Photosynthesis Research 32: 79-94, 1992. © 1992 KluwerAcademic Publishers. Printedin the Netherlands. Regular paper

Characterization of the pet operon of Rhodospiriilum rubrum Savita Shanker ~, Carolyn Moomaw 2, Saadettin Giiner ~'3, Joan Hsu 2, Mariko K. Tokito 4, Fevzi Daldal 4, David B. Knaff 1'* & James G. Harman 1 1Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX 79409-1061, USA; 2Howard Hughes Medical Institute Research Laboratories, University of Texas Southwestern Medical Center at Dallas, Dallas TX 75235-9050, USA; 3Department of Chemistry, Karadeniz Technical University, Trabzon, Turkey; 4Department of Biology, Plant Science Institute, University of Pennsylvania, Philadelphia, PA 19104-6018, USA; *To whom correspondence should be addressed at: Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX 79409-1061, USA. Tel.: 806-742-3067. FAX: 806-742-1289 Received 26 September 1991; acceptedin revised form 30 January 1992 Abstract

The three genes of the pet operon, coding, respectively, for the Rieske iron-sulfur protein, cytochrome b and cytochrome c I components of the cytochrome bc~ complex in the photosynthetic bacterium Rhodospirillum rubrum have been sequenced. The amino acid sequences deduced for these three peptides from the nucleotide sequences of the genes have been confirmed, in part, by direct sequencing of portions of the three peptides separated from a sample of the purified, detergent-solubilized complex. These sequences show considerable homology with those previously obtained for the pet operons of other photosynthetic bacteria. Northern blots of R. rubrum mRNA have established that the operon is transcribed as a single polycistronic message, the start site of which has been determined by both primer extension and nuclease protection. Photosynthetic growth of R. rubrum was shown to be inhibited by antimycin A, a specific inhibitor of cytochrome bc 1 complexes, and antimycin A-resistant mutants of R. rubrum have been isolated. Preliminary results suggest that it may be possible to express the R. rubrum pet operon in a strain of the photosynthetic bacterium Rhodobacter capsulatus from which the native pet operon has been deleted.

Introduction

The cytochrome bc~ complex (ubiquinol:cytochrome C(C2) oxidoreductase) is an oligomeric, membrane bound protein present in the respiratory chains of mitochondria (Weiss and Kolb 1979, Sidhu and Beattie 1982), in the electron transport chains of many purple non-sulfur photosynthetic bacteria (Hauska et al. 1983, Yu et al. 1984, Wynn et al. 1986, Ljungdhal et al. 1987) and aerobic bacteria such as Paracoccus denitrificans (Yang and Trumpower 1981). The cytochrome bc~ complex catalyses electron transfer from ubiquinol to a soluble c-type cytochrome. The redox reaction generates an electrochemical proton gradient which can be cou-

pied to ATP synthesis and other energy requiring processes (Trumpower 1990). The analogous cytochrome b6f complex found in cyanobacteria (Krinner et al. 1982, Kallas et al. 1988a) and in chloroplasts of higher plants and algae catalyzes electron transfer from plastoquinol to the copper-containing protein plastocyanin or cytochrome c553 (Hurt and Hauska 1981, Hauska et al. 1983). Both the cytochrome bc 1 and the b6f complexes contain two protohemes, one c-type heme, and a high potential [2Fe-2S] cluster. Despite functional similarity and the presence of the Rieske iron-sulfur protein, cytochrome b, and cytochrome c I in all cytochrome bcl complexes, the complexes differ with regard to their subunit composition. In photosynthetic purple

80 sulfur bacteria these complexes contain either three (Wynn et al. 1986, Cully et al. 1989, Kriauciunas et al. 1989, Purvis et al. 1990, Majewski and Trebst 1990) or four peptide subunits (Yu et al. 1984, Ljungdhal et al. 1987, Andrews et al. 1990, Purvis et al. 1990, Yu and Yu 1991, Usui and Yu 1991). Cytochrome bc I complexes isolated from mitochondria contain at least nine subunits (Teintze et al. 1982). The cytochrome b6f complexes of chloroplasts and cyanobacteria contain at least four subunits (Widger et al. 1984, Willims et al. 1987). Genes encoding the Rieske iron-sulfur protein, cytochrome b and cytochrome c I from four species of photosynthetic purple non-sulfur bacteria (Gabellini and Sebald 1986, Davidson and Daldal 1987a, Davidson and Daldal 1987b, Verbist et al. 1989, Majewski and Trebst 1990, Yun et al. 1990), the aerobic bacterium Paracoccus denitrificans (Kurowski and Ludwig 1987), the nitrogen fixing bacterium Bradyrhizobium japonicum (Thony-Meyer et al. 1989), and, for the cytochrome b6f complex, from the cyanobacterium Nostoc PCC 7906 (Kallas et al. 1988b), have been cloned and sequenced. The genes for the three catalytic subunits of the cytochrome bc 1 complex of photosynthetic purple non-sulfur bacteria are contained in an operon named pet or fbc (Gabellini and Sebald 1986, Davidson and Daldal 1987a, Verbist et al. 1989, Majewski and Trebst 1990, Yun et al. 1990). The pet operon 5' to 3' gene order is: petA, encoding the Rieske iron-sulfur protein; petB, encoding cytochrome b; and petC, encoding cytochrome cl. We report here the complete DNA sequence of the genes coding for the R. rubrum cytochrome bc I complex. The amino acid sequence predicted from the DNA sequence was confirmed, in part, by peptide sequence analysis of purified, detergent-solubilized R. rubrum cytochrome bc~ complex. The R. rubrum pet operon was shown to be transcribed as a single polycistronic message and the 5' end of the transcript was mapped. Advantage has been taken of the availability of sequence data for an additional petB gene product, i.e., R. rubrum cytochrome b, to analyze the possible involvement of specific cytochrome b amino acids in binding substrate quinone and/or quinone analog inhibitors. R. rubrum mutants resistant to the

cytochrome bc 1 complex inhibitor, antimycin A have been isolated and preliminary experiments designed to express the R. rubrum pet genes in R. capsulatus have been carried out. Materials and methods

Enzymes and chemicals

Restriction endonucleases, T4 DNA ligase and calf intestine alkaline phosphatase were obtained from New England Biolabs or Promega. Exonuclease III and mung bean nuclease were included in a pBluescript II Exo/Mung DNA sequencing kit purchased from Stratagene. Avian myloblastosis (AVM) reverse transcriptase was purchased from Promega. DNA sequencing kits were purchased from United States Biochemicals. T7 DNA polymerase was purchased from Pharmacia. Nytran nylon membranes (0.45/zm pore size) were purchased from Schleicher and Schuell, Inc. a-[32p] dATP (3000 Ci/mmol) and .y.[32p] ATP (6000 Ci/mmol) were purchased from Dupont/NEN. Genius non-radioactive DNA labeling kits were purchased from Boehringer Mannheim. Oligonucleotides were either purchased from The Midland Certified Reagent Company or were synthesized using phosphoramidite chemistry on a Milligen/Biosearch Cyclone Plus DNA Synthesizer using reagents purchased from Milligen. All other chemicals were reagent grade or better and purchased from United States Biochemicals or Sigma Chemical Co. Bacterial strains and plasmids R. rubrum (strain S1) was obtained from the American Type Culture Collection and grown as described previously (Wynn et al. 1986). Rb. capsulatus strain MT-RBC1 (Atta-Asafo-Adjei and Daldal 1991), a strain with the pet operon deleted and incapable of photosynthetic growth was used as the recipient in mating experiments. Escherichia coli strains XL-1 blue (Stratagene) and HB101 were grown in LB medium (Maniatis et al. 1982) containing the appropriate antibiotics. Plasmid pBluescript II SK ÷ (Stratagene) was used as the cloning vector. Plasmid p17-4

81 (Daldal et al. 1987) served as the source of Rb. capsulatus petBC sequences used as the heterologous probe. The broad host range plasmid pRK404 (Ditta et al. 1985) was used as the vector to introduce the R. rubrum petABC genes into Rb capsulatus.

DNA isolation R. rubrum DNA was isolated by the method of Seidman et al. (1987) modified by omitting the CsCI gradient step. Large scale plasmid DNA isolation was accomplished using the alkaline/ SDS protocol described by Maniatis et al. (1982). Plasmid minipreparations were performed according to Maniatis et al. (1982). Plasmids that served as templates in DNA sequencing reactions were purified over Qiagen tip-20 columns (Qiagen, Inc.). Hybridization Colony filter hybridization was performed according to Maniatis et al. (1982). Clones carrying the genes for the cytochrome bc 1 complex of R. rubrum were identified by hybridization with a digoxygenin-labeled heterologous cytochrome b and cytochrome c 1 probe from Rb. capsulatus (Daldal et al. 1987). Southern blotting was performed according to Meinkoth and Wahl (1984) using 20 x SSC (SSC is 0.015 M sodium citrate buffer, pH 7.0, containing 0.15 M sodium chloride) as the capillary transfer medium. DNA hybridizations were carried out overnight at 68 °C using 30 ng/ml digoxygenin labeled, denatured probe DNA in 5 x SSC that was 0.02% in SDS, 0.1% in N-lauroyl sarcosine (Na + salt), and 0.5% in blocking reagent. After hybridization, membranes were washed twice for 5 min with 2 x SSC that was 0.1% in SDS at room temperature and twice for 15 min at 68°C in 0.1 x SSC that was 0.1% in SDS. Immunological detection of the digoxygenin-labeled DNA was carried out according to the protocol provided by Boehringer Mannheim.

