Rhodobacter sphaeroides 2.4.1T respires aerobically via a branched respiratory chain consisting of both cytochrome c oxidases and quinol oxidases. Here, genes from chromosome II encoding two distinct quinol oxidases have been characterized. The qoxBA genes encode a putative heme-copper quinol oxidase, whereas the qxtAB genes encode a quinol oxidase homologous to the cyanide-insensitive oxidase of Pseudomonas aeruginosa. No phenotype was observed for mutations in either oxidase in the wild-type background. A strain containing a qxtA mutation in a cytochrome bc1 complex mutant background was unable to grow aerobically. No role was found for the Qox oxidase, nor was a qoxB::lacZ transcriptional fusion expressed under a variety of conditions. These are the first molecular studies to characterize the quinol oxidases of R. sphaeroides 2.4.1T.
Like many bacteria, the facultative phototroph Rhodobacter sphaeroides 2.4.1T possesses a branched energy-transducing electron-transfer chain (reviewed in ). Under aerobic growth conditions, the respiratory chain consists of both cytochrome c-dependent and -independent branches. The aa3-type cytochrome c oxidase, encoded by the cta genes, is a classic member of the heme-copper superfamily of bacterial oxidases containing the binuclear heme a3-copper (CuB) center as the active site . cta Genes are repressed when the O2 tension is lowered, in accordance with the proposed role of the aa3 oxidase as a low-affinity oxidase functional under high O2 conditions .
A second cytochrome c oxidase is predominantly synthesized under low O2 concentrations as well as photosynthetic growth conditions . This oxidase contains heme b and CuB at the binuclear center, and has thus been termed the cbb3 oxidase. This oxidase lacks CuA and has two membrane-bound cytochromes c in place of subunits II and III. The cbb3 oxidase is encoded by the ccoNOQP gene cluster whose expression is induced by the FnrL protein when the O2 tension in the growth medium is lowered to 2%. A growing body of evidence points to an additional role of the cbb3 oxidase in the generation of a redox-dependent regulatory signal for anaerobic gene expression in R. sphaeroides[6–8].
A cytochrome bc1 mutant of R. sphaeroides is able to grow aerobically, suggesting the presence of a functional quinol oxidase . In addition, a CtaD CcoN double mutant was capable of ubiquinol oxidation in the presence of myxothiazol, a cytochrome bc1 inhibitor . In this study, we have identified and characterized two distinct genetic loci which contain genes homologous to previously sequenced bacterial quinol oxidases.
2 Materials and methods
2.1 Bacterial strains, plasmids and growth conditions
Bacterial strains and plasmids used or constructed in this work are listed in Table 1. Escherichia coli strains were grown at 37°C in Luria–Bertani medium, and R. sphaeroides strains were grown as previously described for the conditions employed here . Antibiotics were used as follows to maintain selection for plasmids or to select for recombinant strains: ampicillin (Amp), 100 μg ml−1 (E. coli); kanamycin, 25 μg ml−1 (R. sphaeroides and E. coli); spectinomycin (Sp), 25 μg ml−1 (R. sphaeroides and E. coli); streptomycin (St) 25 μg ml−1 (R. sphaeroides and E. coli); and tetracycline (Tc), 1 μg ml−1 (R. sphaeroides) and 10 μg ml−1 (E. coli).
