Institut für Mikrobiologie und Molekularbiologie, University of Giessen, Heinrich-Buff-Ring 26-32, D-35392 Giessen, Germany
Correspondence
Gabriele Klug
Gabriele.Klug{at}mikro.bio.uni-giessen.de
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ABSTRACT |
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INTRODUCTION |
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Bacteria of the genus Rhodobacter are facultatively photosynthetic bacteria. At low oxygen tension the formation of photosynthetic complexes is induced, which allows the bacteria to perform anoxygenic photosynthesis if no oxygen is available. As long as oxygen is present Rhodobacter species produce ATP by aerobic respiration. The molecular mechanisms underlying the rapid adaptation to changes in oxygen concentration have been extensively studied in the past (reviewed by Gregor & Klug, 1999, 2002
; Zeilstra-Ryalls & Kaplan, 2004
). In its natural environment Rhodobacter occasionally needs to tolerate very high oxygen concentrations which are produced by micro-organisms performing oxygenic photosynthesis. While some previous studies have addressed the role of superoxide dismutase and thioredoxins in defence against oxidative stress in Rhodobacter (Cortez et al., 1998
; Pasternak et al., 1999
; Li et al., 2003a
, b
), little is known about the role of catalases and their regulation. In this study we have addressed the ability of two Rhodobacter species to detoxify H2O2 and have studied the expression of catalase genes in order to understand better how Rhodobacter can deal with high oxygen concentrations in its environment.
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METHODS |
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Construction of oxyR deletion mutants of R. capsulatus and R. sphaeroides.
R. capsulatus strain SBoxyR : : Sp and R. sphaeroides strain 2.4.1oxyR : :
Sp were generated by transferring the suicide plasmids pPHUSB
oxyR : :
Sp into R. capsulatus SB1003 and pPHU2.4.1
oxyR : :
Sp in R. sphaeroides 2.4.1, respectively, and screening for insertion of the omega (
)-spectinomycin cassette into the chromosome by homologous recombination. Briefly, parts of the oxyR genes of R. capsulatus SB1003 or R. sphaeroides 2.4.1, together with upstream and downstream sequences were amplified by PCR using oligonucleotides SBoxyupEco (5'-GTGTTCGAATTCCCCGCC-3'), SBoxy315Bam (5'-GAAGATAGGATCCAATCGTCG-3') and SBoxy500Bam (5'-GAAGATAGGATCCAATCGTCG-3'), SBoxydownPst (5'-CAGGCGCTGCAGAGGGCG-3') for SB1003 and 2.4.1oxyupEco (5'-CGAATTCTGGTTGTCGGCGATC-3'), 2.4.1oxy290Bam (5'-GGGATCCCGCCCAGATTGAC-3'), 2.4.1oxy600Bam (5'-CGGATCCGCTGCGGCGGTGGCGC-3') and 2.4.1oxydownPst (5'-CCTGCAGGACGGCCGCGTGGA-3') for 2.4.1.
The amplified PCR fragments were cloned into the EcoRIBamHI and BamHIPstI sites of the suicide plasmid pPHU281, respectively, generating plasmids pPHUSBoxy and pPHU2.4.1
oxyR. A 2·0 kb BamHI fragment containing the
-spectinomycin cassette from pHP45
was inserted into the BamHI site of pPHUSB
oxy and pPHU2.4.1
oxyR to generate pPHUSB
oxyR : :
Sp and pPHU2.4.1
oxyR : :
Sp, respectively. Both pPHUSB
oxyR : :
Sp and pPHU2.4.1
oxyR : :
Sp were transferred into E. coli strain SM10, and mobilized from SM10(pPHUSB
oxyR : :
Sp) or SM10(pPHU2.4.1
oxyR : :
Sp), into either R. capsulatus SB1003 or R. sphaeroides 2.4.1 wild-type strain by diparental conjugation. Conjugants were selected on malate minimal salt agar plates containing 10 µg spectinomycin ml1. Southern blot analysis of chromosomal DNA was carried out to confirm the double crossover event of the
-spectinomycin cassette into the Rhodobacter chromosome. By insertion of the
-spectinomycin cassette, 181 bp of the 915 bp R. capsulatus oxyR gene and 247 bp of the 926 bp R. sphaeroides oxyR gene, respectively, were deleted.