DNA sequence analysis Alkaline denatured double stranded DNA was sequenced using the dideoxynucleotide chain ter-

mination methodology of Sanger et al. (1977) according to the protocol provided by E.G. & G., Inc. 32p-labeled DNA was separated on 8% polyacrylamide gels that were 7.0 M in urea and 40% in formamide using an E.G. & G. Acugen 402 automated DNA sequencer. The petA gene and a portion of the petB gene were sequenced using specific oligonucleotide primers (See the legend for Fig. 1). A portion of the petB gene and the entire petC gene were sequenced using a nested set of deletion plasmids generated from pBS9 and pBS5. These deletion plasmids were constructed according to the protocol provided in the Stratagene pBluescript II Exo/Mung DNA sequencing kit. The 3'-nested deletion set was generated from SacI/XbaI digested pBS9. The 5'-nested deletion set was generated from HindlII/KpnI digested pBS5.

Computer analysis of DNA and peptide sequences DNA sequence information collected by the Acugen automated sequencer was accumulated on an IBM PS/2 computer and analyzed using the program Genquest (E.G. & G.). Alignment and analysis of independent sequence files with respect to open reading frames, translation, Hoppe-Woods hydropathy calculations and DNA secondary structure predictions were all performed on a Macintosh SE computer using the program DNA Inspector IIe (Textco).

Characterization of the R. rubrum pet transcript Total cellular RNA was isolated from photosynthetically grown R. rubrum cells by extraction with guanadinium thiocyanate/phenol/chloroform (Chomczynebi and Sacchi 1987) or by the procedure of Summers (1970). The RNA was then dissolved in water and selectively precipitated with 4 M LiCI to remove any contaminating DNA. The RNA was denatured in formaldehyde/formamide and fractionated on 1.2% agarose gels containing 1.1% formaldehyde, 20 mM MOPS (pH 7.0), 5 mM sodium acetate and 1 mM EDTA. Following electrophoresis, the gels were washed with water and then equilibrated with 0.3 M sodium citrate buffer (pH 7.0) that was 3 M in NaCI, transferred to nylon mem-

82 branes and cross-linked by irradiation with ultraviolet light. Prehybridization of the membranes and hybridization with digoxygeninlabeled probe DNA were carried out at 42 °C in 5 x SSC buffer that was 0.2% in SDS, 0.1% in N-lauroyl sarcosine, 2% in blocking reagent and 50% in formamide. Primer extension reactions were performed according to the methods of Seidman et al. (1987) and Grisshammer et al. (1990). A 33 base oligonucleotide complimentary to the 5' end of the petA gene was labeled with [.y_32p] ATP in the presence of T4 polynucleotide kinase (Seidman et al. 1987) and hybridized to 50/xg of total R. rubrum RNA at 30 °C overnight. The primer was extended using AVM reverse transcriptase (24 units) for 90 min at either 45 °C or 50 °C. Reactions were terminated by phenol/chloroform extraction, the DNA was precipitated and washed with ethanol, and dissolved in TE buffer (Seidman et al. 1987). Primer extension reaction products were separated on 6% polyacrylamide sequencing gels that were 7.0 M in urea. X-ray film was exposed to the gels, and the film was analyzed using a Molecular Dynamics Model 300 Computing Densitometer. Mung bean nuclease mapping was carried out using a SphI/ClaI restriction fragment of the R. rubrum pet operon labeled with [3/-32p] ATP in the presence of T4 polynucleotide kinase (Maniatis et al. 1982). The labeled DNA fragment was co-precipitated with 50/xg of R. rubrum RNA and dissolved in 30 ~1 of hybridization buffer (Grisshammer et al. 1990). Samples were heated at 85 °C for 5 min. and incubated overnight at 58°C. The R N A / D N A hybrids were digested with 200 units of mung bean nuclease for 30 min at 37°C, phenol extracted, precipitated with ethanol, dissolved in TE buffer (Maniatis et al. 1982) and analyzed on 5% polyacrylamide sequencing gels that were 7.0 M in urea. Peptide sequencing R. rubrum cytochrome bc I complex was solubilized and purified as described previously (Kriauciunas et al. 1989, G/iner et al. 1991). Amino acid sequence analysis of the subunits of the complex was performed with an Applied Biosystems Model 470A sequencer using the

manufacturer's standard programming and chemicals. The quantities of the proteins subjected to analysis were estimated by comparing the intensity of individual bands with the intensity of stained standard marker proteins of known quantity in Coomassie Brilliant Blue-stained polyacrylamide/SDS gels (Laemmli 1970). Subunits for N-terminal sequencing were isolated from gels loaded with 100 pmol of cytochrome bc 1 complex; the proteins were electroblotted onto lmmobilon paper (Millipore Corp.) located and excised (Matsudaira 1987). The apparent molecular masses determined for the three subunits: 35 kDa (cytochrome b), 31 kDa (cytochrome c 1) and 24 kDa (Rieske iron-sulfur protein); were similar to those reported previously (Wynn et al. 1986, Kriauciunas et al. 1989, Purvis et al. 1990, Majewski and Trebst 1990). The Rieske iron-sulfur protein and cytochrome c 1 were susceptible to Edman degradation and provided N-terminal sequences. Cytochrome b was blocked at the N-terminus, but incubation of this protein in situ with cyanogen bromide yielded peptides that were susceptible to Edman degradation. Internal amino acid sequence information was acquired using subunits separated by electrophoresis through polyacrylamide/SDS gels (Laemmli 1970), electroblotted to nitrocellulose paper and digested with trypsin (Aebersold et al. 1987). Tryptic peptides derived from approximately 300 pmol of R. rubrum cytochrome bcl complex were separated on an Applied Biosystems Model 130A system equipped with a 2.1 x 100 mm RP 300 reverse phase HPLC column. Chromatography was performed for 100 min, at a flow rate of 50 p~l per minute in a gradient formed from 0.1% trifluoroacetic acid to 70% acetonitrile that was in 0.1% in trifluoroacteic acid. Sequences of the cytochrome c I and the Rieske subunits were obtained from homogeneous samples of cytochrome c~ and the Rieske proteins. Polyacrylamide/SDS gel electrophoresis (Laemmli 1970) of samples prepared according to this protocol showed that while the Rieske iron-sulfur protein and cytochrome c~ were stable to storage at - 2 0 °C, the cytochrome b peptide was not. Degradation products of cytochrome b were not completely separated from cytochrome c 1. As a result, sequences of the

83 cytochrome b subunit were derived from a heterogeneous sample of cytochrome b that contained cytochrome c~ as well. Expression of the R. rubrum pet operon in Rb. capsulatus Plasmid p9T1 was constructed by ligating HindlII digested pBS10 into the HindlII site of the broad host range plasmid pRK404, followed by transformation of the plasmid into E. coli strain HB101 and selecting Amp r and Tet r transformants. E. coli HB101/p9T1 was used as a donor in a triparental cross, transferring the R. rubrum pet genes to Rb. capsulatus strain MTRBC1. The triparental mating was performed essentially as described previously for the introduction of the Rb. sphaeroides pet operon into Rb capsulatus (Davidson et al. 1989). Tet r transconjugants were selected under respiratory growth conditions on RCV plates supplemented with 1 /zg/ml tetracycline and tested for photosynthetic growth on various growth media, as described earlier (Daldal et al. 1987).

