweke and Gennis,1991). The epitope for these antibodies has been mappedto an 11 amino acid sequence in a periplasmic loop ofCydA (amino acids 252–262 in E. coli CydA; Fig. 1A). Ithas been noted previously that the epitope is perfectlyconserved between E. coli and A. vinelandii CydA sequ-ences (Moshiri et al., 1991a), and while there is not perfectconservation with CioA, five out of 11 amino acids areidentical and a further three are conservatively substituted(Fig. 1A). The hydrophobicity plots of CioB and E. coliCydB (Fig. 2B) are also very similar and hydrophobicregions corresponding to the eight proposed transmem-brane segments of CydB are present in CioB (Georgiouet al., 1988; Newton et al., 1991). The plot also empha-sizes that CioB is shorter than CydB, and this is mainlydue to shorter hydrophilic regions between the proposedtransmembrane segments in CioB.

Spectroscopic studies of E. coli cytochrome bd suggestthat cytochrome b558 is a low-spin haem while cytochromeb595 and cytochrome d are high-spin haems (Miller andGennis, 1983; Hata et al., 1985; Hata-Tanaka et al.,1987; Meinhardt et al., 1989). Therefore, the protein isexpected to provide four axial ligands; two to the low-spin haem b558 and one each to the high-spin haemsb595 and d. Three out of the four expected ligands havebeen identified with a reasonable degree of certainty bya combination of site-directed mutagenesis and a variety


Fig. 2. Comparative hydropathy plots of the deduced amino acidsequences of the P. aeruginosa cioA and cioB genes with E. colicydA and cydB. The plots were generated using the programPROTEAN from the DNASTAR Lasergene software package whichuses the Kyte and Doolittle algorithm (Kyte and Doolittle, 1982)with a window size of 19 amino acids.A. Hydropathy plots of CioA and CydA.B. Hydropathy plots of CioB and CydB.The proposed transmembrane helices in CydA and CydB based onhydrophobicity analyses and immunological and gene fusion dataare numbered together with the corresponding regions in CioA andCioB. The horizontal bar indicates the periplasmic Q-loop of CydA,which contains the proposed quinol-binding site, and thecorresponding shorter region in CioA.


of spectroscopic methods (Kaysser et al., 1995; Fang etal., 1989; Jiang et al., 1993; Spinner et al., 1995). Thesestudies have identified His-186 and Met-393 of CydA asthe ligands to haem b558. His-186 (which corresponds toHis-196 in CioA) is conserved in all four CydA homologuesshown in Fig. 1A and there is a high degree of similarity inthe surrounding residues; however, this is stronger in E.coli CydA, A. vinelandii CydA and AppC than in CioA.Met-393 is also conserved in all four CydA homologues(Met-329 in CioA) and there is again a high degree ofsequence similarity in the surrounding residues. There-fore, the conservation of the axial ligands and the quinol-binding site suggests that CioAB is a quinol oxidase andsupports previous data (Ray and Williams, 1996). Site-directed mutagenesis of E. coli CydA has also indicatedthat His-19 is essential for oxidase function and it hasbeen proposed as a ligand for haem b595 or haem d (Fanget al., 1989). Recent spectroscopic data lend further sup-port to a histidine being a ligand to b595 (Tsubaki et al.,1993). His-19 is also conserved in all four sequences(His-21 in CioA; Fig. 1A). A number of other amino acidshave been proposed as potential ligands to haem d,including tyrosine residues as is the case in some cata-lases, methionine as in c-type cytochromes, and cysteinewhich is found in cytochrome P450, chloroperoxidase andNO synthases. C214 in CydB has been suggested as apotential haem ligand but it is not conserved in CioB(Hirota et al., 1995). Two of the four histidines present inE. coli CydB are conserved in A. vinelandii CydB but notin CioB or AppB (Fig. 1B). The consensus of recent spec-tral data from electron paramagnetic resonance (EPR),electron nuclear double resonance (ENDOR), Fourier-transform infra-red spectroscopy (FT-IR) and resonanceRaman studies of cytochrome bd is that the proximalligand is not cysteine or methionine, and that if it is a histi-dine or strong nitrogenous ligand then the residue is in anunusual environment (Tsubaki et al., 1993; Hirota et al.,1995).