PLA2917 derivative+ca. 20 kb of R. sphaeroides 2.4.1T DNA containing qoxB qoxA
PLA2917 derivative+ca. 20 kb of R. sphaeroides 2.4.1T DNA containing qxtA qxtB
Kmr, sacB RP4-oriT ColE1-oriV
PLO1 from which two PstI sites are removed
PRK415 containing 5.5-kb BamHI–HindIII fragment (fbcFBC)
PBS II containing 3001-bp fbcABC PCR product
PBS II containing 1.65-kb fragment (fbcAfbcB″fbcC)
PLO1-PstI containing 1.65-kb SphI fragment (fbcAfbcB″fbcC)
PBCΔ3 containing ΩTpr inserted into PstI site of fbcB″fbcC
PBSII containing 531-bp qxtA′ PCR product
PML5 containing qxtA::lacZ
PBS II containing 2.5-kb PstI fragment (‘qoxB qoxA’)
PUI1087 containing 865-bp ′qxtA qxtB′ PCR product
pNMT125 containing ΩStr/Spr cassette inserted into StuI site of qoxA
pNMT126 containing ΩStr/Spr cassette inserted into BamHI site of qxtA
PSUP202 containing 4.5-kb PstI fragment from pNMT129
PSUP202 containing 2.8-kb PstI fragment from pNMT132
2.2 Construction of quinol oxidase mutants
To construct an insertion mutation in qoxA, plasmid pNMT125 was linearized with StuI and was ligated to SmaI-digested ΩSpr/Str cassette from pHP45 ΩSpr/Str to give plasmid pNMT129. The 4.5-kb PstI fragment from pNMT129 was cloned into PstI-digested pSUP202 resulting in plasmid pNMT133.
A qxtA insertion mutation was constructed by first amplifying a 865-bp fragment from pNMT97 using primers QXTMUT1 (5′-GCGAATTCACCATGCTGACCGCG-3′) and QXTMUT2 (5′-GGGCCTGCAGCGCATCTCCTGCG-3′). BamHI-digested ΩSpr/Str cassette was cloned into the BamHI site of qxtA in pNMT126, resulting in plasmid pNMT132. The 3.1-kb EcoRI–PstI fragment from pNMT132 was cloned into EcoRI–PstI-digested pSUP202 to give plasmid pNMT134.
The qoxB::Ω and qxtA::Ω insertion mutation pSUP202-derivative plasmids were conjugated into R. sphaeroides 2.4.1T using triparental matings with the suicide vector pRK2013. The integration sites for the antibiotic-resistance cassettes in the R. sphaeroides genome were confirmed by non-radioactive Southern hybridizations of restriction digests of genomic DNA probed with appropriate DNA fragments. Labeling of DNA probes and detection of hybridized sequences by chemiluminescence were performed using a NEBlot Phototope kit (New England Biolabs Inc., Beverly, MA, USA) according to the manufacturer's instructions.
2.3 Construction of a cytochrome bc1 complex mutant
In order to construct a cytochrome bc1 complex mutant, a 3001-bp fragment from pRK:BC was amplified by PCR using primers BC+ (5′-GTGGGCATGCAGCGGCCCGAGG-3′) and BC− (5′-CCGAGCATGCGCGCCGCTTCGC-3′) and Pfu Turbo polymerase (Stratagene). The PCR product was cloned into EcoRV–SmaI-digested pBSII to give plasmid pBCΔ1. A 1.35-kb PstI fragment containing part of the fbcB and fbcC genes was removed from pBCΔ1 by digestion with PstI, and self-ligation yielded plasmid pBCΔ2. To remove two PstI restriction sites from the suicide vector pLO1, the vector was digested with PstI, blunt-ended by treatment with the Klenow fragment of E. coli polymerase I, and self-ligated to give plasmid pLO1-PstI. A 1.65-kb SphI fragment from pBCΔ2 was cloned into SphI-digested pLO1-PstI generating plasmid pBCΔ3.
Finally, a 2.5-kb PstI fragment containing the trimethoprim-resistance cassette from pUI1680 was cloned into PstI-digested pBCΔ3, resulting in plasmid pBCΔ4. Plasmid pBCΔ4 was transferred from E. coli strain S17-1 into R. sphaeroides strains by conjugation. Heterogenotes of R. sphaeroides, generated by a single recombination event, were selected for their kanamycin resistance on SIS agar plates incubated under aerobic conditions, and homogenotes, containing a double crossover, were obtained from the heterogenotes after a second recombination for sucrose resistance on SIS agar plates containing 15% w/v sucrose, 1% w/v bactotryptone, 0.5% w/v yeast extract, 0.5% w/v NaCl, 0.5% v/v DMSO and trimethoprim under dark-anaerobic growth conditions. The allelic exchange in the homogenotes was verified by Southern hybridization. This mutant strain, as expected, does not grow photosynthetically. A more complete characterization of this mutant is in progress.