Complementation of the oxyR deletion mutants of R. capsulatus and R. sphaeroides.
For complementation of the oxyR deletion mutant of R. capsulatus, a 1·4 kb PCR fragment containing the entire oxyR gene along with approximately 450 bp of the upstream sequence was amplified using the oligonucleotides SBoxyRup450Eco (5'-GACGTTGTCGGGCCAGGAATTCAGC-3') and SBoxydownKpn (5'-GGGGTACCTGGCCTCGGTCAGATTTG-3'). Following digestion with EcoRI and KpnI, the PCR fragment was cloned into the corresponding sites of pRK415, resulting in plasmid pSBoxyR. For complementation of the oxyR deletion mutant of R. sphaeroides, a 1·2 kb PCR fragment containing the entire oxyR gene along with 100 bp of the upstream sequence was amplified using the oligonucleotides 2.4.1oxyupEco (5'-CGAATTCTGGTTGTCGGCGATC-3') and 2.4.1oxydownPst (5'-CCTGCAGGACGGCCGCGTGGA-3'). After digestion with EcoRI and PstI, the PCR fragment was cloned into the corresponding sites of pRK415 resulting in plasmid p2.4.1oxyR. To complement the oxyR deletion in SB1003 and 2.4.1, respectively, plasmids pSBoxyR and p2.4.1oxyR were transferred into E. coli strain SM10 and conjugated into the SBoxyR or 2.4.1oxyR strains by diparental conjugation.
Enzyme assays.
Rhodobacter cells were disrupted by brief sonication, and crude extract was used for enzyme assays. Protein concentration was determined according to Bradford (1976). For total catalase measurement, 30300 µg of total protein were diluted to 1 ml with water. H2O2 (0·5 ml of 59 mM H2O2 freshly diluted in 50 mM potassium phosphate buffer, pH 7·0) was added, and the absorbance of the samples at 240 nm was measured every 10 s for 1 min. The initial linear rates were used to calculate the activities. Specific activity of catalase (µM H2O2 decomposed per minute per milligram of total protein) was calculated using an extinction coefficient of 43·6 M1 cm1 (Hochman & Shemesh, 1987
).
For analysis of H2O2 detoxification, cells were grown exponentially to an OD660 of 0·5 and H2O2 was added to the desired final concentration. Aliquots were drawn at various time points, and the amount of H2O2 was determined by oxidation of Fe2+ in the presence of xylenol orange and measuring the absorbance at 560 nm. The absorbance of standard concentrations of H2O2 served to quantify the amount of H2O2 for each experiment. As previously described by Perelman et al. (2003), the ability to decompose H2O2 is strongly dependent on cell density. Therefore, all of the analyses presented in this study were performed at a fixed cell density.
Measurement of resistance to H2O2.
Exponentially grown cultures of Rhodobacter strains (0·2 ml) were diluted into 2 ml of pre-warmed top agar (0·7 % agar) and layered onto minimal malate agar plates. Filter paper disks were placed on the surface of the agar, and 5 µl of different concentrations (0·0010·2 M) H2O2 were applied on the filter disk. Zones of inhibition were measured after overnight incubation at 32 °C.
RNA extraction and quantitative real-time RT-PCR analysis.
Rhodobacter strains in exponential phase (OD660=0·5), grown under semi-aerobic conditions, were treated with H2O2 (1 mM final concentration) and at certain time-points cells were collected into centrifugation bottles and pelleted by centrifugation. Total RNA was isolated by the hot phenol method, quantified by spectrophotometric analysis (absorbance at 260 nm), and 60 ng of total RNA were used for quantitative real-time PCR.