Analysis of the cioAB genes transcript

Northern blot analysis of total RNA from aerobically grownP. aeruginosa PAO6049 identified a single transcript ofapprox. 2.7 kb when probed with the cioAB gene insertfrom pLC8 (Fig. 3A). This is consistent with cioA andcioB being co-transcribed and forming an operon.

The 58 end of the cioAB gene transcript was determinedfrom RNA extracted from aerobic, exponentially grown P.aeruginosa PAO6049. S1 nuclease mapping showed twoseparate transcription initiation sites. The major site corre-sponded to an A residue 210 bp upstream of the transla-tional initiation codon of cioAB and the second, muchweaker, initiation site was at a C residue at þ213 bp(Fig. 3, B and C). Examination of the sequence at ¹10

relative to the major initiation site indicated a possible pro-moter sequence (TAAGA; Fig. 3C). The region at approx.þ7 to þ20 has a sequence TTGCTct.gcATCAA (Anr Box2 in Fig. 3C), which differs by only 1 bp from the consensusbinding site of the E. coli transcriptional regulator Fnr.Therefore, this could be a binding site for Anr or Dnr,which are P. aeruginosa homologues of Fnr (Sawers,1991; Zimmerman et al., 1991; Arai et al., 1995). A secondputative Anr/Dnr-binding site is found around nucleotides¹44 to ¹57 (Anr Box 1; Fig. 3C). We have preliminarybiochemical evidence, based on CIO activity measure-ments, that Anr may act as a negative regulator of theCIO (A. Ray, I. S. Viney and H. D. Williams, unpublishedresults). It is known that when Fnr acts as a negative regu-lator, e.g. in the case of the E. coli ndh operon, at least twoand sometimes three binding sites are present (Green andGuest, 1994). Low-resolution S1 nuclease mapping showedthe 38 end of the DNA transcript to be approximately 58 bpdownstream of the stop codon (data not shown). However,examination of the sequence in this area showed no regionthat could form a stem-loop structure normally associatedwith the termination of transcription.

The molecular nature of the P. aeruginosa CIO:Does it contain haem d?

Despite early reports that P. aeruginosa might possess ahaem a-containing cytochrome oxidase (Azoulay andCouchound-Beaumont, 1965; Calcott et al., 1975) andthe recent purification of a cytochrome baa3 from oneparticular strain (Fujiwara et al., 1992) Matsushita andco-workers, in detailed studies of the aerobic respiratorychain of this bacterium, found only b- and c-type cyto-chromes under aerobic growth conditions (Matsushita etal., 1980; 1982; 1983; Yang, 1986). The distinctive absorp-tion bands of a cytochrome bd oxidase have never beenobserved in P. aeruginosa and consequently this bacter-ium has not previously been considered to have a cyto-chrome bd-type quinol oxidase. In an earlier study, wefound no differences in the cytochrome composition ofwild-type and cio mutant strains and no evidence of cyto-chrome bd (Cunningham and Williams, 1995). Therefore,we re-investigated the cytochrome composition of P. aeru-ginosa strains using more-carefully defined growth con-ditions particularly in respect of oxygen levels. Thecytochrome bd of E. coli is induced by low oxygen levelswhile the A. vinelandii oxidase is induced at high oxygenlevels under nitrogen-fixing conditions (Cotter et al., 1990;D’Mello et al., 1996; Moshiri et al., 1991b). A variety ofP. aeruginosa strains were grown to mid-exponentialphase at high aeration (dissolved-oxygen tension (DOT)at the time of harvest was approx. 20%) and low aeration(DOT at the time of harvest ¼ 0%). The reduced-minus-oxidized and carbon-monoxide difference spectra of