2.4 Construction of a qxtA::lacZ reporter fusion plasmid
The upstream regulatory sequence (URS) of the qxtA gene was amplified by PCR using the primers QXTP1 (5′-GGGCTGAGGATCCCGATAAACG-3′) and QXTP2 (5′-GGCGGCAAGCTTGAGGTAGG-3′), generating a 531-bp product. The PCR product was purified, digested with BamHI and HindIII and cloned into BamHI–HindIII-digested pBSII, resulting in plasmid pNMT108. The cloned qxtA URS was subcloned into the promoterless lacZ transcriptional fusion vector, pML5, using BamHI–HindIII double digestions, resulting in plasmid pNMT109. pNMT109 was conjugated into R. sphaeroides 2.4.1T by triparental matings with pRK2013.
2.5 Construction of a qoxB::lacZ reporter fusion plasmid
The upstream regulatory region of qoxB was amplified by PCR using primers Eug1 (5′-GTCCTCGCCAAGCCCGATCTGG-3′) and Eug2 (5′-AAGAACAGACGGAGAGCCGTATC-3′) to generate a 513-bp product. The PCR product was purified and cloned into EcoRV-digested pBSII, resulting in plasmid pGak140. The qoxB URS was subcloned into pML5 to give plasmid pGak141.
2.6 DNA sequencing
Automated DNA sequencing was performed using an ABI 373A automatic DNA sequencer (Applied Biosystems Inc., Foster City, CA, USA) at the DNA Core Facility of the Department of Microbiology and Molecular Genetics, The University of Texas Health Science Center, Houston, TX, USA.
2.7 Cell extract preparation and assays of β-galactosidase activity
Preparation of crude cell extracts and β-galactosidase activities were performed as described previously , using reagent-grade o-nitrophenyl-β-D-galactopyranoside as the substrate. Cell extract protein concentrations were determined using the Pierce bicinchoninic acid protein assay reagent (Pierce, Rockford, IL, USA) with bovine serum albumin as a reference standard.
2.8 Nucleotide sequence accession numbers
The nucleotide sequences of the qoxBA and qxtAB genes have been deposited into the nucleotide sequence databases with the respective accession numbers AF111945 and AF084032.
3 Results and discussion
3.1 Sequence of quinol oxidase genes from R. sphaeroides 2.4.1T
As part of our sequencing project of chromosome II (CII) of R. sphaeroides 2.4.1T, we identified several sequences possessing significant homology to previously sequenced genes for quinol oxidases from various bacterial species . We identified two distinct operons, which we term qox and qxt (Fig. 1).
Physical map of (A) the qoxBA operon and, (B) the qxtAB operon of R. sphaeroides 2.4.1T. The large arrows indicate the direction of transcription. The small arrow in front of qxtA indicates a putative promoter. The functions of the gene products are shown below the genes.
3.2 qoxB and qoxA
The qoxBA operon maps to the AseI E-fragment from CII, close to the previously sequenced dorCBA operon and is present on cosmid pUI8656 . The putative qoxBA gene products represent new members of the heme-copper oxidase superfamily . The qoxB gene product consists of 261 amino acids and shares significant homology (32% identity, 53% similarity) with the Cox2 protein of Bacillus firmus which is the subunit II of the aa3 cytochrome c oxidase in that organism . However, Bacillus spp. possess both an aa3-type quinol oxidase and a cytochrome c oxidase. Similar to the subunit II gene products of other quinol oxidases, QoxB lacks the residues required for CuA binding in cytochrome c oxidases.