The following primers were designed for gene amplification, katE: katE-A (5'-CTATCCGCTGATCGAGGT-3') and katE-B (5'-GTCGGCATAGGAGAAGAC-3'); katC: katC-A (5'-GGATGCGGCGATGCTAGCCGCCAAC-3') and katC-B (5'-GAGGGTTCCCGCCCAGCGTCGCC-3'); katG: katG-A (5'-GCCTCGGTCGCCGATGTGAT-3') and katG-B (5'-CACCGGCTCCAGCACGTCAA-3'); rpoZ genes: 2.4.1rpoZ-A (5'-TTCGAGCTGGTGATGCT-3') and 2.4.1rpoZ-B (5'-ACTCGATCTGGGTCTGG-3'); SBropZ-A (5'-GATGATCTGCGCGAGCGTCT-3') and SBrpoZ-B (5'-CCTTGCGCGTCCATCAATGC-3').
The rpoZ gene (encoding the -subunit of RNA-polymerase) of R. capsulatus and R. sphaeroides, respectively, was used to normalize expression values for all other genes. The One-Step RT-PCR kit (Qiagen) was used for reverse-transcription-PCR following the manufacturer's instruction, except that a total volume of 15 µl was used. SYBR Green was used to monitor amplification and to quantify the amount of PCR products using the Rotor-Gene 3000 real-time PCR cycler (LTF). Relative expression of kat and rpoZ mRNA were calculated after the method of Pfaffl (2001)
.
Expression and isolation of the R. capsulatus OxyR protein.
Oligonucleotides SBoxystartBam (5'-CCCCGGATCCTCTCGATGAAACAGC-3') and SBoxydownKpn (5'-GGGGTACCTGGCCTCGGTCAGATTTG-3'), which hybridize to the 5' and 3' regions of the oxyR gene, respectively, were used to amplify the oxyR coding region. The 950 bp PCR product was digested with BamHI and KpnI and ligated into pQE32 to generate pQEoxyR, which was transformed into E. coli JM109. The correct construct as confirmed by sequencing (using a Genetic Analyser 310 sequencer; ABI) was transformed into E. coli M15(pREP4) for overexpression of His-tagged OxyR. For this purpose M15(pREP4 pQEoxyR) was grown in 500 ml of LB medium to an OD600 of 0·70·8 and induced with 1 mM IPTG for 45 h at 32 °C. Following harvest, cells were resuspended in lysis buffer (50 mM Tris, pH 7·5; 250 mM NaCl; 3 mM imidazole; 1 µg lysozyme µl1 and 0·1 mM PMSF) and disrupted by a brief sonication. The lysate was centrifuged at 12 000 r.p.m. for 20 min at 4 °C. The supernatant was loaded onto Ni-NTA agarose and incubated at 4 °C for 45 h. Proteins were washed with washing buffer (0·05 M Tris, pH 7·5 and 0·25 M NaCl) containing 2050 mM imidazole and eluted with imidazole at a concentration between 80 and 100 mM. Aliquots of these fractions were analysed on SDS 15 % polyacrylamide gels, using the buffer system of Laemmli (1970). Fractions containing purified OxyR protein were pooled by using Centricon-10 columns (Amicon) and washed in Z buffer (50 mM HEPES, pH 8·0; 0·5 mM EDTA, pH 8·0; 10 mM MgCl2 and 300 mM KCl).
Gel chromatography (FPLC).
The purified OxyR protein in a 2 ml volume was applied to a Superdex-200 HR 16/60 gel filtration column (Pharmacia Biotech) equilibrated with Z buffer. For reducing conditions 200 mM DTT was added to the Z buffer. The protein was eluted with Z buffer and aliquots of the column fractions were analysed by SDS 10 % PAGE using the buffer system of Laemmli (1970).
Determination of OxyR redox status by using AMS.
For in vitro modification of free thiol groups, 4-acetamido-4'-maleimidylstibene-2',2'-disulfonic acid (AMS; Molecular Probes) was used. The addition of AMS leads to the alkylation of free thiol groups, present in the reduced but not in the oxidized OxyR. The addition of the high-molecular-mass AMS moiety to the reduced but not to the oxidized protein allows separation of the two forms by gel electrophoresis. The purified OxyR protein was first treated with freshly prepared H2O2 (1 mM) or DTT (200 mM) at room temperature for 30 min. The protein was then mixed with 10 % trichloroacetic acid (TCA). Precipitated protein was collected by centrifugation (10 000 r.p.m., 10 min). After complete removal of the supernatant, the pellet was dissolved in a buffer containing 0·1 % SDS; 50 mM Tris/HCl, pH 8·0 and 15 mM AMS (apart from the non-AMS-modified sample which was dissolved in the same buffer without AMS), and incubated for 2 h at 37 °C. The samples were loaded on SDS 10 % PAGE and visualized by silver staining.