Results

The genes for the cytochrome bc 1 complex of R. rubrum were identified through hybridization to a heterologous D N A probe prepared from Rb. capsulatus. Probing BamHI digested R. rubrum D N A with a l k b NaeI fragment of p17-4 (Daldal et al. 1987) identified a 6 kb DNA flag-

® (~) 5 '=-=-~petA "41----0

ment that specifically hybridized to the Rb. capsulatus probe (not shown). A partial genomic library of R. rubrum D N A was constructed by cloning size fractionated BamHI fragments (approximately 3 to 8 kb) into the BamHI site of pBluescript. The library was screened by hybridization to the Rb. capsulatus cytochrome bc l probe and positive colonies were rescreened twice before one was selected for further use. A plasmid, designated pBS9, was isolated from cells grown from this colony. Southern analysis of genomic DNA from R. rubrum digested with EcoRV resulted in hybridization of a hapten-labeled l kb BamH1/PstI fragment from pBS9 with a 5 kb DNA fragment (not shown). A partial genomic DNA library of R. rubrum DNA, consisting of size fractionated EcoRV fragments (approximately 2 to 8kb) cloned into pBluescript, was screened by colony hybridization. After rescreening, one positive clone was isolated and shown to contain a plasmid, designated pBS10, that was later demonstrated to contain the entire pet operon of R. rubrum. Previous work on the R. rubrum pet operon did not include any studies on the nature of its transcript (Majewski and Trebst 1990). To determine if the R. rubrum petA, B and C genes are transcribed as a single polycistronic message, total R. rubrum RNA was fractionated on an agarose gel and hybridized with labeled DNA derived from different portions of the R. rubrum pet operon (see Fig. 1 for the locations of these D N A probes). DNA probes complimentary to

(D

® EcoRV

BamHl [

petB !~i~.:~,~!~I~ i

petC

~'-'3'

Fig. 1. Organization and DNA sequencing strategy for the R. rubrum pet operon. Sequencing primer sequences (circles) were:

AAGGCTCTATGAGATCGoH; AGAGCGCGCAAAACATCoH; TrGTCTCCGAGGGGTCGon; TCCACGGAAGTGGATGoH; GCTGGGCCAGMGGCGGoH ; GGTI'CGATGAGCGGCTToH ; GACGTGAATTACGGCTGoH. Sequences derived from nested deletion plasmids (arrows) were obtained using the M13 universal sequencing primer (GTAAAACGACGGCCAGToH) and the M13 reverse sequencing primer (AGCGGATAACAAqTFCACACAGGAon). Hybridization probes (horizontal lines labeled 1, 2 and 3) utilized for Northern analysis of R, rubrum mRNA were derived from pBS5 and pBS9. The 33 base otigonucleotide (labeled 4) used in primer extension reactions had the sequence CGGTGTOGAGGCGGTGTGTTCGGCTTCAGo~.

84 the petA, petB and petC transcript sequences were found to hybridize with a single R N A species approximately 2.6kb in length. An example is shown for petA and petC probes in Fig. 2 and similar results were obtained with a petB probe (data not shown). This documents that the pet operon of R. rubrum gives rise to a single polycistronic m R N A species. A full length transcript of the R. rubrum pet operon would be expected to be approximately 3 kb in length, which is larger than the 2.6 kb message detected. As both primer extension and nuclease protection experiments (see below) indicate that the 5' end of the transcript is intact, it is possible that a portion of the m R N A at the 3' end has suffered some degradation during isolation. To determine the 5' end of this transcript, two

Fig. 2. Northern analysisof R. rubrum RNA. Northern blots of the RNA isolated from R. rubrum were probed with either: Panel A; digoxygenin-labeledSmaI petA DNA (Fig. 1, primer 3) or Panel B; digoxygenin-labeledPstI-EcoRV petC DNA (Fig. 1, primer 2). RNA isolated by the method of Summers (1970) was loaded onto gels containingethidium bromide (lanes 1, 2, 3 and 5) or premixed with ethidium bromide prior to loading the gel (lanes 4 and 6). Lanes 1, 3 and 5 and lanes 2, 4 and 6 were loaded with RNA obtained from two independent isolations. RNA size standards (Bethesda Research Laboratories) used to calibrate the gels are indicated with an arrowhead; the values for the standard RNA is in kb. The standard RNA solution was run in adjacent wells, cut from the membrane after transfer and stained in acetate buffered methylene blue (0.2%).

different approaches were taken. Primer extension analysis was performed using a 33 base oligonucleotide that was complimentary to the 5'-end of the petA gene (Fig. 1). A 303 bp extension product was detected at both 45 °C (not shown) and 50 °C (Fig. 3). This 5' end for the transcript corresponds to a guanine at position 191 (Fig. 4), located 269 bp upstream of the petA initiator A U G sequence. Additional extension products were also observed (Fig. 3). The most abundant of these was located at a position 190 bp upstream from the initiation codon. The occurrence of these products was dependent upon the temperature used during the extension reactions and most likely results from secondary structure-dependent extension termination. Independent confirmation of the transcript start point was obtained from mung bean nuclease mapping conducted using a 7-[32p]-labeled ClaI/ Sphl D N A fragment of the R. rubrum petA gene. A 392-+ 3 base product was observed, corresponding to the position 269 + 3 bases upstream of the petA gene initiator codon (data not shown). A minor 190-+ 3 base product was also detected. Translation of the first gene of the operon, petA, initiates at an A U G codon, includes 549 bases and terminates at an U A G codon. The A U G start codon is preceded by a sequence complimentary to the 16S r R N A of R. rubrum (Gibson et al. 1979); a presumptive ribosome binding site. The Rieske iron-sulfur protein is 182 amino acids in length with a molecular mass of 19 402 Da, lower than the apparent 22 400 Da mass estimated from polyacrylamide/SDS gels (Kriauciunas et al. 1989, Majewski and Trebst 1990, Purvis et al. 1990). R. rubrum synthesizes a form of the Rieske iron-sulfur protein that lacks a leader peptide sequence. Amino acid sequencing of the N-terminal portion of the Rieske peptide indicates that only the initiating methionine is cleaved, leaving alanine at the amino terminus (Fig. 4). Comparison of the amino acid sequence of the R. rubrum Rieske protein with those from other purple non-sulfur bacteria shows that the Cterminal amino acid regions are highly conserved (Fig. 5A). This region contains four cysteines, and three histidines. Of these, two cysteines and two histidines are thought to bind the iron-sulfur

85 1.5

1.3 f f ~ 1.1 Band Intensity 0.9 0.? 0.5 0.3 269

190

Position Fig. 3. petABC transcript 5'-end mapping by primer extension. Primer extension was carried out, as described in Methods at 50 °C and the X-ray film exposed to the gel analyzed using a Molecular Dynamics Model 300 Computing Densitometer. Shown are densitometer traces for the primer extension reaction (lower trace) and the T lane from DNA sequencing reactions primed with the same primer (upper trace). Arrows indicate the positions 190 and 269, respectively, upstream from the A U G start site of the petA gene.

cluster (Gurbiel et al. 1989, Kulia et al. 1987, Telser et al. 1987, Britt et al. 1991, Gurbiel et al. 1991). The petA nucleotide sequence determined in this study differs from that reported by Majewski and Trebst (1990) in three of the 549 nucleotides. These differences result in amino acid differences at positions 89 and 90, where we find aspartate and glutamate residues, respectively, instead of the two consecutive valines reported by Majewski and Trebst (1990) and at position 98, where we have observed threonine rather than serine. The presence of threonine at position 98 was confirmed through peptide sequencing (Fig. 4), but independent confirmation for the presence of aspartate and glutamate at positions 89 and 90 is not available. A hydrophobicity plot of the R. rubrum Rieske protein (not shown) is essentially identical to that previously reported by Majewski and Trebst, except for the local change in the polarity that occurs at positions 89 and 90 because of the differences in the two sequences at these positions. Translation of the petB coding sequence mRNA initiates at an AUG codon and terminates at a U G A codon. The petA, petB intercistronic region is 13 nucleotides long and the putative ribosomal binding site of the petB mRNA is located 12 bases upstream from the start codon. The amino acid sequence deduced from the petB gene sequence indicates that cytochrome b contains 405 amino acids and has a

molecular mass of 46 185 Da. As the cytochrome b peptide, separated from the samples of the detergent-solubilized R. rubrum cytochrome bct complex used in this study, had a blocked aminoterminus (see above), we were unable to determine whether cytochrome b is synthesized with a leader sequence. However, Trebst and Majewski (1990) have reported that R. rubrum cytochrome b is not processed. Apparent molecular masses between 35 000 Da and 37 000 Da have been estimated for cytochrome b from the results of polyacrylamide gel electrophoresis in the presence of SDS (Kriauciunas et al. 1989, Majewski and Trebst 1990, Purvis et al. 1990). Similar discrepancies between the molecular masses calculated from the amino acid sequences and the apparent molecular masses for this hydrophobic protein estimated from electrophoresis data have been observed for the cytochromes b of other photosynthetic bacteria (Gabetlini and Sebald 1986, Davidson and Daldal 1987a, Verbist et al. 1989, Yun et al. 1990) The R. rubrurn petB nucleotide sequence presented here and that reported by Majewski and Trebst (1990) are identical. The two protohemes of cytochrome b are thought to be bound to the protein by four conserved histidines present in all cytochrome b and cytochrome b 6 proteins for which amino acid sequences are available (Hauska et al. 1988, Yun et al. 1991a,b). These histidines (H94, H108, H195 and H209) are conserved in R. rubrum (Fig. 5B). Four positively