The qoxA gene is predicted to encode a protein of 825 amino acids which shares significant homology (39% identity, 58% homology) with the CoxI protein of B. firmus. This protein represents subunit I of the aa3 cytochrome c oxidase in B. firmus and possesses conserved residues for binding of the CuB-heme binuclear center. Interestingly the conserved Lys residue (Lys362 in the R. sphaeroides aa3 oxidase) shown to be important for proton translocation to heme aa3 via the ‘K-channel’ in the aa3 oxidase, is replaced by a Gln residue in QoxA . The implications of this substitution on the activity of the Qox enzyme are unknown at present.
3.3 qxtA and qxtB
The qxtAB operon is located on the AseI D-fragment from CII, approximately 200 kb from the qoxBA operon, and is present on cosmid pUI8736. The products of both genes possess extensive homology to the cyanide-insensitive quinol oxidase (Cio) of Pseudomonas aeruginosa[15,16]. This enzyme is related to the cytochrome bd oxidases of E. coli and Azotobacter vinelandii, enzymes which are not members of the heme-copper oxidase superfamily and which do not possess a heme-copper bimetallic center [2,15]. The qxtA gene encodes a putative product of 465 residues which has approximately 61% identity and 74% similarity with the CioA protein of P. aeruginosa. Residues implicated in quinol binding are conserved in QxtA, as are the His and Met residues implicated in the binding of heme b in the related enzymes. The conservation of all these sites suggests that the Qxt oxidase is a quinol oxidase.
qxtB is predicted to encode a 335 amino acid protein with 53% identity and 70% similarity to the CioB gene product from P. aeruginosa. Similar to CioB, QxtB lacks the conserved Cys and His residues suggested to be involved in the binding of a heme ligand in the cytochrome bd quinol oxidases from E. coli and A. vinelandii.
The start and stop codons between qxtA and qxtB overlap, suggesting that the qxtAB genes are transcribed in a single transcriptional unit. 3′ To qxtB is a stem-loop structure, similar to those associated with rho-independent transcriptional termination in bacteria, suggesting that the qxtAB genes form a complete transcriptional unit.
3.4 Preliminary characterization of quinol oxidase mutants
To investigate the roles of the Qox and Qxt quinol oxidases, mutations in genes encoding each of the quinol oxidases were introduced into R. sphaeroides wild-type and cytochrome bc1 mutant backgrounds.
3.5 Doubling times
Doubling times were calculated from triplicate cultures grown at high O2 tension. The results are presented in Table 2. No differences were seen for single oxidase mutants compared to the wild-type strain. A cytochrome bc1 mutant had a doubling time >2-fold from that of the wild-type strain. When a qoxA mutation was introduced into the cytochrome bc1 mutant, the resulting strain grew somewhat better than the cytochrome bc1 mutant itself. Although this effect is quite reproducible, it is unclear as to the cause. However, when a qxtA mutation was introduced into the cytochrome bc1 mutant, the resulting strain was unable to grow aerobically. This suggests that in the absence of a functional cytochrome bc1 complex, oxidative respiration proceeds via the Qxt quinol oxidase and not the Qox quinol oxidase, under the conditions employed here.
aDoubling times are the mean of three independent growth experiments for each strain. Deviation from the mean was no more than 10% in each case. NG, no growth.
3.6 Quinol oxidase gene expression
To determine under which conditions the Qxt and Qox quinol oxidases are expressed, we constructed transcriptional lacZ fusions to the apparent upstream regulatory sequences (URS) of the qxtA and qoxB genes. Each of the fusions was introduced on a low copy number plasmid into the wild-type strain, the cytochrome bc1 complex mutant BC1, and strain CCM which lacks all c-type cytochromes. Expression of qxtA::lacZ increased approximately 2-fold when cells were grown under low O2 tension, compared with the β-galactosidase activity measured after growth under high O2 tension (Table 3). A similar 2-fold increase was observed when qxtA::lacZ expression was measured under high O2 tension in strains BC1 and CCM, which lack the cytochrome c respiratory branches. This is reminiscent of R. sphaeroides strains lacking the cytochrome cbb3 oxidase in which expression of respiratory genes, such as the photosynthetic apparatus and DMSO reductase, is increased under strictly aerobic growth conditions [7,11]. The results suggest that qxtA expression may be under the control of an as yet unknown regulatory system that responds to redox alterations.