membranes from the strains were examined, together withspectra of cytochrome bd-containing membranes of E. coli(Fig. 4). The respiratory activities of the strains are shownin Table 1 and the quantification of the cytochrome levelsin Table 2. The E. coli membranes (Fig. 4, A and B) had thecharacteristic signals of cytochrome bd in reduced-minus-oxidized spectra; a weak maximum at 595 nm, a prominentmaximum at 628 nm due to reduced cytochrome d and atrough at 650 nm due to the oxygenated form (Miller andGennis, 1983; Poole et al., 1983). The CO-differencespectrum of E. coli had a characteristic maximum at644 nm from the d 2þ-CO species and a minimum at621 nm (Fig. 4B). These signals were clearly absent fromthe membranes of P. aeruginosa cells grown under highand low aeration (Fig. 4, C, D, E and F). Although growingP. aeruginosa with low aeration led to an 11-fold increasein CIO activity (Table 1) and a more than twofold increasein the levels of b- and c-type cytochromes (Table 2)there were no detectable cytochrome d signals in these

membranes (Fig. 4, E and F). However, membranes fromPAO6049/pLC2 (high aeration) had weak signals thatcould be due to a cytochrome d as there was clearly aweak but distinctive absorbance at approx. 623 nm (Fig.4G). This signal was present even though the CIO activityof this strain was only 67% of that in PAO6049 (low aera-tion) membranes (Table 1). This suggests that the appear-ance of a cytochrome d-like signal may be due toincreased levels of the CioA and CioB polypeptides result-ing from expression from the multicopy plasmid pLC2. Thisconclusion is reinforced by examination of the spectrafrom membranes of PAO6049/pLC2 (low aeration) whichhave prominent cytochrome d-like signals (Fig. 4I). Quan-tification of the relative amounts of cytochrome d in thisstrain indicated a >11-fold increase concomitant with justa fourfold increase in the CIO activity over the levels inmembranes from the same strain grown with high aeration(Tables 1 and 2). Membrane preparations that hadcytochrome d-like signals in reduced-minus-oxidized


Fig. 3. Transcriptional analysis of the cioAB genes.A. Northern blot of RNA extracted from P. aeruginosa PAO6049and probed with an NdeI fragment from pLC8 carrying the whole ofthe cioA and cioB genes.B. High resolution S1 nuclease mapping of the 58 end of cioABmRNA.C. The nucleotide sequence of the proposed promoter region ofcioAB. A potential ribosome-binding site (RBS) and putativepromoter and Anr-binding sites are indicated. The small solidrectangles indicate the sites of transcription initiation, the larger ofthe two being the major site. M indicates the proposed initiationcodon of cioA.


difference spectra also had haem d-like signals in CO-difference spectra and this was most clearly seen forPAO6049/pLC2 (low aeration, Fig. 4J). The E. coli d 2þ–CO complex has a maximum at 644 nm and a minimumat 621 nm (Fig. 4B). In contrast, in P. aeruginosa mem-branes the maximum was at 635 nm and the minimum at594 nm (Fig. 4J). This suggests that the haem or thehaem environment is different in this P. aeruginosahaem protein compared to that of E. coli cytochrome bd.It is also noticeable that the cytochrome d-like maximumin the reduced-minus-oxidized spectrum was at approx.623 nm in PAO6049/pLC2 (low aeration) membranes(Fig. 4I), which represents a small but reproducible shiftfrom the 628 nm maximum of E. coli cytochrome bd (Fig.4A). Similar spectra were seen when NADH was used asthe reductant (data not shown). We also recorded spectraof membranes from PAO7720, a mutant of P. aeruginosathat does not make c-type cytochromes and in which allthe available evidence points towards the CIO being thesole oxidase (Ray and Williams, 1996). PAO7720 (high

aeration) had similar CIO activity to PAO6049/pLC2(high aeration) but no clear cytochrome d-like spectral sig-nals (Table 1; Fig. 4, K and L). When more-sensitive spec-tral measurements were made it was possible to see a hintof signals at 623 nm and 645 nm, but the signals were notas prominent as the weak signals in membranes ofPAO6049/pLC2 (high aeration; Fig. 4, G and H).