86 ~GTTTGACCTTCGTCATT~CAAGG~GTGCCAGAGCGCGCAA~CAT~TTTGTT~TTTTCAGAA~CAGGGATTTGCTTTATATCCGCGCCC ~9~T~C~GCCGC~TGGTCCGTGCTTGCCAGCACGGGGTAT~GGAGGGCCGTTT~GGGGGCTCCGTCCCGAGTGTCAAAGGGGCGGACTGGA ••8•ACGGAATATGG•GGT•GCGGCC•GGTTGATCGTGGGGGATTATCGGAGTCGACTCGG•ATCCAGGGCGGGGCAG•CCCTCATATCACAGG ~27~GGGGGCGTCCTATA~CT~TTTCTTGCCGCGGTTGTCTCCGA~GGTCGAGA~TTGTCCTCTAGGCG~GG~AAGAGAGAGGAAACCAGG M A E A E H T A S T P G G E S S R R D F L I Y G T T A V G A •36•ATGGCTGAAGCCGAACACACCGCCTCGACACCGGGCGGAGAGTCATCACGCCGCGAcTTCCTGATTTATGGCACGACCGCCGTGGGCGCC V G V A L A V W P F I D F M N P A A D T L A L A S T E V D V ~45~GTTGGCGTCGCCCTGGCCGTTTGGCCGTTcATCGATTTCATG~TCCCGCCGCTGACACGCTGGCTCTCGcCTCCACGG~GTGGATGTG S A I A E G Q A I T V T W R G K ~ V F V R H R T Q K E I D * E *

054~TCGGCCATTGCCG~GGTCAGGCGATTACCGTTACCTGGCGCGG~ACCGGTTTTcGTCCGCCACCGCAC~C~AAGG~ATCGACGAG A R A V D P A T * L R D P O T D Z A R V Q Q A Q W L V M V G V ~63~GCGCGCGCGGTCGATCCCGCGACGCTGCGCGATCCGC~ACCGACGAGGCCCGGGTGCAACAGGCCC~TGGTTGGTCATGGTCGGCGTC C T H L G C I P L G Q K A G D P K G D F D G W F C P C H G S 072~TGCACGCATCT~GCTGCATTCCGCTGGGCCAGAAGGCGGGCGACCCC~AGGCGACTTCGATGGGTGGTTCTGCCCCTGCCATGGGTCG H Y D S A G R I R K G P A P L N L P V P P Y A F T D D T T V •8••CATTACGATTCCGCCGGCCGTATCCGCAAGGGTCCCGCCCCCCTGAACCTCCCGGTGCCGCCGTATGCTTTCACGGACGACACCACGGTT L I G " M Y T P P R W N N K A L K W F D E R L P V ~9~CTGATCGGTTAGGAGCTGCCCGACGATGTATACCCCTCCGCGTTGGAACAAC~GGCCCTCAAGTGGTTCGATGAGCGGCTTCCGGTcTT L T V A H K E L V V Y P A P R N L N Y F W N F G S L A G I A 099~GACCGTGGCGCACAAGG~CTGGTCGTCTACCcGGCTCCGCGCAACCTCAATTACTTTTGGAATTTCGGCTCGCTGGCCGGTATCGCCAT M I I M I A T G I F L A M S Y T A H V D H A F D S V E R I M ••8•GATCATCATGATCGCCACGGGTATTTTCCTGGCGATGAGCTATACCGCCCATGTCGACCACGCCTTCGATTCCGTCGAGCGCATCATGCG R D V N Y G W L M R Y M H A N G A S M F F I V V Y V H M F R ••7•CGACGTGAATTACGGCTGGCTGATGCGCTACATGCACGCCAATGGCGCTTCGATGTTCTTCATCGTCGTCTATGTGCACATGTTCCGCGG G L Y Y G S Y K P P R E V L W W L G L V I L L L M M A T A F •26•CCTCTATTACGGATCCTACAAGCCGCCCCGCGAAGTTCTGTGGTGGCTGGGTCTGGTCATTCTGCTGCTGATGATGGCGACCGCCTTCAT M G Y V L P W G Q M S F W G A T V I T N F L S A I P V V G D ~35~GGGCTATGTCTTGCCCT~GGCCAGATGTCGTTCTGGGGCGCCACGGTGATCACCAATCTGTTCTCGGCGATTCCCGTCGTCGGCGACGA D I V T L L W G G F S V D N P T L N R F F S L H Y L F P M L •44•CATCGTGACCTTGCTCTGGGGTGGCTTCAGCGTTGATAACCCGACGCTCAACCGCTTCTTCTCGCTGCACTATCTGTTCCCGATGCTGTT L F A V V F L H M W A L H V K K S N N P L G I D A K G P F D ~53~GTTCGCGGTCGTGTTCCTGCACATGTGGGCGCTGCACGTGAAGAAGTCGAACAACCCCCTGGGCATCGACGCC~GGGTCCGTTCGATAC T I P F H P Y Y T V K D A F G L G I F L M V F C F F V F F A •62•CATCCCCTTCCACCCGTACTACACGGTGAAGGATGCCTTCGGTCTTGGCATCTTCCTGATGGTATTCTGCTT•TTTGTCTTCTTCGCCCC P N F F G E P D N Y I P A N P M V T P T H I V P E W Y F L P •7••CAATTTCTTTGGCGAACCCGACAACTACATCCCGGCCAACCCGATGGTGACGCCGACCCACATCGTTCCGGAATGGTACTTCCTGcCGTT F Y A I L R A V P D K L G G V L A M F G A I L I L F V L P W

•8••CTA•G•CATCTTG•GGGCCGTTCCCGACAAGCTGGG•GGCGTG•TGGCGATGTTCGGGGCCATCTTGAT•TTGTTCGTGCTGCCCTGGCT L D T S K V R S A T F R P V F K G F F W V F L A D C L L L G ~89~CGATACCTCGA~GTGCGCTCCGCGACCTTCCGTCCGGTGTTCAAGGGCTTCTTCTGGGTCTTCCTGGCCGACTGCCTGCTGCTCGGTTA Y L G A M P A E E P Y V T I T Q L A T I Y Y F L H F L V I T •98•CCTGGGCGCCATGCCCGCCGAGGAGCCCTATGTGACCATCACCCAGCTCGCGACCATCTATTATTTCCTGCACTTCCTGGTCATCACCCC P L V G W F E K P K P L P V S I S S P V T T Q A "

2•7•GCTGGTGGGTTGGTTCGAGAAGCcCAAGCCGCTGcCGGTGAGCATCAGCTCCcCGGTGACGACCCAGG•CTGACGGCAGCACGAGAGGAT M

T

T

I

V

K

R

A

L

V

A

A

G

M

V

L

A

I

G

G

A

A

Q

A

N

E

G

2~6~CGA~GAGAG;~T~.~A~TA~GATCGT~AAA~GGGC~cTAGTGGC~G~GGcATGGT~TGGC~AT~GGcGG~G~GGCC~AGG~AACG~GG~ ~ V S L H K O D W S W K G I F ~ R Y D O P O L O R G F Q V F 225•GGGGTTTCCCTGCACAAGCAGGATTGGAGCTGGAAGGGCATCTTCGGACGCTATGACCAGCCCCAGCTTCAGCGCGGCTTCCAGGTTTTC H E V C S T C H G M K R V A Y R N L S A L G F S E ~ R * I K ~ 234~CATGAGGTCTGCAGCACCTGCCATGGCATG~GCGCGTGGCCTATCGC~CCTGAGCGCCCTTGGTTTCTCCGAGGACCGCATCAAGGAG L A A E K E F P A G P D D N G D M F T R P G T P A D H I P S 243•CTGGCCGCCGAGAAGGAATTCCCGGCCGGTCCCGACGACAACGGCGATATGTTCACCCGTCCGGGCACCCCGGCCGACCATATCCCCTCG P F A N D K A A A A A N G G A A P P D L S L L A K A R P G G 252•CCCTTCGCCAACGACAAGGCCGCCGCCGCGGCCAATGGCGGGGCGGCGCCGCCCGACCTGTCGCTGCTTGCCAAGGCCCGCCCCGGCGGT P N Y I Y S L L E G Y A S D S P G E P A E W W V K Q Q Q E K

26~CCGAACTACATCTA~AGC~TG~TTGAAGGCTATGCGTCCGACAGCCCGGGCGAGCCGGCCGAATGGTGGGTC~GCAG~AGCAGGAGAAG G L E V A F N E A K Y F N D Y F P G H A I S M P P P L M Q D 270~GGTCTCGAGGTCGCCTTcAACGAGGCGAAGTACTTC~CGACTACTTCCCCGGCCACGCCATCTcGATGCCGCCGCCGCTGATGGACGAC L T T Y E D G T A A T K D Q M A Q D V V A Y L N W A A E P E 279•CTCATCACCTATGAGGACGGCACCGCCGCCACCAAGGATCAGATGGCTCAGGACGTCGTCGCCTATCTGAACTGGGCGGCCGAGCCGGAA L D A R K S L G L K V L L F L G V L T A M L L A L K L A I W 288•cTCGATGCCCGCAAGTCGCTGGGTCTCAAGGTTCTGCTGTTCCTGGGCGTTCTGACCGCCATGTTGCTGGCGCTGAAGCTGGCGATCTGG R D V K H ° 297•CGCGACGTCAAGCATTAAGAAACCGCTTTAACCGCCATCCTGCGCTAAACGGCCGCCGGCCCCCACCGGCGGCCGTTTTTTATTCGCCGC

3~6~CCCTCCC~G~GACGGGCTC~T~GCCTTGGTGGCTTTTCATC~GGG~GGTGGCGCGCTAAGGTGCCCCA~CCGCAAAAGGGTGAGCCAG 3~5~CCAGG~GAGGGGAAGACATGT~GAGGCGTATCGGCAGCCGGT~TTGGGGTC~cGGCGGAT~GGGGTTT~GATATC

Fig. 4. R. rubrum pet DNA and peptide sequence. The putative promoter sequence (underlined nucleotides), experimentally determined transcription start site (bold, underlined nucleotide), and potential transcription terminating secondary structures (doubly underlined nucleotides) are illustrated. The standard one letter amino acid lettering was used to designate protein primary sequences. Amino acids that differ from those reported by Majewski and Trebst (1990) are identified with an asterisk. Peptide sequences confirmed by amino acid sequence analysis are underlined.

