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The terminal oxidases of denitrificans Gier, Jan-Willem L. de; Lübben, Mathias; Reijnders, Willem N.M.; Tipker, Corinne A.; Slotboom, Dirk-Jan; Spanning, Rob J.M. van; Stouthamer, Adriaan H.; Oost, John van der Published in: Molecular Microbiology

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Publication date: 1994

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Citation for published version (APA): Gier, J-W. L. D., Lübben, M., Reijnders, W. N. M., Tipker, C. A., Slotboom, D-J., Spanning, R. J. M. V., Stouthamer, A. H., & Oost, J. V. D. (1994). The terminal oxidases of Paracoccus denitrificans. Molecular Microbiology, 13(2), 183-196.

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Download date: 02-10-2021 Molecular Microbiology (1994) 13(2), 183-196

The terminal oxidases of Paracoccus denitrificans

Jan-Willem L. de Gier,^ Mathias Lubben,^ possession of alternative respiratory branches enabies a Willem N. M. Reijnders,^ Corinne A. Tipker,^ bacterium to adapt its efficiency of oxidative phosphoryl- Dirk-Jan Slotboom,^'^ Rob J. M. van Spanning,^ ation to changes in the environmentai conditions. Adriaan H. Stouthamer^ and John van der Oost^* An extensiveiy studied prokaryotic cytochrome c oxidase ^Department of Molecular and Cellular Biology. is cytochrome aa3 from Paracoccus denitrificans and BioCentrum Amsterdam, Vrije Universiteit, De Boelelaan Rhodobacter sphaercides. The primary sequences of its 1087, 1081 HV Amsterdam. The Netherlands. three subunits show strong homology to the - ^European Molecular Biclcgy Laboratory. encoded eukaryotic cytochrome oxidase subunits i, ii and ill Meyerhofstrasse 1, 69018 Heidelberg, Germany. (Raitio etai. 1987; 1990; Steinrucke etai., 1987; Cao etai, 1991; Shapleigh and Gennis 1992; Saraste, 1990). Subunit I of cytochrome aa^ contains two haems A: one is in the low- Summary spin (6-co-ordlnate) configuration and the other is in the Three distinct types of terminal oxidases participate in high-spin (5-co-ordinate) configuration. The catalytic site the aerobic respiratory pathways of Paracoccus of reduction is formed by a binuciear centre of the denitrificans. Two alternative genes encoding subunit high-spin haem A and a copper ion, CUB {Wikstrbm et ai, i of the aaa-type cytochrome c oxidase have been iso- 1981). Subunit II harbours another copper site, CUA (van lated before, namely ctaDi and ctaDil. Each of these derOost e^a/,, 1992; Lappalainen efa/,, 1993; von Wachen- genes can be expressed separately to complement a feldt etai, 1994), which is probably involved in the oxidation double mutant (ActaDi, ActaDIl), indicating that they of the substrate cytochrome c (Hill, 1993). Like the mito- are isoforms of subunit i of the aas-type oxidase. The chondrial cytochrome c oxidase (Wikstrom, 1977; Krab genomic locus of a quinol oxidase has been isoiated: and Wikstrbm, 1978), the bacterial cytochrome aa^ is a cyoABC. This protohaem-containing oxidase, called redox-driven proton pump: the reduction of oxygen is cytochrome bb^, is the oniy quinoi oxidase expressed coupled to the translocation of protons across the under the conditions used, in a tripie oxidase mutant membrane (van Verseveld et ai, 1981; Soiioz et ai, 1981; (sctaDI, ActaDII, cyoS::Km") an aiternative cyto- Hosier efa/., 1993), chrome c oxidase has t>een characterized; this ebbs- The best-characterized prokaryotic quinol oxidase is type oxidase has been partiaiiy purified. Both cytochrome bcz from Escherichia ccli As the name sug- cytochrome ass and cytochrome b/>3 are redox-driven gests, a low-spin haem B and a high-spin haem 0 are proton pumps. The proton-pumping capacity of cyto- located in the major subunit, in addition to CUB (Minghetti chrome cbb^ has been analysed; arguments for and etai. 1992). Like the cytochrome coxidases, cytochrome against the active transport of protons by this novel bO3 functions as a proton pump (Puustinen et ai, 1989). oxidase compiex are discussed. Sequence analysis has revealed that cytochrome 603 is ciosely related to cytochrome aa^ (Saraste et ai. 1988; Chepuri et ai. 1990). However, in contrast to the aas- Introduction type cytochrome c oxidases the quinot-oxidizing cyto- In mitochondriai respiration the reduction of oxygen to chrome bo^ does not contain a CUA site (Puustinen et water is catalysed by cytochrome c oxidase. This ai, 1991; van der Oost et ai, 1992). An alternative type membrane-bound complex is the iast component of the of quinol oxidase from E, coli, cytochrome bd, is not respiratory chain, in which electrons from a reductant related to the haem-copper oxidases (Anraku and Gennis, (NADiH, succinate) are transferred in subsequent 1987; Green etai, 1988). redox reactions to oxygen (Babcock and Wikstrbm, Apart from the aa3-type cytochrome c oxidase (John and 1992). in aerobic respiration is often a more Whatley, 1975), P. denitrificans has been reported to compiex branched pathway. For many bacterial species express alternative terminal oxidases. Several ubiquinol more than one terminai oxidase has been described, cata- oxidases (cytochrome b^. Cox ef ai, 1978; cytochrome iysing the oxidation of either cytochrome c or quinol. The d, Henry and Vignais, 1979; cytochrome a^,, van Verse- veld ef ai, 1983; cytochrome ba3, Ludwig, 1992) and cytochrome c oxidases (cytochrome Q,, Bosma, 1989; Received 14 January, 1994; revised 1 April, 1994; accepted 5 April, 1994,'For correspondence, Tel. (20) 5485665; Fax (20) 6429202, cytochrome aa^ isoenzyme, Raitio et ai, 1990) have 184 J.-W. L deGieretal been either demonstrated or suggested. However, in Cloning and mutagenesis of a new oxidase locus contrast to cytochrome aaj the alternative oxidases from Degenerated oligonucleotides derived from conserved Paracoccus have not been described in detail. regions in subunit I of haem-copper oxidases have been In this paper a molecular genetic approach is used tc designed as described by M. Lubben etal. (submitted). A acquire more detailed information on the terminal oxi- polymerase chain reaction (PCR) has been perfomied on dases from Paracoccus. A new oxidase locus {cyoABC) genomic DNA from the cytochrome aa3 double mutant has been cloned and its product identified as a protohaem- {ActaDI, ActaDil), in conditions essentially as before (M. containing quinol oxidase. Single and multiple mutants Lubben et al., submitted). A PCR product of the expected have been generated for cytochrome aas, the putative size has been cloned into an Ml3 derivative and cytochrome aa^ isoenzyme, and the quinol oxidase. For sequenced. The predicted amino acid sequence turned the first time expression of the ctaDI gene has been out to be very similar to that of subunit I of cytochrome observed and the isc-cytcchrome aa3 has been character- bOa from E. coti (Chepuri et al., 1990). Using the PCR frag- ized. After mutagenesis of the cyoABC locus no alternative ment as a probe, a genomic Sph\ fragment of approx. 5 kb quinol oxidase activity is detectable. In a triple oxidase has been isolated (Fig. IB). mutant a c/>type cytochrome c oxidase has been analysed in more detail. Sequence analysis of the cyoABC locus