In summary, there is no evidence for the classical cyto-chrome bd oxidase spectral signals in wild-type P. aerugi-nosa PAO6049.

Discussion

We have previously described the isolation and character-ization of mutants defective in the CIO of P. aeruginosaand the cloning of the complementing genes. We foundthat there was no net loss of any spectral signals followingmutation and there were certainly no signals to suggestthe presence of a cytochrome bd oxidase in P. aeruginosa(Cunningham and Williams, 1995). Therefore, it was intri-guing in this current study to find that the cio locus encodestwo polypeptides which are homologous to the subunits ofthe cytochrome bd quinol oxidases of E. coli and A. vine-landii. In E. coli the induction of cytochrome bd occurs atlow O2 levels that are consistent with its high-oxygen affi-nity. In contrast, A. vinelandii induces cytochrome bd athigh oxygen levels consistent with its lower oxygen affinityand a role in protective respiration under N2-fixing condi-tions (D’Mello et al., 1994; 1996; Moshiri et al., 1991b;Kelly et al., 1990). It is clear from our data that membranesof wild-type P. aeruginosa do not contain a spectrallydetectable cytochrome d signal under high- or low-aera-tion conditions. Cytochrome d-like signals were only seenwhen the cioAB genes were present in multiple copieson the plasmid pLC2 and, in very much lower amounts,when the CIO was present as the sole oxidase in P. aeru-ginosa PAO7720. These signals were atypical in theirabsorbtion maxima indicating that the haem or haem


Table 1. Respiratory activities of membranes from various P. aeru-ginosa strains.

NADH-dependent CIO activityb % CIOStrain O2 uptakea (relative level) activityc

PAO 6049 12 3.4 (1) 28high aeration

PAO6049 61 39 (11.5) 64low aeration

PAO6049/pLC2 28 26 (7.6) 93high aeration

PAO6049/pLC2 112 104 (30.6) 93low aeration

PAO7720 ccm 35 35 (10.3) 100high aeration

a. Units are nmol O2 min¹1 mg¹1 protein.b. NADH-dependent O2 uptake in the presence of 1 mM KCN, givenin nmol O2 min¹1 mg¹1 protein.c. CIO activity as a percentage of total NADH-dependent O2 uptake.

Table 2. Cytochrome levels in membranesfrom various P. aeruginosa strains. Strain Cytochromes c a Cytochromes b a Cytochrome d a

PAO6049 0.017 0.013 NDhigh aeration

PAO6049 0.049 0.033 NDlow aeration

PAO6049/pLC2 0.019 0.015 0.0005high aeration

PAO6049/pLC2 0.051 0.038 0.0057low aeration

PAO7720 ccm 0.006 0.019 6high aeration

a. Cytochrome levels given as DA (mg¹1 protein) and calculated from reduced-minus-oxidizeddifference spectra using the following wavelength pairs: cytochromes c, 545–551 nm; cyto-chromes b, 558–575 nm; cytochromes d, 625–645 nm.ND, no detectable signal; 6, spectral signal visible but too small to accurately quantify.


environment was different in the haem protein presentin PAO6049/pLC2 membranes to that in E. coli cyto-chrome bd. As no haem d-like signal was present inwild-type P. aeruginosa we feel that the balance of theavailable evidence indicates that the CIO does not containhaem d.

P. aeruginosa does make haem d1, a haem d-likemolecule, under semi-anaerobic and anaerobic conditionsas a part of the periplasmic cytochrome cd1-type nitrite

reductase (Zannoni, 1989; Weeg-Aerssens et al., 1991;Kuronen and Ellfork et al., 1972). Haem d1 is structurallydistinct from haem d but when bound in the cytochromecd1 it does give rise to spectral signals at wavelengths> 610 nm (Timkovich et al., 1985; Yap-Bondoc et al.,1990). Therefore, an explanation for the presence of cyto-chrome d-like signals in PAO6049/pLC2 is that the CIO ispromiscuous with respect to its haem type and that a frac-tion of the overexpressed CioA and CioB polypeptides