Expression of qxtA::lacZ and qoxA::lacZ transcriptional fusions in R. sphaeroides
β-Galactosidase activity after growtha
aCultures were grown to mid-log phase under high oxygen tension (30% O2) or low oxygen tension (2% O2). Cell extracts were assayed for β-galactosidase activity. Units of activity are micromoles of o-nitrophenol (ONP) formed min−1 mg of protein−1. Values represent the mean values±the standard deviations from triplicated assays of at least three independent growths and are corrected for activity from the vector alone under the same conditions (pML5, <35 μmol of ONP formed min−1 mg of protein−1). ND, not determined. NA, no activity.
Our experiments using a transcriptional lacZ fusion to the URS of qoxB demonstrated a total lack of β-galactosidase activity for the growth conditions assayed, including photosythetic and dark-anaerobic conditions in the presence of DMSO (Table 3). The fusion present in pGak141 contains 513 bp of the URS of qoxB. Other fusions were constructed which respectively contain 444 bp, 590 bp, 758 bp and 1116 bp upstream of qoxB. Only the fusion containing the 1116-bp fragment showed any β-galactosidase activity, but at a very low level (7.9±0.3 μmol ONP formed min−1 mg protein−1). These results, and our phenotypic observations above, suggest that under the growth conditions employed here, the Qox oxidase does not function in respiration. DNA sequence analysis of the region upstream of qoxBA does not reveal any obvious ORFs or other identifiable genetic elements. It is also possible that the qox operon is expressed only under very specific growth conditions or is expressed in response to specialized regulatory elements. In either case, more work needs to be performed on the role of the Qox quinol oxidase. In more recent experiments involving direct measurement of quinol oxidase activity reveals that all measurable activity can be accounted for by the Qxt oxidase (Gak, E., Chang, K.-T. and Kaplan, S., unpublished observations).
If both the qox and qxt genes do encode functional oxidases, then R. sphaeroides 2.4.1T will possess four distinct oxidases, two quinol oxidases and two cytochrome c oxidases. This resembles the situation in the closely related Bradyrhizobium japonicum, another member of the α-3 subgroup of the Proteobacteria, which possesses four terminal oxidases, each belonging to the heme-copper oxidase superfamily . However, it is clear that the Qxt oxidase is by itself sufficient to permit aerobic growth of R. sphaeroides under conditions where all other oxidative respiratory activity is eliminated.
Furthermore, we have previously characterized the dor gene cluster of CII, which encodes components of the DMSO reductase in this organism [11,13]. Both the quinol oxidases and DMSO reductase derive their electrons directly from the quinone pool, without the requirement of the cytochrome bc1 complex. Thus, we have now described three energy-yielding electron transport systems whose genes are encoded on CII that use quinol as their direct electron donor. In contrast, to date we have not found genes on CII for any electron transport complex which utilizes the cytochrome bc1 complex as an electron donor. All systems so far identified that use cytochrome bc1 are encoded by genes present on CI, such as the photosynthetic apparatus and the two cytochrome c oxidases. We suggest that this may be an important functional difference between the products derived from genes of CI and CII, and this may raise important questions as to the nature of the origin of CII versus that of CI.
We would like to thank Dr. T. Donohue for providing us with R. sphaeroides 2.4.1 mutant strain CCM, prior to publication. This work was supported by US Public Health Service Grant GM55481 to S.K.
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