87 A

Rieske protein

131 G V C T H L G C V P

140

150

160

M G D K S G D F G G

W F C P C H G S H Y

G V C T H L G C V P

I G G V S G D F G G

W F C P C H G S H Y

R.S

A S C T H L ~ C I P

L G H Q

W F C P C H G S Q Y

R.v

G V C T H L G C I P

L G Q K A G D P K ~ F D G W F C P C H G S H Y

B

Cytochrome

Qol

domain

140 M G

T

A

F M G

M :

A T A F M G Y V L : : : : : : :

M : M

b

R.r

protein

150 L P

YV

G D W G G

R.C

S

F

WG

W G Q M S : : : :

:

F W G A T : : : :

V

163 I T

R.c

V I T : : :

R.S

:

A T A F MG Y V L P : : : : : : : : : :

W G Q M S F WG A T : : : : : : : : : :

V I T : : :

R.v

A T A F M G Y V L P

W G Q M

V

R.r

QoII

:

:

P :

160 A T

W G Q M

S

F W G A T

I

T

domain

280 NY : : N Y

290 V Q A N P L S TP A : : : : : : : : I E AN P L S T P A

H : H

N Y : :

I P AN P G : : : : :

H I V P E W Y L : : : : : : :

L P : :

F Y A I L K A: : : : : : :

-

NY

I P

H

LP

F

--

A N P

V T P A : -" :

M V T

311 D V W V V I L V D G : : : : :

P

320

D V W V V Q I A N F

T

300 I VP E W Y F LP : : : ; : : : I : I VP E W Y F L P

I

VP

E W Y F

I

L

K A -

336 F G V I A M ::: ::

T S F G I I D A K F

F G V L A M .. :::

R.S

G G V L A M

R.r

. . . . . . . . . . . . . . .

V P D K L

-

R.c

domain

33 40 I M I P T P KN : : : : : : :

L N W W W : : :

I M

L N

I P

T

P

RN

FV~PV~N ~

YA

330 L T F G I V D A K F :--__ : : : : :

Qi I

310 I L R A F A A : : : : : : I L R A F T A

F YA : : : F YA

-

QIIX

~

~

50 I WG I V -_-. : : :

W M W

I W

G

VV

52 LA : :

R.C

L

R.S

A

I ~ Y ~ ; A I

LA

a.v

~

AG

~.~

F

~

s

~

domain

213 I WA F H T : : : : :

220 T G : :

N N N P : : :

I

T G : •

230 T G V E VR : : • : : : :

RT S K AD : : : : :

240 KD : :

AE :

N N N P T G V E v R : : : : : : :

R T S K A E A Q K D : :

V W A L ~ V ~ : : : : :

Q ~ P T G V E V K • . :

. . . . . .

S

E~D

M W A L

S

. . . . . .

G

P

W A : ,

F

H S :

H

V K K

241 T L P F W P Y F V I : : : : : : :

NN

250

P

L

G

I D

: AK

255 K D L F A ~: ::

R.C

T V P F W P Y F I I

K D V F A

R.S

T V ~ F T P F A L T

K D A V A

K.v

T I P F H P Y Y T V

K D A F G

R~r

:

FD

Fig. 5. Comparison of the amino acid sequences of portions of the R. rubrum cytochrome bc I complex subunits with the corresponding portions of analogous subunits from of other organisms. (A) Rieske iron-sulfur protein. (B) Cytochrome b. In both cases, the numbering is based on the Rb. capsulatus sequence. R.c., Rb. capsutatus; R.s., Rb. sphaeroides; R.v., Rps. viridis and R.r., R. rubrum.

88 charged residues R91, R i l l , R190, H214, likely to be in the vicinity of heme binding sites, are also conserved in R. rubrurn. The sequences PEWY, beginning at amino acid 285 and ILR, beginning at amino acid 295, that have been proposed to be important in quinol oxidation (Hauska et al. 1988) are also conserved. Translation of the petC coding sequence initiates at an A U G codon and terminates at a U A A codon. The petB, petC intercistronic region is 26 nucleotides in length and the putative ribosomal binding site is located 10 bases upstream from the start codon. The amino acid sequence of the petC gene product, deduced from the DNA sequence, predicts that the protein contains 272 amino-acids with a molecular mass of 29 560 Da. N-terminal amino acid analysis of the mature protein (Fig. 4) indicates that the first 24 residues constitute a hydrophobic leader peptide that is subsequently cleaved. Similar leader sequences have previously been found in the cytochromes c 1 of photosynthetic bacteria (Gabellini and Sebald 1986, Davidson and Daldal 1987a, Verbist et al. 1989, Yun et al. 1990, Majewski and Trebst 1990). The mature protein contains 248 amino acids with a molecular mass of 27 567 Da. This value is significantly lower than the value of 31 000 Da estimated from polyacrylamide electrophoresis of the mature R. rubrum cytochrome c I peptide (Kriauciunas et al. 1989, Purvis et al. 1990, Majewski and Trebst 1990). Hydrophobicity analysis indicates the likely presence of a single membrane-spanning helix near the C-terminus of cytochrome c 1. This C-terminal hydrophobic domain is thought to be involved in anchoring the protein to the membrane (Gabellini and Sebald 1986, Davidson and Daldal 1987a, Verbist et al. 1989, Yun et al. 1990, Majewski and Trebst 1990, Konisihi et al. 1991). Cytochromes c a contain a covalently bound heme attached to the protein by two cysteines, with histidine and a methionine serving as axial ligands to the heme iron (Simpkin et al. 1989, Hobbs et al. 1990). The CSTCH sequence beginning at amino acid 61 is likely to contribute the cysteines and the histidine involved in heme binding, with either methionine 200 or 224, which are well conserved among all the purple non-sulfur bacteria, serving as the other axial ligand (Gabellini and Sebald

1986, Davidson and Daldal 1987a, Verbist et al. 1989, Yun et al. 1990, Majewski and Trebst 1990). A comparison of the petC nucleotide sequence reported here with that sequence reported by Majewski and Trebst (1990) reveals one difference in 819 nucleotides. This difference occurs in the codon for amino acid 84 where we identify a sequence coding for arginine of the codon coding for glycine reported by Majewski and Trebst (1990). This assignment was confirmed by sequencing of both DNA strands of the petC gene and by the observation that treatment of cytochrome c~ with trypsin yields a peptide that includes amino acid residues 73 through 83. Arginine at position 84 produced no significant change in the hydropathy profile of the protein (data not shown) compared to that previously reported (Majewski and Trebst 1990). Analysis of the region downstream from the 3' end of the petC gene showed two regions of dyad symmetry which could serve as transcription terminators. One of the regions is 28 bp downstream from the stop codon of petC gene. It contains a G - C rich stretch followed by poly T, a sequence which shows strong resemblance to rho-independent transcription terminators in other bacteria. Two similar stable hairpin structures were observed in the pet operon of Rb. capsulatus (Gabellini and Sebald 1986, Davidson and Daldal 1987b) and were noted by Majewski and Trebst (1990) in their analysis of the R. rubrum pet operon. Codon usage in the R. rubrum pet operon, which has been previously reported by Trebst and Majewski (1990), shows considerable bias towards G or C in the third position, as expected from the high G + C content of the genes coding for the R. rubrum cytochrome bc~ complex. Ten codons ending with an A or T are not used in these genes. A similar pattern of codon usage has also been observed for the pet genes of other purple non-sulfur photosynthetic bacteria (Davidson and Daldal 1987a, Yun et al. 1990, Verbist et al. 1989). Interspecies complementation for the cytochrome bcI complex between the two purple non-sulfur species Rb. capsulatus and Rb. sphaeroides has been demonstrated (Davidson et al. 1989). The pet genes of Rb. sphaeroides