Fig. 4. Difference spectra of membranes from various P. aeruginosa strains and E. coli. Spectra A, C, E, G, I and K are reduced (withdithionite)-minus-oxidized (with persulphate) difference spectra. Spectra B, D, F, H, J and L are reduced þCO-minus-reduced differencespectra. All samples were at a protein concentration of 10 mg ml¹1 except E. coli which was at 5.9 mg ml¹1.A and B. E. coli MG1655.C and D. PAO6049 high aeration.E and F. PAO6049 low aeration.G and H. PAO6049/pLC2 high aeration.I and J. PAO6049/pLC2 low aeration.K and L. PAO7720 high aeration.The vertical bar represents a DA of 0.08 in (A) and 0.04 in (B) and a DA of 0.27 in (C), (E), (G), (I) and (K) and a DA of 0.027 in (D), (F), (H),(J) and (L).


incorporate haem d1, leading to the appearance of theatypical haem d-like signals. It is possible that CIO doesnaturally contain haem d but that the oxidase is presentat such low levels as to render the haem d spectral signalsundetectable. However, this explanation is inconsistent withthe observation that some strains, such as PAO6049 (lowaeration) and PAO6049/pLC2 (high aeration), differ enor-mously in cytochrome d content but have similar CIO activ-ities. The low-spin haem site of cytochrome bo from E. coliis promiscuous with respect to haem type. The enzyme iso-lated from wild-type E. coli has predominantly haem b in thelow-spin site, whereas enzyme isolated from various over-expressing strains contains up to 70% haem o in the low-spin site (Puustinen et al., 1992).

If CIO does not contain haem d then what haem(s) doesit contain? The amino acid ligands to haem b558 of theE. coli cytochrome bd (His-186 and Met-393) are con-served in the CIO as is the proposed ligand to the high-spin haem b595 (His-19). Therefore CIO may contain ahaem b558 and b595. The spectral features of P. aeruginosasuggest that, if there is an analogue to the haem b595-haem d site of cytochrome bd, the second haem is mostlikely to be another high-spin haem b. We have carriedout some preliminary low-temperature photodissociationstudies of P. aeruginosa membranes (H. D. Williams andR. K. Poole, unpublished results). These provide evidencefor a photodissociable CO complex of high-spin b-typecytochromes but none due to haem c. Photodissociationof CO-liganded cytochromes in the presence of O2 didnot lead to the formation of the characteristic d650 speciesof E. coli cytochrome bd but provided evidence of O2 bind-ing to a high-spin haem b (H. D. Williams and R. K. Poole,unpublished results; Poole et al., 1983). This hypothesisneeds testing following the purification and biochemicalcharacterization of the P. aeruginosa CIO.

The E. coli appCB genes encode homologues to thesubunits I and II of cytochrome bd. It has recently beendemonstrated that the product of the appC and appBgenes is a cytochrome bd-type of quinol oxidase (Sturret al., 1996). The purified enzyme contains the classicalspectral properties of a cytochrome bd including maximadue to haem d. The authors suggested that the appCBgenes be renamed cbdAB and this oxidase be referredto as cytochrome bd-II and the product of the cydABgenes now be called cytochrome bd-I. The spectral prop-erties of the P. aeruginosa CIO are clearly different fromcytochrome bd-II which is spectrally indistinguishablefrom cytochrome bd-I and has a clearly identifiablehaem d component.

The broader significance of this study lies in the possibi-lity that there is a family of cytochrome bd-like quinol oxi-dases, perhaps a haem–haem oxidase family as opposedto the haem–copper oxidases. They would have in com-mon protein sequence homology to cytochrome bd-I of

E. coli and a haem–haem binuclear centre, but differ intheir biochemical and regulatory properties (A. vinelandiicytochrome bd, E. coli cytochrome bd-II) or haem compo-sition (CioAB) from the E. coli paradigm. Interestingly, ahomologue of E. coli cydA has been found in Campylobac-ter jejuni, a bacterium thought to have only b- and c-typecytochromes and for which there are no reports of a cyto-chrome bd-type oxidase (J. M. Cox, J. M. Ketley and P. H.Williams, personal communication). Therefore, it is quiteprobable that cytochrome bd-like quinol oxidases are pre-sent in a much wider range of bacteria than had previouslybeen thought but that they have remained undetectedbecause of the absence of the haem d spectral signals.