Gel mobility-shift assay.
Binding of the OxyR protein to the katG and katE upstream region, respectively, was determined by gel mobility-shift assay. For this, a DNA fragment containing the katG or the katE promoter region was generated by PCR. The following oligonucleotides were used to generate the PCR fragments. katG: katGupBspEI (5'-CGTCCGGACCCGCGGCACCATC-3') and katGStuI (5'-AGGCCTTCATCGCGCCATGCAT-3'); katE: katEupEcoRI (5'-GTAGAATTCCTGCCGCAG-3') and katEPstI (5'-GTCTGCAGTCGGCGCGCCGGCCGT-3'). The PCR fragments with a length of 350 bp were cloned into T-vector (Promega), and isolated from the vector by using enzymes BspEI/StuI and EcoRI/PstI, respectively. The restricted DNA fragments were then radioactively labelled in a fill-in reaction with [-32P]dCTP using the Klenow fragment.
Binding reactions were carried out in a final volume of 20 µl and contained an appropriate amount of protein, [-32P]CTP-labelled DNA probe, and binding buffer (10 mM Tris/HCl, pH 8·0; 50 mM NaCl; 1 mM DTT; 1 mM EDTA and 5 % glycerol). Since purified OxyR protein is predominantly in the oxidized form, we added 200 mM DTT to the binding reaction to reduce OxyR. Binding incubations were carried out for 30 min at room temperature (25 °C) before the samples were loaded onto a 4 % polyacrylamide gel in 0·5x Tris Borate EDTA (TBE) buffer and run at 130 V for 2 h.
DNaseI footprinting assay.
A BamHIStuI DNA fragment containing the katG promoter region was 5'-end labelled at the BamHI site as follows. Plasmid TkatGup (Table 1) was linearized with BamHI, dephosphorylated using alkaline phosphatase (NEB) and labelled with polynucleotide kinase (NEB) and [
-32P]ATP. The DNA was then digested with StuI and the 361 bp BamHIStuI fragment was purified from a 6 % non-denaturing polyacrylamide gel. The end-labelled DNA fragment (approx. 10 000 c.p.m.) was incubated with different amounts of purified OxyR protein, using the same reaction conditions as described for the gel mobility-shift assay. After 30 min of incubation at room temperature, 5 µl of a buffer containing 0·01 M CaCl2 and 0·01 M MgCl2 were added. DNaseI (1 µl, 0·1 U µl1; Promega) was then added for 2 min at room temperature. The reaction was stopped by adding 250 mM EDTA. After phenol/chloroform extraction, the samples were dissolved in formamide dye and loaded onto a 6 % sequencing gel. The DNA fragment containing the katG promoter region was sequenced with the primer SBkatG300seq (5'-GCACCATCGCCGCGCCCAG-3') by the dideoxy chain-termination method (Sanger et al., 1977
) using the T7 sequencing kit from USB.
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RESULTS |
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Regulation of katC gene expression
In contrast to the gene encoding katE of R. sphaeroides, the katC gene showed only a weak response when H2O2 was added to exponentially growing cultures of R. sphaeroides (Fig. 3c). The low H2O2-dependent induction of katC expression in R. sphaeroides was independent of OxyR (Fig. 3c
). In contrast to the katE and katG basal expression levels, respectively, oxygen tension had no effect on the basal katC expression level (Fig. 4
). To determine whether expression of the katC gene is induced in the stationary phase, as it was reported for Sinorhizobium meliloti (Sigaud et al., 1999
), expression of the gene was measured by real-time RT-PCR during growth (Fig. 5
a, b). An 11- to 38-fold increase in expression of katC was observed when cells reached stationary phase, suggesting a function of the katC gene product in this growth stage. This finding is consistent with the results of Terzenbach & Blaut (1998)
, who observed a twofold higher catalase activity in R. sphaeroides cells grown in stationary phase. Expression of katE did not increase after cells entered stationary phase (data not shown).