89 introduced into a cytochrome bcl-Rb, capsulatus strain yield cells that express the Rb. sphaeroides cytochrome bc 1 complex and exhibit photosynthetic growth (Davidson et al. 1989). The ability of the pet genes from R. rubrum to complement the Rb. capsulatus cytochrome bc I deletion mutant MT-RBC1 was tested. Tet r merodiploids obtained from a mating between E. coli HB101/ p9T1 and Rb. capsulatus MT-RBC1 were tested on RCV (Weaver et al. 1975) and MedA (Sistrom 1960) minimal media (malate and succinate based, respectively) as well as on MPYE rich media (Davidson et al. 1989). Although the strain Rb. capsulatus MT-RBC1/p9T1 grew well on these media in the presence of oxygen, it did not grow under photosynthetic growth conditions. This finding indicates that perhaps either the pet genes of R. rubrum do not initially produce active cytochrome bc 1 complex in Rb. capsulatus or that an active complex is present but does not interact with its reaction partners effectively enough to overcome the photosynthetically defective phenotype. The basis for this lack of complementation is presently unknown. Prolonged incubation (6-9 days) of Rb. capsulatus MT-RBC1/p9TI under photosynthetic growth conditions yielded rare colonies that gained the ability to grow photosynthetically on rich media. Transformation of Rb. capsulatus MT-RBC1 with plasmid isolated from Rb. capsulatus MT RBC1/p9T1 cells that had gained the ability to grow photosynthetically produced a similar pattern; i.e., rare colonies that grew photosynthetically only after prolonged incubation (S.-Y. Park and F. Daldal, unpublished observations). These results suggest that one or more mutational events in the Rb. capsulatus chromosome are required to obtain photosynthetically competent merodiploids.

Discussion

Previous studies of the R. rubrum cytochrome bc~ complex conducted in our laboratory had focused on the properties of the purified, detergent-solubilized complex (Wynn et al. 1986, Kriauciunas et al. 1989, Hobbs et al. 1990, Britt et al. 1991, Gfiner et al. 1991). In order to be able to analyze the functions of the complex in

terms of its structure at the molecular level, it was important to know the amino acid sequences of the three prosthetic group-containing subunits of the complex. After our efforts to sequence the genes coding for these peptides were well underway, sequences for the three genes of the R. rubrum pet operon were reported by Majewski and Trebst (1990). We have completed our sequencing of this operon and our data agree with the results of Majewski and Trebst (1990) at all but four nucleotide positions (see below). The earlier report of Majewski and Trebst (1990) presented no data on the R. rubrum pet operon transcript. Thus, our demonstration that in R. rubrum, as in Rb. capsulatus (Gabellini and Sebald 1986), transcription of the pet operon produces a single polycistronic transcript coding for all three genes of the operon has provided the first information on transcription of the pet operon in R. rubrum. Although additional experiments will be required to define the 3' termination point of the transcript, we have determined the 5' start of the transcript by both primer extension and nuclease protection. Not only have we also extended the characterization of the R. rubrum pet operon by analyzing the mRNA resulting from transcription of this operon, but we have also obtained preliminary results indicating that it may be possible to express the R. rubrum pet operon in Rb. capsulatus. Although considerable additional evidence must be obtained before it is conclusively established that the R. rubrum cytochrome bc 1 is actually present in the membranes of photosynthetically growing cells of a pet- Rb. capsulatus deletion strain transformed with a plasmid containing the R. rubrum pet operon, we have made significant preliminary progress in establishing a system for studying interspecies complimentation. We have also demonstrated that R. rubrurn mutants resistant to a quinone analog inhibitor of the cytochrome bct complex, antimycin A, can be isolated, something that has not been previously possible with other photosynthetic purple bacteria (see below). The amino acid sequence obtained for the R. rubrum cytochrome b in this study is identical to that reported previously (Majewski and Trebst 1990). However, the amino acid sequences that we have obtained for the R. rubrum cytochrome

90 c 1 and Rieske iron-sulfur protein subunits differ from those reported by Majewski and Trebst (1990) at four positions, three in the Rieske protein and one in cytochrome c~. In the case of the difference in cytochrome c~, we have confirmed our assignment with sequence data from both DNA strands and by an analysis of the tryptic digestion pattern obtained with the purified cytochrome c 1. In one position of the Rieske protein, where the difference is a conservative change from serine (Majewski and Trebst 1990) to threonine (this work), we have confirmed our assignment by amino acid sequencing. In the other two positions in the Rieske protein where the two sequences differ, both of which involve the replacement of an uncharged residue (Majewski and Trebst 1990) by a charged residue (this work), DNA sequence data from only one strand is available and, unfortunately, peptides that could have provided confirmatory sequence information were not obtained. We have no explanation for the differences in the sequences obtained by the two groups except to attribute them to possible differences in the R. rubrum strains used in the two laboratories. Majewski and Trebst (1990) have provided a complete discussion of possible correlations between the structure and function of the cytochrome c 1 and Rieske iron-sulfur protein subunits of the R. rubrum complex and of several aspects of the structure of the R. rubrum cytochrome b. We will confine our analysis to the possible location of quinone and inhibitor binding sites on cytochrome b. Previous genetic studies of spontaneous quinol oxidation (Q0) inhibitor-resistant mutations in yeast (diRago and Colson 1988, diRago et al. 1989), mouse (Howell and Gilbert 1988) and Chlamydomonas reinhardtii mitochondria (Bennoun et al. 1991) and in the photosynthetic bacterium Rb. capsulatus (Daldal et al. 1989) indicated that they were located in two discrete regions of cytochrome b delimited by the amino acid residues 140 to 163 for the Q01 and 279 to 336 for the QolI domains (Fig. 5B), both thought to be located on the same, outer (periplasmic) side of the lipid bilayer. (Numbering based on the Rb. capsulatus cytochrome b sequence will be used throughout this discussion.) A comparison

of the Q01 domain of the cytochrome b subunit of the cytochrome bc I complex from the four purple non-sulfur bacteria, Rb. capsulatus (Davidson and Daldal 1987), Rb. sphaeroides (Yun et al. 1990), Rps. viridis (Verbist et al. 1989) and R. rubrum (Majewski and Trebst 1990, this work) indicates that the inhibitorresistance conferring positions M140, F144, G152 and T163, as well as the Q0-defective site G158 (Daldal et al. 1989), are entirely conserved within these species. Furthermore, 23 of the 24 residues located between positions 140 and 163 of the Q01 region are identical, with the only difference occurring at position 141 (Fig. 5B). At this latter position glycine is found in Rb. capsulatus instead of the alanine found in the three other species. The cytochrome b QoII domain residues N279, V333 and M336, where mutations were observed to confer resistance to Q0 inhibitors in several organisms (diRago et al. 1989, Daldal et al. 1989), are entirely conserved between the four phototrophic bacteria compared here (Fig. 5B). At position 298, where changes from the wild type leucine were observed to provide resistance to myxothiazol in yeast mitochondria, only the Rps. viridis cytochrome b among the photosynthetic bacteria contains leucine, while it is replaced by phenylalanine in the remaining three other species. In the QoII domain amino acid sequence variations are observed in the positions 281,282, 286, 287, 290, 326-28, 330-31 and 334 (see Fig. 5B), while the other positions located between the residues 279 and 336 of cytochrome b are entirely conserved. The overall high degree of conservation of the amino acid residues located in these specific Q01 and QoII regions of cytochrome b and the available data on the nature and location of spontaneous Q0-inhibitor resistant mutations in various systems allow one to predict that the cytochrome bc 1 complex of Rps. viridis and R. rubrum should be sensitive to the Q0-inhibitors like those of Rb. capsulatus and Rb. sphaeroides. In the case of R. rubrum, this prediction based on the analysis of genetic experiments, is in complete agreement with previous biochemical experiments which established that purified complex from R. rubrum (Wynn et al. 1986, Kriauciunas et al. 1989, G/iner et al. 1991) is sensitive to Q0 inhibitors in vitro. The