Results The genomic Sph\ fragment has been subcloned and sequenced. Independently, the same locus has been Mutagenesis of cytochrome aa3 and iso-cytochrome aa^ isolated by O. Preisig and B. Ludwig (personal communi- The genomic loci encoding the aas-type cytochrome c oxi- cation); their complete sequence has been submitted to dase (cfaCSGEand ctaDll), as well as the gene encoding the EMBL Data Library and they will publish their data else- a putative subunit I isoenzyme [otaDI] have been isolated where. Upstream of the subunit I gene (oyoB) a subunit II previously (Raitio ef ai, 1987; 1990; Steinrucke et ai, (cyoA) homologue was found, downstream of a cyoB 1987; van Spanning et al., 1990). In the course of this subunit Ill-like gene {cyoC) (Fig. IB), Screening of the study deletions have been generated in both subunits I data library revealed highest homology with E. coli [tiCtaDt, ActaDII) as shown in Fig. 1A, using a gene- cyo>lSCDE(Chepuri etat., 1990) and Baciltus subtitis qox- replacement method as described by van Spanning >ASCD(Santana etal., 1992), encoding the quinol oxidases et al. (1991). In this method the target gene is first cytochrome bo^ and aa3, respectively. inactivated by insertion of a kanamycin-resistance marker; Primary sequences have been analysed in order to the subsequent removal of this insertion cassette in a obtain information on the metal-binding sites. In subunit I second recombination event results in an unmarked of haem-copper oxidases six Invariant histidines are the deletion. established ligands for the redox centres, i.e. two haems

Fig. 1. Genomic oxidase loci of P. denitri- ficans. A. Subunit I genes of iso-cytochrome aaa (ctaDI) and cytochrome aa^ {ctaDliy, the latter mwmwm 500 bp , is iocated adjacent to the cycA gene, encod- i>rf4 CtaDI ing cytochrome C550. B. The cyoABC locus, encoding the b-type quinol oxidase (cytochrome btjj). Sites ot deletion and/or insertion mutagenesis are indicated.

cycA CtaDll B Terminal oxidase of Paracoccus 185 A subunit I helix II helix VI p.d. CtaDI .WTYHGILMMFFWIPALFG. . . .HILWFFGHPEVYIIILPGFG... P.d. CtaDll .MITYHGVLMMFFWIPALFG. . . .HILWFFGHPEVYIIILPGFG... B.s. CtaD .VMTMHGTTMIFLAAMPLLFA... .HLFWIFGHPEVYILILPAFG... B. j. FixN .LRPLHTSAVIFAFGGNVLIA... .MFQWWYGHNAVGFFLTAGFF... P.d. CyoB .IFTAHGVIMIFFVAMPFITG... .NLIWIWGHPEVYILILPLFG... E.c. CyoB .IFTAHGVIMIFFVAMPFVIG... .NLIWAWGHPEVYILILPVFG... S.a. SoxA .ALTIHGWAAMIAFVPMAAAA... .ILFWFYGHPWYYVPFPLFG. . . 102 121 277 296

helix VII helix X P.d. CtaDI .MAAIAFLGFIVWAHHMYTAG... .YHDTYYIVAHFHYVMSLGAV.., P.d. CtaDll .MAAIGILGFWWAHHMYTAG. . , .YHDTYYWAHFHYVMSLGAV. . . B.s. CtaD .AIVLGFLGFMVWVHHMFTTG.., .FHDTYFWAHFHYVIIGGW. . . B. j. FixN .FWALIFLVIWAGPHHLHYTA... .SHYTDWTIGHVHSGALGWVG... P.d. CyoB .TVCITVLSYLVWLHHFFTMG.. .LHNSLFLIAHFHNVIIGGVL... E.c. CyoB .TVCITVLSFIVWLHHFFTMG.. .LHNSLFLIAHFHNVIIGGW. . . S.a. SoxA .IYLLAIGTMGVWVHHLQTWP... . FHNSYYWGHFHLMIWTLII . . 320 339 410 429

B subunit II

P.d. CtaC . . .TATDVIHAWTIPAFAVKQDAVPGRIAQLWFSVD QEGVYFGQCSELCGINHAYMPIWKA . . . B.s. CtaC ...KASDVKHSFWIPSVGGKLDTNTDNENKFFLTFDSKRSKEAGDMFFGKCAELCGPSHALMDFKVKT... P.d. CyoA ...TSTSVMNAFYIPAMAGMIYAHPGMETKLNGVLN HPGKYKGIASHYSGHGFSGMHFKAHA... E.C. CyoA ...TSNSVMNSFFIPRLGSQIYAMAGMQTRLHLIAN EPGTYDGISASYSGPGFSGMKFKAIA... 166 225

Fig. 2. Alignment of metal-binding amino acid sequences of four putative membrane-spanning helices of subunit I (A), and the C-terminai peri- piasmic domain of subunit Ii (B). Ligands are in bold. P. denitnficans CtaDI, CtaDii, CtaC (Raitio et ai. 1987; 1990), B. subtilis CtaD, CtaC (Saraste etai. 1990), fi. yaponicum FixN (Preisig etai, 1993), P. denitrificans CyoB. CyoA (this study), E. co//CyoB, CyoA (Chepuri etat.. 1990), Sulfolobus acidocatdarius SoxB (Lubben et ai, 1992). Sequence numbering refers to E. coli proteins. and a copper ion, CUB (reviewed by Hosier et ai, 1993). The single, double, and triple oxidase mutants gener- Alignment of Paracoccus CyoB sequences with corre- ated in this study have been analysed spectroscopically sponding fragments from several haem-copper oxidases using whole cells, grown in batch culture on minimal clearly indicates similar metal-binding sites (Fig. 2A). A medium with succinate (Fig. 3). As expected, the fourth redox centre resides in subunit II of cytochrome c cytochrome aa^ double mutant [ActaOt, ActaDII) has a oxidase, i.e. a binuclear copper centre, CUA (van der Oost et ai, 1992; Lappalainen etai, 1993; von Wachen- feldt et ai, 1994). Five amino acids in subunit II (2 cys- teines, 2 histidines and 1 methionine) have been identi- fied as a copper ligand (Kelly ef ai, 1993). In quinol- oxidizing members of the haem-copper oxidase family this copper centre is absent {Puustinen et at., 1991; van der Oost et ai, 1992). In Paracoccus CyoA four of the five CUA ligands are substituted (Fig. 2B), indicating that the cyoABC genes code for a quinol oxidase.