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In order to define the binding sites of reduced and oxidized OxyR to the katG promoter region better, we performed footprint analysis. Oxidized OxyR protein bound to AT-rich sequences close to the translational start of oxyR (Fig. 6c, d). When reduced OxyR was applied, the footprint was significantly extended. A long DNA stretch comprising sequences well within the oxyR gene was protected from DNaseI digestion.
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DISCUSSION |
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Our results reveal that the two Rhodobacter strains show significant differences in their ability to cope with peroxide stress, which is correlated to strong differences in catalase activities. Both strains showed much faster detoxification of H2O2 under aerobic growth conditions than during growth under low oxygen tension, again correlating to higher catalase activities in both strains when grown under high oxygen tension. The basal expression of genes encoding catalases as determined by real-time RT-PCR was indeed higher under aerobic conditions, in both wild-type strains. Growing at high oxygen tension, bacteria will produce more ROS than during growth at low oxygen tension. Therefore, bacteria adapt to the presence of high oxygen levels by increasing the expression of genes involved in the detoxification of ROS, such as catalases. These higher expression levels result in higher resistance to peroxide stress at high oxygen levels. The assumption that the higher resistance of R. sphaeroides to H2O2 is caused by the presence of two catalases, KatE [encoded by RSP2779 (katE)] and KatC [encoded by RSP2380 (katC)] was not supported by our findings. While the expression of katE was strongly induced by oxygen or peroxide stress, expression of katC remained unaffected (Fig. 3b, c and Fig. 4
). The possibility that peroxidases could be responsible for the higher resistance of 2.4.1 can be excluded, since R. sphaeroides does not harbour peroxidase activity (Terzenbach & Blaut, 1998
). Hochman & Shemesh (1987)
, however, observed two different peroxidase activities in R. capsulatus. Thus, the higher resistance of R. sphaeroides to peroxide stress in comparison to R. capsulatus is probably not due to the presence of additional enzymes, but rather to the high activity of KatE. Nevertheless, even the low expression levels of katC might contribute to increase the resistance of R. sphaeroides to H2O2.
A key regulator of the response to H2O2 is the OxyR transcriptional regulator, which induces the expression of antioxidant activities in response to H2O2 stress (Storz & Imlay, 1999). oxyR mutants of both Rhodobacter species were more sensitive to the exposure to H2O2 and showed slower kinetics in the detoxification of this agent compared to the parental strains, confirming a role of OxyR in the oxidative stress response of Rhodobacter. While very little detoxification of H2O2 was observed in strain SBoxyR, the R. sphaeroides mutant 2.4.1oxyR was able to detoxify 3·5 mM H2O2 within 300 s, but with slower kinetics than the isogenic wild-type (Figs 1b and 2c
). This indicates that OxyR is more important for defence against H2O2 in R. capsulatus than in R. sphaeroides. Unexpectedly, we found that the oxyR mutation resulted in a decrease of total catalase activity in both Rhodobacter species. This finding is in contrast to observations in E. coli, where the deletion of oxyR did not reduce the basal activity of catalases during exponential growth, but only in cells induced with H2O2 (Visick & Clarke, 1997
). The lower levels of catalase activity in strain SBoxyR compared to strain 2.4.1oxyR are in agreement with the very poor detoxification of H2O2 by the R. capsulatus mutant. The addition of H2O2 to semi-aerobically grown wild-type cultures resulted in an induction of catalase activity, whereas no induction was observed in both oxyR mutants. This again is in agreement with findings in other bacterial systems, where OxyR acts as an activator of H2O2-inducible genes. Our observations suggest an effect of OxyR on the catalase activity in unstressed cells as well as an important role of OxyR in the adaptation of Rhodobacter to H2O2.