91 sensitivity of the isolated Rps. viridis cytochrome bc 1 complex to these inhibitors has not yet been tested. Finally, it should be noted that an 18 amino acid long sequence located between the positions 307 and 326 is present in Rb. capsulatus and Rb. sphaeroides but is absent in Rps. viridis and R. rubrum (Fig. 5B). The structural and functional significance, if any, of this insertion is currently unknown. Among the non-photosynthetic bacteria, this insert is also present in the cytochrome b of the P. denitriticans cytochrome b c I complex (Kurowski and Ludwig 1987), but absent in that of B. japonicum (Thony-Meyer et al. 1989). Mitochondrial mutations affecting the Qi site of the cytochrome bcl complex were also found in two discrete regions located between residues 33-52 and 213-255 (Fig. 5B) of the cytochrome b polypeptide, referred to as the QiI and Q~II domains (diRago and Colson 1988, diRago et al. 1989, Howell and Gilbert 1988). These regions are thought to be located on the same inner (cytoplasmic) side of the membrane. It should be noted that, like the QoII domain, the Q i l I region of cytochrome b contains a short insertion of 7 amino acid residues of unknown role between the positions 231-237 in Rb. capsulatus, Rb. sphaeroides and P. denitrificans which is absent in Rps. viridis, R. rubrum and B. japonicum. Previous genetic studies aimed at isolating spontaneous mutations resistant to Qi-inhibitors in photosynthetic bacteria (Daldal et al. 1989) revealed that, although the cytochrome bc I complexes of Rb. capsulatus and Rb. sphaeroides are known to be inhibited by Q~ inhibitors in vitro (Dutton 1986), cells of these bacteria are naturally resistant to various Q~ inhibitors (e.g., antimycin and funiculosin) under photosynthetic growth conditions in vivo. This finding, which has prevented the isolation of the Qi mutations in these bacteria, has limited our current knowledge about the location of the Q~ site to information gained from studies on mitochondrial cytochrome b. A comparison of the Q~I and QilI regions of the cytochrome b from these four phototrophic species indicate that, of the residues at positions 33, 46 and 52 at the Q~I and 213, 248, 251 and 255 at the QilI regions known to provide resistance to Qi inhibitors in mitochondria, only position 251 is absolutely con-

served (Fig. 5B). A further comparison of amino acid residues in this region of cytochrome b indicates that at positions 46, 52, 251 and 255 (N, G, K and G, respectively) R. rubrum is the only photosynthetic bacterium that contains the same amino acid residues found in the mitochondrial cytochromes. This resemblance between R. rubrum and mitochondria suggested that if the molecular basis of Qi-inhibitor resistance in vivo observed in Rb. capsulatus and Rb. sphaeroides is due to single amino acid substitutions, then R. rubrum, unlike Rb. capsulatus or Rb. sphaeroides, might be sensitive to some Qi-inhibitors in vivo. This hypothesis was tested, and it was found that 10 -6 M antimycin inhibited photosynthetic growth of wild type R. rubrum strain S1 on MPYE rich media. Several antimycinresistant mutants of R. rubrum have been isolated, but it is not yet known whether these mutants carry mutations directly affecting the cytochrome bc 1 complexes (S.-Y. Park and F. Daldal, unpublished observations). Work designed to characterize these antimycin-resistant mutants is currently in progress in our laboratories. Antimycin-resistant mutants of R. rubrum have also been isolated in the laboratory of A. Trebst (personnel communication). Should these antimycin-resistant mutants contain amino acid changes in cytochrome b, they should prove helpful in further defining the structure and location of the 0 i site.

Acknowledgements The authors gratefully acknowledge Prof A. Trebst for sharing DNA sequence information prior to its publication, Ms Ida Schaefer for the synthesis of oligonucleotides and Dr Clive Slaughter for his assistance with the peptide sequencing. Supported by grants from NSF (DMB-8806609 to D.B.K), the U.S. Department of Agriculture (9101230 to D.B.K.), the Texas Advanced Research Program (003644-041 to J.G.H.), and NIH (GM 37238 to F.D.).

References Aebersold RH, Leavitt J, Saavendra RA, Hood LE and Kent SBH (1987) Internal amino acid sequence analysis of

92 proteins separated by one- or two-dimensional gel electrophoresis after in situ protease digestion on nitrocellulose. Proc Natl Acad Sci USA 84:6970-6974 Andrews KM, Crofts AR and Gennis RB (1990) Large-scale purification and characterization of a highly active foursubunit cytochrome bG complex from Rhodobacter sphaeroides. Biochemistry 29:2645-2651 Atta-Afaso-Adjei E and Daldal F (1991) Size of the amino acid side chain at position 158 of cytochrome b is critical for an active cytochrome bc~ complex and for photosynthetic growth of Rhodobacter capsulatus. Proc Natl Acad Sci USA 88:492-496 Bennoun P, Delosme M and Kiick U (1991) Mitochondrial genetics of Chlarnydomonas reinhardtii: Resistance mutations marking the cytochrome b gene. Genetics 127: 335343 Britt RD, Sauer K, Klein MP, Knaff DB, Kriauciunas A, Yu CA, Yu L and Malkin R (1991) Electron spin echo envelope modulation spectroscopy supports the suggested coordination of two hisitidine ligands to the Rieske Fe-S centers of the cytochrome b6f complex of spinach and the cytochrome bc~ complexes of Rhodospirillum rubrum, Rhodobacter sphaeroides R-26 and bovine heart mitochondria. Biochemistry 30:1892-1901 Chomczynebi P and Sacchi N (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenolchloroform extraction. Anal Biochem 162:156-159 Cully M, Jay FA, Gabellini N and Oesterhelt D (1989) Characterization of the bcx complex in Rhodopseudomonas viridis. In: Barber J and Malkin R (eds) Techniques and New Developments in Photosynthesis Research, pp 287- 289. Plenum Press: New York Daldal F, Davidson E and Cheng S (1987) Isolation of the structural genes for the Reiske Fe-S protein, cytochrome b and cytochrome G, all components of the ubiquinol: cytochrome c 2 oxidoreductase complex of Rhodopseudomonas capsulata. J Mol Biol 195:1-12 Daldal F, Tokito MK, Davidson E and Faham M (1989) Mutations conferring resistance to quinol oxidation (Qz) inhibitors of the cyt bc~ complex of Rhodobacter capsulatus. EMBO J 8:3951-3961 Davidson E and Daldal F (1987a) Primary structure of the bG complex of Rhodopseudornonas capsulata. J Mol Biol 195:13-24 Davidson E and Daldal F (1987b) fbc operon, encoding the Rieske Fe-S protein, cytochrome b and cytochrome c~ apoproteins, previously described from Rhodopseudomonas sphaeroides, is from Rhodopseudornonas capsulata. J Mol Biol 195:25-29 Davidson E, Prince RC, Haith CE and Daldal F (1989) The cytochrome bc~ complex of Rhodobacter sphaeroides can restore cytochrome c2-independent photosynthetic growth to a Rhodobacter capsulatus mutant lacking cytochrome bc~. J Bact 171:6059-6068 diRago JP and Colson A-M (1988) Molecular basis for resistance to antimycin and diuron, Q-cycle inhibitors acting at the Q~ site in the mitochondrial ubiquinol-cytochrome c reductase in Sacchrornyces serevisiae. J Biol Chem 263:12564-12570 diRago JP, Coppee J-Y and Colson AM (1989) Molecular basis for resistance to myxothiazol, mucidin (strobilurin A) and stigmatellin. Cytochrome b inhibitors acting at the

center o of the mitochondrial ubiquinol-cytochrome c reductase in Sacchromyces serevisiae. J Biol Chem 264 14543-14548 Ditta G, Schmidhauser T, Yakobson E, Lu P, Liang X, Finlay D, Guiney D and Helinsky D (1985) Plasmid related to the broad host range vector, pRK290, useful for gene cloning and monitoring gene expression. Plasmid 13: 149-153 Dutton PL (1986) Energy transduction in anoxygenic photosynthesis. In: Staehlin LA and Arntzen CA (eds) Encyclopedia of Plant Physiology new series, Vol. 19, Photosynthesis III, pp 197-237. Springer-Verlag, Berlin Gabellini N and Sebald W (1986) Nucleotide sequence and transcription of the fbc operon from Rhodopseudornonas sphaeroides. Eur J Biochem 154:569-579 Gibson J, Stackebrant E, Zablen LB, Gupta R and Woese CR (1979) Curr Microbiol 3:59-64 Glaser EG, Meinhardt SW and Crofts AR (1984) Reduction of cytochrome b-561 through the antimycin-sensitive site of the ubiquinol-cytochrome c 2 oxidoreductase complex of Rhodopseudomonas sphaeroides. FEBS Lett 178:336-342 Grisshammer R, Wiessner C and Michel H (1990) Sequence analysis and transcriptional organization of the Rhodo° pseudornonas viridis cytochrome c 2 gene. J Bact 172: 50715078 Giiner S, Robertson DE, Yu L, Qiu Z, Yu C-A and Knaff DB (1991) The Rhodospirillum rubrum cytochrome bc 1 complex: redox properties, inhibitor sensitivity and proton pumping. Biochim Biophys Acta 1058:269-279 Gurbiel RJ, Batie CJ, Sivaraja M, True AE, Fee JA, Hoffman BM and Ballou DP (1989) Electron-nuclear double resonance spectroscopy of 15N-enriched phthalate dioxygenase from Pseudomonas cepacia proves that two histidines are coordinated to the [2Fe-2S] Rieske-type clusters. Biochemistry 28:4861-4871 Gurbiel RJ, Ohnishi T, Robertson DE, Daldal F and Hoffman BM (1991) Q-band ENDOR spectra of the Rieske protein from Rhodobacter capsulatus ubiquinol:cytochrome c oxidoreductase show two histidine ligands coordinated to the [2Fe-2S] cluster. Biochemistry 30:11579-11584 Hanahan D (1983) Studies of transformation of Escherichia coli with plasmids. J Biol Chem 166:557-580 Hauska G, Hurt E, Gabellini N and Lockau W (1983) Comparative aspects of quinol-cytochrome c/plastocyanin oxidoreductases. Biochim Biophys Acta 726:97-133 Hauska G, Nitschke W and Herrmann RG (1988) Amino acid identities in the three redox center-carrying polypeptides of cytochrome bG/b6f complexes. J Bioenerg Biomembr 20:211-228 Hobbs JD, Kriauciunas A, G/iner S, Knaff DB and Ondrias MR (1990) Resonance Raman spectroscopy of cytochrome bG complexes from Rhodospirillum rubrum: initial characterization and reductive titrations. Biochim et Biophys Acta 1018:47-54 Howell N and Gilbert K (1988) Mutational analysis of the mouse mitochondrial cytochrome b gene. J Mol Biol 203: 607-618 Hurt E and Hauska G (1981) A cytochrome fib 6 complex of five polypeptides with plastoquinol-plastocyanin-oxidoreductase activity from spinach chloroplasts. Eur J Biochem 117:591-599 Kallas T, Spiller S and Malkin R (1988a) Primary structure of