Optical spectra, oxygen consumption Deletion of the Paracoccus ctaDt gene, proposed tc encode subunit I of cytochrome aa^ (Raitio et ai, 1987), 500 550 600 630 did not affect the cytochrome aa3 biosynthesis. This result wavelength (nm) has led to the isolation of a second gene, ctaDIt (Raitio et Fig. 3. Optical spectra of cells of Paracoccus. Cells were grown in ai, 1990). Deletion cf the latter gene (ActaDII) results in a batch cultures on minimal medium with succinate, and harvested complete loss of cytochrome aa3 absorption, with typical during late-exponential growth. The figure presents absolute spectra of dithionite-reduced cell suspensions: wild type (Pd1222), cyto- maxima at 445 nm and 605 nm (de Gier efa/., 1992; Raitio chrome bb3 mutant (Pd2621). cytochrome aa3 double mutant and Wikstrbm, 1994). (Pd9220), and the triple oxidase mutant (Pd9311). 186 J.-VJ. L deGiere\a\.

Table 1. Oxygen-consumption measurements on whole-cell spectrum, with characteristic maxima at 445 nm and suspensions of different P, denitrtftcans strains: Pd1222 (wild type), 605 nm (not shown). For the first time the spectrum of cyte- Pd9220 (aag double mutant), Pd262i (bb^ mutant), and Pd93ii triple mutant). chrome aa^ with subunit I expressed from cfaD/has been observed (Fig. 4); it is obvious that it cencems a cyto- Ascorbate/ chrome aa3 iseenzyme, as originally proposed by Raitio Substrate Endogenous Succinate TMPD era/. (1990). Strain Inhibitor - AA myx - AA myx - The quinol oxidase has been complemented using the Sph\ fragment shown in Fig. IB. In this case the effect Wild type 1.2 0,9 0,9 3,8 1,0 1-0 4,3 aa^ double 1,2 1,2 1,2 3,8 3,2 3.3 3,1 was most clearly demonstrated by analysis of the oxygen mutant consumption. As stated above, the respiration of the triple bth mutant 1,1 0 0 3,6 0 0 4,3 mutant was inhibited completely by antimycin A and myxo- aajjbb^ triple 1,2 0 0 3,7 0 0 3,4 mutant thiazol (Table 1), In the CyoABC-cemplemented clone, however, the direct oxidation ef ubiquinel (independent ef Values are the means of three independent assays (nmol cytechrome c oxidase) is restored (not shown). protein ' s ^), Inhibitors: antimycin A (AA), myxothiazol (myx). cytechrome aa3-minus phenetype. The spectrum of the Haem analysis cyoB mutant (cyoS::Km"), en the ether hand, did not differ Haems have been extracted frem wild-type and mutant significantly from that of the wild type. In the optical spec- cells and subsequently analysed by high-perfemnance trum of the triple mutant (AcfaD/, ActaDII, cyo6::Km") a liquid chromatography (HPLC) reversed-phase chroma- clear increase of cytechrome b and c is observed (Fig. 3), tegraphy (Table 2). In extracts ef the wild type and the The oxygen consumption of wild-type and mutant cells quinol oxidase mutant, haem B (protohaem) and haem A has been measured pelaregraphically, both without are present abundantly. In the cytochrome aa3 double supplemented substrate (endogenous) and after addition mutant and in the triple mutant the haem B quantity is of succinate (Table 1). In separate assays the electron not altered significantly; in contrast, haem A is just detect- flow via cytochrome c reductase has been inhibited by able in the absence ef cytochreme aa^ (the concentration addition ef either antimycin A or myxethiazol. The endo- decreased 10-15-fold). At least under the cultivation genous respiration of the wild type and the aa3 double cenditiens used, no haem O is detected in membranes mutant is only slightly affected by these inhibitors. The ef Paracocous. This implies that the cloned quinel inhibition becomes relatively mere preneunced when oxidase is a b-type cytochrome, and that the alternative succinate is used as substrate. In centrast, the oxygen cytochreme c exidase is a civtype cytechrome. consumption is blocked cempletely in the cyoB mutant and in the triple mutant. This is in agreement with the analysis of the primary structure, and implies that CyoABC encodes a quinel oxidase. In addition, the oxidation of the artificial electron-donating ceuple ascerbate and W, W, W, W-tetramethyl-p-phenylenediamine (TMPD) has been assayed; mere or less equal values indicate the presence of cytechreme cexidase(s) in all strains.

Mutant complementations All three oxidase mutants have been complemented using an expression vector and the triple exidase mutant as a hest. Although the lac premeter is theught to be inactive in Paracoccus, the orientation of the exidase genes in the expression censtructs is such that they are enceded by the cemplementary strand, indicating that transcription has been under the control of the native promoters. An 500 550 600 650 interesting observation was that all three complemen- wavelength (nm) tation constructs, but not the vector alone, gave rise to a Fig. 4. Optical spectra of cells of Paracoccus. The figure presents decline of the be peak (Fig, 4), absolute spectra of dithionite-reduced cell suspensions (as described in legend Fig, 3) of the triple oxidase mutant Pd9311, The plasmid-encoded expression of CtaDII was demen- and triple oxidase mutant after complementation with CtaDI: strated by the restoration ef the cytochreme aas optical Pd9311(pEG,ctaDI), Terrninai oxidase of Paracoccus 187

Table 2. Haem analysis by reversed-phase HPLC (Deltapak C18 with substrate, either ascorbate and TMPD or succinate; column. Waters) of different P. denitrificans strains: Pd1222 (wild a subsequent oxygen pulse induces the proton efflux, type), Pd9220 (ass double mutant), Pd262i (OO3 mutant), and which is measured as acidification of the medium (Fig. 6, Pd9311 (aa.Jbt}2 triple mutant). Table 3). Haem Ascorbate donates electrons via TMPD to cytochrome c (van Verseveld et ai., 1981) (Fig. 7), When ascorbate is Strain B 0 A oxidized to dehydro-ascorbate one 'scalar" proton is Wild type released per two electrons; when a proton-pumping aa3 double mutant cytochrome c oxidase is operative, a maximum of two /)b3 mutant triple mutant