In many bacteria, genes encoding hydroperoxidases (HP) are members of the OxyR regulon. Gene expression analysis in oxyR mutant strains revealed that both katG of R. capsulatus and katE of R. sphaeroides are regulated by the OxyR protein. In E. coli the katG gene is strongly induced by H2O2, while the katE gene is not (Schellhorn, 1994). A regulatory effect of OxyR on katG expression has been shown for many bacteria (Loprasert et al., 2003
; Ochsner et al., 2000
). Our observation of fast kinetics in the induction of kat gene expression by H2O2 as well as the reduction 30 min after addition of H2O2 can be explained by the very fast kinetics of activation of OxyR by H2O2. Aslund et al. (1999)
were able to examine the kinetics of OxyR oxidation and reduction in vivo and in vitro. OxyR oxidation by H2O2 was completed within 30 s, and the half-time of deactivation was 1030 min. Compared to katE and katG expression, respectively, expression of katC was not affected by OxyR in R. sphaeroides. This is in agreement with results for the E. coli katE gene (Schellhorn, 1994
). Both the E. coli katE gene (Schellhorn, 1994
) and the R. sphaeroides katC gene are induced during stationary phase. The expression of the E. coli katE gene is known to be regulated by the stationary phase sigma factor RpoS, while the regulator of the R. sphaeroides katC gene is presently unknown. So far, no homologue of rpoS has been reported for the
-subclass of proteobacteria, which includes the Rhodobacter species (Rava et al., 1999
; Roop et al., 2003
).
The oxyR family is widespread among prokaryotes and nearly all known oxyR genes share overlapping promoters with other genes (Kim & Mayfield, 2000). The majority of genes located adjacent to oxyR are involved in oxidative stress protection, such as aphC, dps and oxyS, and are regulated by OxyR (Nakjarung et al., 2003
). In both Rhodobacter species the katE and katG genes, respectively, are located adjacent to the oxyR gene on the chromosome. Both genes are separated by approximately 100 nt (katE/oxyR 101 nt; katG/oxyR 98 nt) of untranslated region and are transcribed divergently. Thus, the oxyR and katE or katG genes, respectively, share a common upstream DNA sequence and may also share cis regulatory elements. This kat/oxyR gene organization is found in many
-proteobacteria [Rhizobium etli (del Carmen Vargas et al., 2003
); Brucella abortus (Kim & Mayfield, 2000
); Agrobacterium tumefaciens (Nakjarung et al., 2003
)], suggesting a general mechanism of regulation of these genes in
-proteobacteria.
Since it is reported that the OxyR protein regulates expression of genes by direct binding to the promoter region (reviewed by Schell, 1993), we tested the ability to bind DNA of the R. capsulatus OxyR protein. The OxyR protein showed strong binding to the katG promoter region and our data indicate that different conformations of DNAprotein complexes are formed with oxidized or reduced OxyR. Footprint analysis of the E. coli OxyR protein showed that OxyR binding is different under oxidizing and reducing conditions (Kullik et al., 1995
). As described for E. coli (Kullik et al., 1995
), the oxidized and reduced forms of R. capsulatus OxyR were predominantly tetrameric as revealed by size exclusion chromatography. As a member of the LysR family of bacterial regulators, OxyR acts as an activator of a regulon of genes and a repressor of its own expression (Storz & Altuvia, 1994
). Toledano et al. (1994)
, looking in E. coli, found that only oxidized OxyR binds katG, ahpC, dps and gorA promoters, whereas both the oxidized and the reduced protein bind the oxyRS promoter. They proposed that, by remodelling its DNA contacts, OxyR can impose opposite regulatory effects on the divergent oxyR and oxyS promoters.
Our results show that oxidized as well as reduced OxyR binds to the katG/oxyR promoter region, but the reduced protein makes extended contacts to the DNA compared to the oxidized protein. Our data also show that katG expression is strongly increased by H2O2, and that H2O2 leads to oxidation of the OxyR protein (Fig. 7c). Without the addition of H2O2, the level of katG expression is similar in the wild-type and the oxyR mutant. Thus OxyR acts as an activator of katG expression in the presence of H2O2. We propose that this activation includes the release of OxyR from binding sites within the oxyR gene. Our findings also suggest that reduced OxyR has little repressing effect on katG expression, which is low in the absence of oxidized OxyR.
Future work will be aimed at the identification of additional OxyR-regulated genes and additional OxyR-binding sites to understand better the OxyR signalling mechanism in Rhodobacter.
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ACKNOWLEDGEMENTS |
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Received 6 May 2004;
revised 22 July 2004;
accepted 25 July 2004.
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