93 cotranscribed genes encoding the Rieske Fe-S and cytochrome f proteins of the cyanobacterium Nostoc PCC 7906. Proc Natl Acad Sci USA 85:5794-5798 Kallas T, Spiller S and Malkin R (1988b) Characterization of two operons encoding the cytochrome b6-f complex of the cyanobacterium Nostoc PCC 7906. J Biol Chem 263: 14334-14342 Konishi K, Van Doren SR, Kramer DM, Crofts AR and Gennis RB (1991) Preparation and characterization of the water-soluble heme-binding domain of cytochrome c~ from the Rhodobacter sphaeroides b G complex. J Biol Chem 266:14270-14276 Kriauciunas A, Yu L, Yu C-A, Wynn RM and Knaff DB (1989) The Rhodospirillum rubrum cytochrome bG complex: peptide composition, prosthetic group content and quinone binding. Biochim Biophys Acta 976:70-76 Krinner M, Hauska G, Hurt E and Lockau W (1982) A cytochrome f-b 6 complex with plastoquinol-cytochrome c oxidoreductase activity from Anabaena variabilis. Biochim Biophys Acta 681:110-117 Kulia D, Fee JA, Schoonover JR, Woodruff WH, Batie CJ and Ballou DP (1987) Resonance Raman spectra of the [2Fe-2S] clusters of the Rieske protein from Thermus and phthalate dioxygenase from Pseudornonas. J Am Chem Soc 109:1559-1561 Kurowski B, and Ludwig B (1987) The genes of the Paracoccus denitrificans b G complex. Nucleotide sequence and homologies between bacterial and mitochondrial subunits. J Biol Chem 262:13805-13811 Ljungdahl PO, Pennoyer J, Robertson D and Trumpower (1987) Purification of highly active cytochrome bc 1 complexes from phylogenetically diverse species by a single chromatographic procedure. Biochim Biophys Acta 89l: 227-241 Majewski C and Trebst A (1990) The pet genes of Rhodospirillurn rubrum: Cloning and sequencing of the genes for the cytochrome bG-complex. Mol Gen Genet 224: 373382 Maniatis T, Fritsch E and Sambrook J (1982) Molecular cloning: A laboratory manual. Cold Spring Harbor, New York Matsudaira P (1987) Sequence from picomole quantities of proteins electroblotted onto polyvinylidene diflouride membranes. J Biol Chem 262:10035-10038 Meinkoth J and Wahl G (1984) Hybridization of nucleic acids immobilized on solid supports. Anal Biochem 138: 267284 Purvis DJ, Theiler R and Niederman RA (1990) Chromatographic and protein chemical analysis of the ubiquinolcytochrome c 2 oxidoreductase isolated from Rhodobacter sphaeroides. J Biol Chem 265:1208-1215 Robertson DE and Dutton PL (1988) The nature and magnitude of the charge-separation reactions of ubiquinol cytochrome c, oxidoreductase. Biochim Biophys Acta 935: 273-291 Sanger F, Nicklen S and Coulson AR (1977) DNA sequencing with chain termination inhibitors. Proc Natl Acad Sci USA 74:5463-5467 Seidman J, Smith A and Struhl K (1987) Current Protocols in Molecular Biology. John Wiley and Sons. Inc, New York Sidhu A and Beattie D (1982) Purification and polypeptide

characterization of complex III from yeast mitochondria. J Biol Chem 257:7879-7886 Simpkin D, Palmer G, Devlin F, Mckenna M, Jensen G and Stephens P (1989) The axial ligands of hemes in cytochromes: A near-infrared magnetic circular dichroism study of yeast cytochromes c, c I , b and spinach cytochrome f. Biochemistry 28:8033-8039 Sistrom WR (1960) A requirement for sodium in the growth of Rhodopseudomonas sphaeroides. J Gen Microbiol 22: 778-785 Summers WC (1970) A simple method for extraction of RNA from E. coli using diethyl pyrocarbonate. Anal Biochem 33:459-463 Teintze M, Slaughter M, Weiss H and Neupert W (1982) Biogenesis of mitochondrial ubiquinol:cytochrome c reductase (cytochrome b G complex) J Biol Chem 257: 1036410371 Telser J, Hoffman BM, Lobrutto R, Ohnishi T, Tsai AL, Simpkin D and Palmer G (1987) Evidence for N coordination to Fe in the [2Fe-2S] center in yeast mitoehondrial complex III. FEBS Lett 214:117-121 Thony-Meyer LD, Stax D and Henneke H (1989) An unusual gene cluster for the cytochrome b G complex in Bradyrhrizobium japonicum and its requirement for effective root nodule symbiosis. Cell 57:683-697 Trumpower BL (1990) The protonmotive Q cycle. J Biol Chem 265:11409-11412 Usui S and Yu L (1991) Subunit IV (M r = 14384) of the cytochrome b-c~ complex from Rhodobacter sphaeroides: Cloning, DNA sequencing, and ubiquinone-binding domain. J Biol Chem 266:15644-15649 Verbist J, Lang F, Gabellini N and Oesterhelt D (1989) Cloning and sequenci,g of the fbcF, B and C genes encoding the cytochrome b/c t complex from Rhodopseudomonas viridis. Mol Gen Genet 219:445-452 Weaver PF, Wall JD and Gest H (1975) Characterization of Rhodopseudomas capsulatus. Arch Microbiol 195:207-216 Weiss H and Kolb HJ (1979) Isolation of mitochondrial succinate-ubiquinone oxidoreductase, cytochrome c reductase and cytochrome c oxidase from Neurospora crassa using non-ionic detergent. Eur J Biochem 99:139-149 Widger WR, Cramer WA, Herrmann RG and Trebst A (1984) Sequence homology and structural similarity between cytochrome b of mitochondrial complex Ill and the chloroplast b6-f complex: Position of the cytochrome b hemes in the membrane. Proc Natl Acad Sci USA 81: 674-678 Willims I, Malkin R and Chain RK (1987) Oxidation-reduction reactions of cytochrome b6 in a liposome-incorporated cytochrome b~:f complex. Arch Biochem Biophys 258: 248-258 Wynn RM, Gaul DF, Choi W-K, Shaw RW and Knaff DB (1986) Isolation of cytochrome bG complexes from the photosynthetic bacteria Rhodopseudomonas viridis and Rhodospirillum rubrum. Photosynth Res 9:181-195 Yang X and Trumpower BL (1986) Purification of a threesubunit ubiquinol-cytochrome c oxidoreductase complex from Paracoccus denitrificans. J Biol Chem 261: 1228212289 Yu L and Yu C-A (1991) Essentiality of the molecular weight 15 000 protein (Subunit IV) in the cytochrome b G complex of Rhodobacter sphaeroides. Biochemistry 30:4934-4939

94 Yu L, Mei Q-C and Yu C-A (1984) Characterization of purified cytochrome bc I complex from Rhodopseudomonas sphaeroides R-26. J Biol Chem 259: 57525760 Yun C-H, Beci R, Crofts AR, Kaplan S and Gennis RB (1990) Cloning and DNA sequencing of the fbc operon encoding the cytochrome bcl complex from Rhodobacter sphaeroides. Eur J Biochem 194:399-411 Yun C-H, Van Doren SR, Crofts AR and Gennis RB (1991a)

The use of gene fusions to examine the membrane topology of the L-subunit of the photosynthetic reaction center and the cytochrome b subunit of the b c I complex from Rhodobacter sphaeroides. J Biol Chem 266:10967-10973 Yun C-H, Crofts AR and Gennis RB (1991b) Assignment of the histidine axial ligands to the cytochrome b H and cytochrome b L complex from Rhodobacter sphaeroides by sitedirected mutagenesis. Biochemistry 30:6747-6754