Haems were extracted from isolated membranes and eluted on an Table 3. Proton-translocation measurements on whole-cell sus- acatonitrile gradient in 0.05% trifluoroacitic acid, as described by pensions of different P. denitrificans strains. H*/2e ratios are Sone and Fujiwara (1991). Relative quantities of extracted haem: avarages± standard deviation. ++, abundantly present; ±, just detectable; -, not detectable. succinate succinate ascorbate 0. Fe(CN)|- O2 Partiai purification of alternative cytochrome c oxidase Strain {H*^/2e ) (H*/2e ) (H72e )

From membranes of the triple mutant {ActaDI, ActaDII, Wild type(s) 2.40 ±0.11 cyoB::Km"), grown on minimal medium with succinate, aa^ double mutant(s) 1.21 ±0.16 the altemative cytochrome c oxidase has been extracted. bba mutant(s) 5.15±0.21 3.99 ±0.11 2.01 ±0.18 aa^bbz triple mutant(s) 4.15±0,14 4.05±0.10 l,12±0.10 Partial purification of the oxidase complex has been aa^lbbj thple mutant. 2.20 ±0.36 accomplished by column chromatography, and the result- CtaDi compl.(s) ing preparation oxidizes both TMPD and horse-heart cyto- aa^bb^ triple mutant. 2,12±0.10 cfaD//compl.(s) chrome c at high rates (M. Saraste and J. van der Oost, unpublished). The optical spectrum shows maxima at Wild type, aerobic/ 2.81 ±0.14 551 nm and 559 nm, indicating the presence of the cyto- -limited (m/ch) Wild type, aerobic/02- 1.21 ±0.35 chromes c and b (Fig, 5). limited (s/ch) Wild type, anaerobic/ 1.19±0.07 N03-limited (s/ch) Proton transiocation Cells were cultivated as aerobic batch cultures, in minimal medium The coupling of respiratory electron (e") transfer to with either succinate (s) or methanol (m); data are compared with analyses from chemostat-cultured cells (ch) of P. denitrificans NCIB the translocation of protons (H*) across the bacterial 8944 (wild type) with limitations as indicated, reported by van cytoplasmic membrane drives the oxidative phosphoryl- Verseveld et ai (1981; 1983). The electron donor was either succin- ation. The efficiency of the latter phenomenon in the differ- ate or ascortiate; anaerobic suspensions were pulsed wtth oxygen (O2) or ferncyanide (Fe(CN)| ). A control pulse with ferricyanide of ent mutants has been studied by determination of the H"/ the ascorbate mixture routinely resulted in HV2e" =1.1 ±0.1, both 2e" stoichiometry. A cell suspension is supplemented in the presence and in the absence of cells (not shown). The assay was as described in the legend to Fig, 5; for details see the Experi- mental procedures.

'vectorial' protons is translocated to the medium in addi- tion (Wikstrbm et al., 1981), In the wild type and the quinol oxidase mutant an HV2e ratio of 2.0-2.4 is measured upon ascorbate oxidation; in the aa3 double mutant and the triple oxidase mutant an HV2e" ratio of 1.1-1.2 (Table 3) is found. Apparently, vectorial proton transloca- tion is detected only in the presence of cytochrome aa3, which is an established proton pump (van Verseveld et aL, 1981); in its absence, however, only the scalar proton release is measured. This obsen/ation is confirmed by MO J« analysis of the triple oxidase mutant, in which cytochrome Wavelength (nm) aa^ is reintroduced by complementation with either CtaDII Fig. 5. Absolute absorbance spectrum of the altamative cyto- or CtaDI. The induced increase of the HV2e" ratios (2.1 - chrome c oxidase (cytochrome cbt>3) complex after reduction with 2.2) also indicates that both isoforms of cytochrome aa^ Na-dithionite; the oxidase has been extracted from membranes of the triple oxidase mutant Pd9311. are proton-pumping cytochrome c oxidases (Table 3). 188 J.-W. L deG/eretal. A. quinoi oxidase mutant B. iriple oxidase mutanl Fig, 6. Respiration-driven proton translocation in cells of Paracoccus. Anaerobic cell sus- 5.3 H+/2C- pensions were pulsed with oxygen (Oj) by injection of air-saturated KCI, Traces were calibrated by injecting an anaerobic solution of 1 mM HC!, The calculated H*/2e " ratio is shown for each oxygen pulse. 120 sec.

2,1

t t O2 HCI HC! ascorbaie/TMPD

Succinate-dehydrogenase catalyses the oxidation of suc- Discussion cinate and the reduction of ubiquinone. Subsequently, ubi- P. denitrificans contains a number of attemative respiratory quinol is oxidized either by a quino! oxidase {cytochrome pathways, branching at the level of ubiquinol. One route 6i>3; HV2e" =4.0), or by cytochrome c reductase (cyto- closely resembles the mitochondrial respiratory chain in chrome be,; H72e =4,0), When eiectrons fiow from the which ubiquinol is oxidized by the so-called supercomplex latter complex, via cytochrome c, to a proton pumping cyto- consisting of cytochrome c reductase (cytochrome be,), chrome c oxidase the energy-transducing efficiency cytochrome c^^ and cytochrome c oxidase (cytochnDme increases (cytochrome aa^', H72e =6.0). In the actuai aa^) (Berry and Trumpower, 1985). In a commonly experiments with succinate the same tendency is observed expressed altemative pathway ubiquinol is oxidized directty wrth cells of the aa^ double mutant and the triple oxidase by a quinoi oxidase called cytochrome bo by Cox et al. mutant: in the presence of cytochrome aa3 the HV2e (1978). stoichiometry is 5.0-5.3, but in the cytochrome aa^ When Paracoccus is cultivated under microaerobic or mutants it never exceeds an H*/2e" ratio of 4.0-4,3 anaerobic conditions, the expression level of cytochrome (Table 3). As an intemal control, the proton efflux associ- aa^ and, to some extent, that of cytochrome C552 appears ated with the succinate oxidation has been measured with significantly lower (Bosma et ai, 1967a). Instead of the ferricyanide as terminai electron acceptor.