Experimental procedures

Bacterial strains, plasmids and growth conditions

The strains of P. aeruginosa and E. coli and the plasmids thatwere used in this study are listed in Table 3. P. aeruginosastrains were grown aerobically at 308C (500 ml of medium in2 l conical flasks, shaken at 250 rpm) in LB broth or in succinateminimal medium (SMM; 5% (w/v) sodium succinate, 1%(NH4)2SO4, 1% KH2PO4, 0.5% yeast extract, FeSO4.7H2O,0.03% MgSO4 and 1 mM methionine). Media were solidifiedwith 1.5% agar and, when required, antibiotics were addedat the following concentrations (mg ml¹1): carbenicillin, 500;tetracycline, 300. Larger-scale growths were carried out in10 l of SMM medium in a Magnaferm fermenter (New Bruns-wick Scientific Inc.) or in 2 l of SMM medium in a Bioflow II


Table 3. Strains and plasmids used in this work.

Strain/Plasmid Relevant characteristics Source/Reference

Strain

E. coliXL1-Blue supE44 hsdR17 recA1

gyrA46 thi relA1 lac ¹ F8[proAB þ lacI q lacZ–M15Tn10 (TetR)]

MG1655 prototroph

P. aeruginosaPAO6049 met-9011 amiE200 strA D. HaasPAO7701 PAO6049 cio Cunningham and

Williams (1995)PAO7703 PAO7701 (pLC2), Cioþ Cunningham and

Williams (1995)PAO7705 PAO7701 (pLC8), Cioþ This workPAO7720 PAO6049 ccm Ray and Williams

(1996)

Plasmid

pUCP18 CbR/ApR Schweizer (1991)pLC2 pUCP18 derivative with

5.2 kb HindIII fragmentcontaining cioAB

Cunningham andWilliams (1995)

pLC8 pUCP18 derivative with2.7 kb NdeI fragmentcontaining cioAB

This work


fermenter. A 2% inoculum of an overnight culture grownaerobically in SMM was used to inoculate the fermenter. Forhigh-aeration growths the culture was sparged with air at aflow rate of 1 l air l¹1 medium min¹1 and stirred at approx.500 rpm. The culture was harvested in late-exponentialphase after approx. 7–8 h of growth, at which time the DOTwas no lower than 20% and had been at this level or higherthroughout the growth. For low-aeration growths the air-flowrate was 0.1 l air l¹1 medium min¹1 and the culture was stirredat approx. 100 rpm. Within 3 h of inoculation the DOT was zeroand remained so until the culture was harvested in late-exponential phase. Cultures were harvested by centrifugationat 10 000 ×g, washed in 50 mM potassium phosphate buffer(pH 7.0) and stored at –20oC until needed. E. coli strainswere grown in LB medium at 378C and antibiotics wereadded as required at the following concentrations (mg ml¹1):tetracycline, 25; ampicillin,100.

Plasmid pLC8 was constructed using PCR to amplify 2.7 kbof DNA carrying the cioAB genes from pLC2. The primers58-GAACAACCGACCACAG-38 and 58-GTCCTGTGCTGA-TCTTTT-38 were used in buffer containing: 30 mM Tricine,pH 8.4, 20 mM MgCl2, 50 mM b-mercaptoethanol, 0.1% gela-tin, 0.5% Tween 200, 200 mM dNTPs, 0.1 pmol primers and0.5 U Taq polymerase. The conditions for PCR were 10 cyclesof 958C for 1 min, 508C for 1 min, 728C for 5 min followed by10 min at 728C (Ponce and Micol, 1992). The amplified frag-ment was cloned into a pGEM-T vector (Promega Inc.) fol-lowed by NdeI digestion and ligation of the 2.7 kb NdeIfragment into the unique NdeI site of pUCP18 (Schweizer,1991) to generate pLC8.