Paracoccus respiratory pathways: sites of proton-translocation Fig. 7. Respiratory pathways in Paracoccus. Indicated here is the theoretical stoichiometry of the respiration-driven proton translocation (H*/2e ), detectable in the medium, for each ascorbate —> dchydm-ascorbatc of the contributing respiratory complexes: I +1 H+ NAOH-dehydrogenaSQ (NADH.dh), succinate nH+ 2H+ dehydrogenase (succdh), cytochrome c reductase (cyt,t>c,). cytochrome C552 iO, aa3-type cytochrome c oxidase (cyt,aa3), type cytochrome c oxidase {cyt,cb£i3) and the btb-type quinoi oxidase (cyt,W^), Ascorbate H (with TMPD) is an artificial reductant of cyto- chrome c. and ferricyanide (Fe(CN)|") an oxi- 0-2 H+ dant. The chemical conversion of ascorbate is NADH NADH.dh indicated (open arrow) to show generation of ubiquinol the "scalar* proton. For discussion, see text.

succdh Terminal oxidase of Paracoccus 189 aferementiened supercomplex, a quinel-exidizing complex Puustinen ef al., 1989); we propose te call it cytechreme has been isolated from such oxygen-limited cells in which bb3, according te the classical terminology ef the mito- cytechreme bc^ is associated with an alternative c£>-type chondrial cytechrome c oxidase in which the 02-binding cytechrome c exidase, called cytechreme Co by Bosma haem is denoted by the subscript 3 (Puustinen and Wik- (1989). strbm, 1991). An alternative quinel oxidase cemplex, cytochrome ba2, has been purified frem a Paracoccus cytechrome c reduc- Ubiquinot oxidase tase mutant (Ludwig, 1992), The genemic lecus of cyte- A new exidase locus {cyoABC) has been isolated from a chrome ba3 has been isolated (O, Richter and B, Ludwig, P. denitrificans genomic library. The predicted protein persenal communication), and comparison of these data sequences share a high degree ef hemelogy with subunits with the partial sequences as presented in Fig. 2 revealed I, II and III ef the quinel-exidizing cytechreme bo^ from E. that cytochreme ba^ and cytechrome bbj are encoded by CO//(Fig. 2, Chepuri etai, 1990). In subunit I (CyeB) the the same set ef genes {cyoABC). As far as we are six conserved histidines are present, Indicating that it con- aware, the cytochreme bc^ mutant is the only Paracoccus cems a haem-cepper exidase (Saraste, 1990; Hesler et strain in which this mixed haem exidase is detectable. ai. 1993), As in E. co//CyoA, subunit II of this Paracoccus Hence, it may be mere likely that the genotype rather oxidase has substituted four out ef five residues that have than the growth conditions gives rise te the ebserved vari- been identified as Cu^ ligands (Kelly ef ai, 1993), This able haem incerporation into the CyeB ape-cytechreme. suggests that CyeABC is a quinel oxidase (Saraste et Similar variability has recently been reperted for the cyte- ai, 1991). Moreover, the oxygen censumptien of the chreme c exidase from the thermophilic bacterium PS3 cyoB mutant was completely inhibited by either antimycin (Sone and Fujiwara, 1991), and for quinol oxidases from A or myxothiazol, whereas the wild-type Paracoccus E. coli and Aoetobacter aceti as well (Puustinen et ai, strain was hardly affected by these compounds (Table 1992; Matsushita etai, 1992), 1). This cenfinns that the cyoABC lecus cedes for a quinol In the quinol oxidase mutant and in the triple mutant, no oxidase. alternative quinel exidase activity could be demonstrated The E. coli cyo operon is a cluster ef five genes; (Table 1). Moreover, extensive attempts te generate a cyoABCDE. The cyoD gene preduct is theught to be a cytechreme c reductase/quinol oxidase deuble mutant fourth subunit (Minghetti et ai. 1992), which may be have net succeeded (net shewn), which indeed might absent from the Paracoccus quinel exidase cemplex. indicate that the Paracoccus genome contains enly one The cyoE preduct is prepesed to be a pretohaem IX quinol oxidase lecus. Previously, the existence of twe famesyl transferase, invelved in the conversion of haem mere terminal oxidases was deduced frem eptical spectra B into haem O (Saiki et ai, 1992). Deletion of the latter ef whole cells. First, a cyanide-telerant exidase with a high gene in E. coli resulted in an exidase with two B haems, affinity fer oxygen and an absortiance maximum at 629 nm hewever, without oxidase activity (Hill etai, 1992; Saiki has been called cytechreme d by Henry and Vignais etai, 1992). (1979), Hewever, cytechrome bbg might have been In well-aerated, succinate-grewn cells of Paracoccus respensible for the reported cyanide-telerant respiration the contribution of the quinol exidase to the endogenous of Paracoccus, in a membrane preparation ef the cyto- respiration varies: it is 75% in wild-type cells and 100% chrome aa3 deuble mutant a K, for cyanide of apprex. in the cytochreme aa^ deuble mutant. Apparently, rela- 250 nM has been determined, whereas the triple mutant tively mere electrons flow via the cytochrome c branch is extremely sensitive te cyanide (J,-W. L, de Gier and when succinate is added as a substrate (Table 1). J, van der Oost, unpublished). The ebserved abserbance Haem analysis indicates that wild-type membranes, band may have originated from the nitrite reductase cyto- with quinol oxidase activity, contain haem B and haem A; chrome cdi (Timkevitch etai, 1982). Second, an exidase in the cytochrome aas deuble mutant haem B and only that is expressed microaerobically, with an absorbance traces of haem A could be detected (Tabie 2). Unlike maximum at 590 nm, has been called cytechreme a^ by haem A and haem B, haem O has never been detected van Verseveld ef ai (1983), It is net unlikely, hewever, in membrane extracts frem the Paracoccus strains grewn that the ebserved cytochreme belongs te another class under these conditions. We conclude that the CyeABC ef enzymes, e.g. the CO-binding catalase-peroxidase cyte- quinel oxidase is a cytochrome with two protohaems. chreme iJ59o (Appleby and Poole, 1991), which was origin- This implies that the hydrexyethylfarnesyl side-chain, ally proposed to be the high-affinity oxidase (cytochrome which is present in haems A and O but not in haem B a^) frem nitrogen-fixing Bradyrhizobium japonicum. (Wu et ai, 1992), is not essential for catalytic activity or Indeed, a cytechreme i)595-associated peroxidase activity for proton translocation. Previously this exidase has has been ebserved in periplasmic fractions of Paracoccus been referred to as cytechreme o er bo (Cox ef ai, 1978; (J. Ras, unpublished). 190 J.-W. L de Gier etal.

aa3-type cytochrome c oxidase alternative cytochrome c oxidase has been studied in vivo. The optical spectrum of this mutant indicates the Expression of the Paracoccus cytochrome aas has been absence of cytochrome aa3 and an increase in the be studied extensively. When Paracoccus is growing on pool (Fig. 3). This increased amount of cytochromes b succinate, either microaerobically or anaerobically, cyto- and c turned out to be reversible: upon complementation chrome aas is undetectable or barely detectable in the opti- of the triple mutant in trans with either one of the three cal spectrum (Bosma, 1989; Stouthamer, 1991). In oxidases, the absorbance peak decreased to a wild-type contrast, this cytochrome c oxidase plays an active role level (Fig. 4). The aitemative cytochrome c oxidase has during autotrophic growth on the Ci substrates methanol recently been partially purified from the membranes of and methyiamine (reviewed by Harms and van Spanning the triple mutant; the optical spectrum indicates the pre- 1991). On the other hand, a cytochrome aa^ mutant of sence of cytochromes c and b (Fig. 5). It is tempting to Paracoccus (Willison ef ai, 1981) is able to grow on assume that an expression induction/repression of this methanol (Harms et at., 1985), and a cytochrome c reduc- cb-type oxidase is responsible for the observed increase/ tase/cytochrome aa3 double mutant of Paracoccus is able decrease in the absorbance spectra of the triple mutant to grow on methyiamine (de Gier et ai, 1992). This sug- (Figs 3and4).Analysisofthe haems extracted from mem- gests the presence of an alternative cytochrome c oxidase branes of the triple mutant only indicates the presence of (see below). haem B (Table 2). Haem A and haem O are absent; The genomic loci encoding the cytochrome aa^ iso- haem C is not extracted, owing to its covalent binding. enzymes have been isolated previously (reviewed by van Oxygen-consumption measurements indicate that respir- der Oost et ai, 1991). In the triple oxidase mutant ation in the triple mutant could be inhibited completely at (ActaDI, ActaDII. cyoB:.Km^) we show that it is possible the level of cytochrome bci (Table 1). It may be that the to restore the optical spectrum of cytochrome aa3 by com- alternative cytochrome c oxidase is the only remaining plementation with either CtaDI or CtaDll (Fig. 4). This has terminal oxidase in this mutant. been confirmed by Western blotting, using antibodies raised against the three-subunit cytochrome aa^ from Recently, we obtained additional genetic information on Paracoccus (not shown). No spectral/immunological evi- this c£>-type oxidase. Using peptide sequences from the dence has been found so far for expression of CtaDI in aforementioned purified cytochrome cb complex, a DNA the ActaDII mutant. The successful expression in trans fragment has been isolated from a P. denitrificans geno- might indicate that a repression of the ctaDI promoter activ- mic library; preliminary sequence analysis indicates the ity has been (partially) overcome by the elevated copy presence of a new oxidase locus (M. Saraste and J. van number. The physiological conditions under which the der Oost, unpublished). The isolated gene cluster is very expression of the cytochrome aa^ isoenzyme is induced similar to fixNOQP from S. japonicum (Preisig et at., remain unknown. 1993). The genes from Bradyrhizobium have been demon- strated to encode an alternative oxidase that is expressed microaerobically or anaerobically; this is in good agree- ment with the finding that the gene cluster is located adja- cb-^pe cytochrome c oxidase cent to the genes of the oxygen-sensing regulators FixJL. Cells of wild-type Paracoccus grown anaerobically/micro- The predicted protein sequences suggest that FixN is the aerobically on succinate are still able to oxidize ascorbate/ haem- and copper-binding subunit I homologue, FixO a TMPD although no aa3-type cytochrome c oxidase is monohaem cytochrome c, and FixP a dihaem cytochrome detectable in the optical spectrum (van Verseveld et ai, c (Preisig et at., 1993). The alignment of the Bradyrhizo- 1983). The latter authors have reported 'the presence of bium FixN sequence (Fig. 2A) suggests that all six a cytochrome oxidase with a very high affinity for oxygen invariant histidines, typical of the haem-copper oxidase and a low affinity for CO'. This high-affinity cytochrome c superfamily, are present in the cb-type cytochrome c oxi- oxidase has been characterized in more detail by Bosma dase; it probably concerns a 6-type haem-copper oxi- (1989), who partially purified it from anaerobically grown dase (Table 2, see below) with associated cytochrome(s) cells. The alternative cytochrome c oxidase was isolated c. In accordance with classical cytochrome oxidase termin- as a complex with cytochrome c reductase, but without ology (Puustinen and Wikstrbm, 1991), we propose to cytochrome C552. Spectroscopic analysis revealed that it name this cd-type oxidase cytochrome cbb^. concerns a cb-type oxidase complex, consisting of a cyto- !n addition, a i)-type cytochrome c oxidase has been chrome b, a 30kDa cytochrome 0 and perhaps a loosely described in Rhodobacter capsulatus (Hudig et ai, 1987). membrane-associated 45kDa dihaem cytochrome c Regulation has been demonstrated at two levels: the (Bosma etai. 1987b; Bosma 1989). expression as well as the activity of this oxidase respond In the present study a triple oxidase mutant (ActaDI, to oxygen and tight. Upon a shift from phototrophic con- ActaDIt, cyoS::Km") has been generated, in which an ditions to chemotrophic conditions (microaerobically) the Terminal oxidase of Paracoccus 191 activity of this oxidase increases fourfold. Garcia-Horsman difference in the method used in this study (HEPES etai (1994) have purified a c6-type cytochrome c oxidase (0.5 mM) and glycylglycine (1,5 mM), respectively). Where- from a Rhodobacter sphaeroides cytochrome aaj mutant. as the proton translocation linked to the cytochrome aa3 The oxidase consists of three subunits: two cytochromes activity is easily detectable when the cell suspension is c and a cytochrome b. Metal and haem analyses indicate buffered by either glycylglycine or HEPES, proton trans- that this cdd3-type oxidase contains haem C, haem B, and location by an alternative oxidase, which is likely to be copper in a ratio of 3:2:1. The presence of a binuclear cytochrome cbt>3, is apparently only detectable in the centre has been demonstrated, including a high-spin HEPES-buffered cell suspension. haem B and a CUB (Garcia-Horsman etaL, 1994). It has been reporfed previously that maxima! rates of oxidative phosphorylation in Paracoccus were measured only in the presence of cytochrome aa3, e.g. after growth Proton translocation on methanol but not after microaerobic growth (Boogerd et Analysis of proton translocation by the generated set of ai., 1981; van Verseveld etai, 1983) (Table 3). Stouthamer Paracoccus oxidase mutants has been performed in order (1991) has recently reviewed the results obtained by his co- to determine whether or not the altemative cytochrome c workers over the last decade, on Paracoccus cultivated in oxidase is a proton-pumping oxidase. During succinate oxi- chemostats under a wide variety of growth conditions. dation, when ubiquinol is oxidized directly by cytochrome Calculations of growth yields and proton-translocation bbs, a minimum H*/2e stoichiometry of 4 is expected rates (assayed in glycylglycine buffer) led him to suggest theoretically; when ubiquinol is oxidized via cytochrome that cytochrome aa3 is a proton-translocating cytochrome bc^ (HV2e"=4) and cytochrome aa^ (HV2e =2) the c oxidase, unlike the altemative cytochrome c oxidase. In maximal HV2e" stoichiometry is 6, An obvious conse- view of the aforementioned variations of the c6b3-catalysed quence of the deletion of cytochrome aa^ is a decreased proton translocation, it can be imagined that the in situ H*/2e"' stoichiometry (Table 3), When ferricyanide is energy-transducing efficiency of this oxidase might also used as tenninal electron acceptor instead of oxygen, vary under different external conditions. Chemostat experi- equal values are obtained because ferricyanide is ments with the available set of oxidase mutants are reduced directly by the cytochrome c pool (Table 3, intended to address this point. Fig. 6). A similar tendency is observed when ascorbate/ Understanding the of the differences observed in TMPD is used as substrate, donating electrons at the the proton pumping behaviour of the cij/>3-type oxidase as level of cytochrome c. Apart from the scalar proton (H*/ compared to, for example, the aa3-type oxidase will be the 2e" = 1), an additional acidification is expected in the goal of future experiments. With current knowledge, it is case of a proton-translocating cytochrome c oxidase, anticipated that cytochrome ebbs is indeed capable of such as cytochrome aa3 (H*/2e" = 2); the maximal HV2e" pumping protons, perhaps only under specific external stoichiometry would be 3. Again, in the absence of cyto- conditions. A detailed study of this 'distant' member of chrome aa^ only the scalar ascorbate proton is measured the haem-copper oxidase family will contribute substan- (Table 3). When cytochrome aa3 is present, an intermedi- tially to a model of the redox-driven proton pump. ate value of 2.0-2.4 is commonly detected. This indicates that, unlike cytochrome aa3 (van Verseveld etai, 1981) and cytochrome bbs (Puustinen et ai., 1989), the cbt>3- Experimental procedures type cytochrome c oxidase does not couple the reduction Bacteriai strains, plasmids, and growth conditions of oxygen to the active transport of protons across the membrane, at least under these conditions (see below). The strains of P. denitrificans and E. coli, as well as the plas- mids, that were used in this study are listed in Table 4. Cells of The proton translocation data as presented in this study the wild-type P. denitrificans (Pd1222) and the mutant strains are consistent with the absence of a proton-pumping were cultivated in aerobic batch cultures (0.51 bottles with oxidase in cytochome aa^ deletion strains. However, 100ml of culture, on a rotary shaker at 30 C), either with Raitio and Wikstrbm (1994) recently presented experimen- brain-heart infusion broth (BHI) or minimal medium supple- tal data that strongly suggest that the alternative cyto- mented with 25 mM succinate, as described previously (van Spanning et ai, 1991). E. coli strains were cultivated in YT chrome c oxidase is able to pump protons. These medium at 37'C. When appropriate, antibiotics were added: authors report an H*/2e" ratio of 6, with whole cells rifampicin(Rif,40mgr''), streptomycin (Sm, SOmgl ''), tetra- (wild-type, cytochrome aa^ double mutant), succinate as cycline (Tc, 12,5mgl ''), kanamycin (Km, 50mgl ^) and substrate, and oxygen as electron acceptor. With ascor- ampiciilin (Amp, ^ bate/TMPD as the electron-donating system, on the other hand, hardly any pumping was observed (H*/2e~ =1.2- DNA manipulations 1.5). The buffer composition of the proton translocation assay as presented by the latter authors is the main General cloning techniques were carried out as described by 192 J.-W. L deG/eretal.