Preparation of membranes

Membranes were prepared from cells as described previously(Cunningham and Williams, 1995).

DNA manipulations and sequencing

Cloning, restriction digests and plasmid isolations were per-formed according to standard protocols (Sambrook et al.,1989). Plasmids were electroporated into E. coli or P. aerugi-nosa using standard methods (Farinha and Kropinski, 1990).

Restriction fragments of pLC2 were cloned into M13mp18and M13mp19 to allow single-stranded DNA production forsequencing. DNA sequencing was carried out using the Taqtrack kit (Promega Inc.) following the manufacturer’s proto-cols using [a-35S]-dATP and either the universal M13 sequen-cing primer or custom-synthesized internal sequencingprimers (Oswel DNA Services). The nucleotide sequencedata were compiled and analysed using the Lasergene pro-gram suite (DNASTAR Inc.). The nucleotide sequenceswere compared to the prokaryotic subset of the GenBankdatabase using the BLAST algorithm from the GCG package.

S1 nuclease and Northern blot analysis of RNA

Total RNA was isolated from P. aeruginosa PAO6049 grownto an OD600 of 1.0 in LB broth using the hot-phenol method(Aiba et al., 1981). For Northern hybridization analysis20 mg of RNA was separated in a 1.5% agarose gel containing

formaldehyde. Transfer of the RNA to a nitrocellulose mem-brane, prehybridization and hybridization were performedexactly as described by the manufacturer (Amersham). TheNdeI fragment from pLC8 carrying the cioAB genes wasradioactivity labelled with [a-32P]-dATP using the Ready toGo labelling kit (Pharmacia Biotech). S1 nuclease mappingof the 58 and 38 ends of cioAB transcripts was performedaccording to Aiba et al. (1981). Single-stranded DNA probeswere produced by primer extension. Primers, 58-AAATCTTC-GACCAGAAA-38 for the 58 end of cioAB and 58-GTCC-TGTGCTGATCTTTT-38 for the 38 end were used to primesynthesis of labelled probe from appropriate single-strandedDNA templates (Sambrook et al., 1989). Digestion of syn-thetic products using an appropriate restriction enzyme pro-duced a probe of uniform size. The probes were isolatedfollowing electrophoresis through a denaturing polyacryla-mide gel (Sambrook et al., 1989) and used in S1 nucleasemapping exactly as described by Aiba et al. (1981) and theprotected products were analysed on a 5% sequencing geltogether with a sequencing reaction generated from thesame primer on a single-stranded template using the Taqtrack sequencing kit (Promega Inc.).

Biochemical analyses

Measurements of NADH-dependent O2-uptake rates andCIO activities were carried out as described previously (Cun-ningham and Williams, 1995; Ray and Williams, 1996). Allenzyme assay data represent the mean values from threeexperiments and there was less than 10% variation betweenexperiments. The cytochrome content of membranes wasanalysed using washed membranes resuspended in potas-sium phosphate buffer (pH 7.0) to a protein concentration of10 mg ml¹1. Room-temperature-difference spectra (reduced-minus-oxidized and reduced-plus carbon monoxide-minus-reduced) were recorded essentially as previously described(Jones and Poole, 1985; Williams and Poole, 1987). Sampleswere reduced either with a few grains (<5 mg) of sodiumdithionite or by adding NADH to a concentration of 1 mM,and oxidized in the presence of a small amount (<5 mg) ofammonium persulphate. Carbon monoxide was introducedby gently bubbling the sample with CO for 3 min. Spectrawere recorded using a Shimadzu MPS2000 double-beamspectrophotometer. Protein assays were carried out usingthe method of Markwell et al. (1978).

Acknowledgements

We would like to thank D. Haas for strain PAO6049 and H.Schweizer for plasmid pUCP18. This work was supportedby a grant from the Wellcome Trust to H.D.W.

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the cioab genes from pseudomonas aeruginosa code for a novel cyanide-insensitive terminal oxidase...

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