Table 4. Strains and plasmids. Strain/Plasmid Relevant characteristics Reference

Strain E. coli TGI supE, hsdD5, thi, A(lac~proAB). f. Sambrook etal. (1989) {traD36proAB lacl"^ lacZAM^5) HB101 F~, hsdS20, iTB'me'). lacYI, proA2, Boyer etal. (1969) recA 13 S17,1 Sm", pro. (re'mB*), RP4-2, Simon etal. (1983) integrated (Tc::Mu) (Km;:Tn7)

P. denitrificans Pd1222 Rif", enhanced conjungation De Vries efa/. (1989) frequencies, (re'mB*) Pd2521 Pd1222 derivative, c(aD/::Km" This work Pd2541 Pd2521 derivative, ActaDI This work Pd9218 Pd2541 derivative, ActaDI. This work

Pd9220 Pd9218 derivative, ActaDI. ActaDII This work Pd2621 Pd1222 derivative, cyoS::Km" This work Pd9311 Pd9220 derivative. ActaDI ActaDII Thi."? work cyoS: Km"

Plasmid pUC18 Amp", lacZ' Yanisch-Perron ef al. (1985) pUC4K Km" (Tn903) Pharmacia pUC4KISS Km" (Tn903) Pharmacia M13mp18 pUC18 mcs, lacZ! Sanger et al. (1980) pEG400/401 IncP. Sm", Spec", pUC12/13 nncs, Gerhus st al. (1990) lacl PRK2020 Tc", pRK2013Km::Tnr0 Dittaefa/, (1985) pGRPdi oriV, (colEI), Amp", oriT, Snn", Van Spanning et al. (1990) (Tn 1631) pRVSI onV(colEI), Amp", oriT, Sm" Van Spanning ef al. (1991) (Tn/fi3T), Tn5p, /acZ pMR3 pUC18 derivative, ctaDI M, Raitio, unpublished pMR3,Km" pMR3 derivative, cfaD/::Km" This work pMR3D pMR3 derivative, ActaDI This work pKiC-cyoABC pUCi8 derivative, cyoABC This work pUG.cyo>ieC,Km" pUC,cyoASC dehvative, cyoS:;Km" This work pRT2521 pGRPdi derivative, cfaD/::Km" This work PRT2541 pRVSI derivative, ActaDI This work pRT2321 pGRPdi dehvative. ctaDlh.Km" This work pRT2341 pRVSI derivative, ActaDII This work pRT2621 pRVSI derivative, cyoS::Km" This work pEG,cfaD// pEG400 derivative, ctaDII This work pEG,cfaD/ pEG401 derivative, ctaDI This work pEG.cyoABC pEG401 derivative, cyoABC This work

Ausubel et al. (1992), Conjugations were performed as conjugated to P. denitrificans Pd1222 to give Pd2521, Excon- described previously (de Vries et al.. 1989). The matings of jugants were selected for kanamycin and rifampicin resistance Paracoccus host strains were performed either directly with and streptomycin sensitivity; genomic Southern blofs were E CO//S17-1 transformed with the plasmid of interest, or via used to confirm the mutations (not shown). a triparental mating using any E. coli strain transformed with From the ctaDI gene a Sal\-Sma\ fragment was deleted the plasmid of interest, in combination vtfith E. coli HB101/ (Fig, 1A); sticky ends were filled in using Klenow DNA (pRK2020) containing the 'helper plasmid', polymerase fragment and religated (pMR3A), The in-frame P. denitrificans mutant strains were constructed by gene- deleted locus was cloned as a Pvull fragment in the Smal replacement techniques as described previously (van site of the suicide vecfor pRVS1 (pRT2541), which was trans- Spanning et al.. 1991), First, a kanamycin-resistance cas- formed to E co//S17,1 and conjugated to the insertion mutant sette from pKISS (Boehringer) was inserted in the Smal site Pd2521, Paracoccus colonies were initially selected by rifam- of pMR3, a pUC18 derivative carrying a cfaD/-containing picin-, kanamycin- and streptomycin resistance. Subsequent genomic SamHI fragment (a generous gift of Dr M, Raitio, restreaking revealed two distinct phenotypes: (i) integrants Fig. 1A), From the resulting construct (pMR3.Km) a Pvull turned blue on plates supplemented with Xgal (pRVS1 carries fragment was ligated in the Smal site of pGRPdi the p-galactosJdase gene) and are streptomycin- and kanamy- (pRT2521), which was transformed to E. coli S17.1 and cin resistant, and (ii) double recombinant colonies stayed Terminal oxidase of Paracoccus 193 white and had lost streptomycin- and kanamycin resistance. batch cultures (minimal medium with succinate) were har- The deletions in the latter clones (Pd254i) were verified by vested, washed, and resuspended in 1,5mM glycylglycine genomic Southern blotting (not shown). From the cteD/dele- buffer (pH7.0), lOOmM KSCN, 50mM KCl (approx. 25mg tion mutant, a ctaDI/ctaDII double deletion mutant was dry weight cells ml ^), incubated at 25 C, and bubbled with derived, using the same methods. As an intermediate an nitrogen. In the reaction vessel, 150^1 of cell suspension insertion mutant (Pd9218) was generated by conjugation was mixed with 3.1 ml of buffer. With succinate (25 mM) as with a pGRPdi derivative (pRT2321), carrying a BamH\- electron donor, rotenone (30jiM) was added and incubated Sph\ fragment of the ctaDtl locus with the Km-resistance for 30 min in order to inhibit endogenous respiration. With gene from pUC4K inserted in the Pst\ site; in the subse- ascorbate/TMPD (0.4mM/0.1 mM) antimycin A (6|iM) was quently derived cfaD//deletion mutant (Pd9220) the HincW- added (final concentrations); in the presence of the quinol oxi- Nco\ fragment was removed after a double recombination dase, rotenone (30 |JM) was added as well. The anaerobic cell event with pRT2341 (Fig. 1A). suspension was pulsed with oxygen (O2) by injecting 20^1 of The degenerated primers used in the PCR experiment air-saturated KCl (9.4 ng atoms of oxygen), or with ferricya- were: #151 (5-GCGCGGAATTC-CAT/CGGG/CGTG/CATT- nide by injecting 20 nl of an anaerobic solution of 1 mM CATGATT/CTTT/CTT) and #152 (5-GCGCGGAATTC-TAG/ K3Fe(CN)6. Traces were calibrated by injecting 20|il of an CACT/CTCG/CGGA/GTGG/CCCA/GAAA/GAACCA). Using anaerobic solution of 1 mM HCI. genomic DNA from the cytochrome aa^ double mutant and these primers a PGR was performed under conditions essen- tially like those described before (M. Lubben etai, submitted); Haem anatysis in this case the annealing temperature was 37C. A PCR The analysis of the haem composition after extraction from product of the expected size was digested with EcoRI (sites membranes was performed as described by Sone and Fuji- introduced in primers, underlined) and cloned into wara (1991). Haems were extracted from isolated mem- M13mp18, Using the PCR fragment as a probe, a clone was branes of different P. denitrificans strains, separated by isolated (p\JCcyoABC) that appeared to contain the com- reversed-phase HPLC (Deltapak C18 column. Waters) on plete oxidase locus. This plasmid was digested with EcoRI, an acetonitrile gradient in 0.05% trifluoroacitic acid. removing the Pst\ site from the pUC multiple cloning site, and religated. Subsequently the internal Pst\ fragment was substituted for the kanamycin-resistance box from Acknowledgements pUC4KISS. The EcoRI-SpAil insert was cloned in pRVS1, transformed to E. coti S17.1, and conjugated to the wild-type We thank Matti Saraste (Heidelberg) for critical reading of the Paracoccus Pd1222 and the ctaDI/ctaDII double deletion manuscript. Oliver Richter and Bernd Ludwig (Frankfurt), mutant (Pd9220). Conjugants were selected by kanamycin Arthuro Garcia-Horsman and Robert B. Gennis (Urbana), resistance; colonies of integrants turned blue on plates Mirja Raitio and Marten Wikstrom (Helsinki) are gratefully supplemented with Xgal, but colonies of double recombinants acknowledged for communicating data prior to publication. remained white. The insertions in the latter clones were This research was supported by the Netherlands Foundation verified by genomic Southern blotting (not shown). for Chemical Research, with financial aid from the Nether- lands Organization for the Advancement of Science. M.L. had a long-term EMBO Fellowship, and J.v.d.O. has a Fellow- Optical spectroscopy ship of the Royal Dutch Academy of Science. Spectra of whole-cell suspensions were recorded at room temperature on an Aminco/SLM DW2 UV/Vis Spectro- photometer, essentially as decribed previously (de Gier et References ai. 1992). Anraku, Y., and Gennis, R.B. (1987) The aerobic respiratory chain of Escherichia coti. Trends Biochem Sci 12: 262- Oxygen-consumption analysis 266. Appleby, C.A., and Poole, R.K. (1991) Characterization of a Oxygen consumption by bacterial cell suspensions was ana- soluble catalase-peroxidase hemoprotein f>-590, previously lysed polarographically at 30 C in a biological oxygen moni- identified as 'cytochrome aV from Bradyrhizobium japoni- tor. Model 53 (Yellow Springs Instrument Co.). Succinate cum bacteroids. FEf\4S Microbiot Lett 78: 325-332. (25 mM), or ascorbate (0.4 mM) and TMPD (0.1 mM) were Ausubel. F.M., Brent, R., Kinston, R.E., Moore. D.D., Smith, added as external substrates; electron flow via cytochrome c J.A., Seidman, J.C., and Struhl, K. (1992) Current Protocots reductase was inhibited by antimycin A (6^M) or myxothiazol in Motecutar Biotogy. New York: John Wiley and Sons, Inc. (6|iM). Assays were repeated at least three times and were Babcock, G.T., and Wikstrom, M. (1992) Oxygen activation found to be reproducible within 10%. No auto-oxidation of and the conservation of energy in cell respiration. Nature ascorbate/TMPD was detected when solutions were pre- 356: 301-309. pared freshly. Berry, E.A,, and Trumpower, B.L, (1985) Isolation of ubiquinol oxidase from Paracoccus denitrificans and resolution into Proton-translocation analysis cytochrome fac, and cytochrome c-aaj complexes. J Biot Chem 260: 2458-2467. Measurement of H* transiocation in intact cells was performed Boogerd, F.C, van Verseveld, H.W., and Stouthamer, A.H. essentially as described by Boogerd et ai (1981). Cells from (1981) Respiration driven proton transiocation with nitrate 194 J.-W. L. de GiereXal

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