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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Present address: Institut für Mikrobiologie, TU Braunschweig, Spielmannstr. 7, 38106 Braunschweig, Germany.
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INTRODUCTION |
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The electron transfer through disulfide bond exchange reactions in the cytoplasm recycles essential enzymes such as ribonucleotide reductase (Orr & Vitols, 1966), which provides deoxyribonucleotides for DNA synthesis. Other metabolic enzymes that are recycled by thiol-disulfide reactions are phosphoadenosine-phosphosulfate reductase (Lillig et al., 1999
), methionine sulfoxide reductase (Boschi-Muller et al., 2000
, 2001
) and arsenate reductase (Shi et al., 1999
).
Furthermore, thioredoxin is an essential subunit of the DNA polymerase of bacteriophage T7 (Huber et al., 1987; Mark & Richardson, 1976
) and is essential for the assembly of several filamentous phages (Russel & Model, 1985
). In eukaryotic organisms, many additional roles of thioredoxin have been reported, among which are the regulation of transcription factors such as NF-
B (Schulze-Osthoff et al., 1995
) and the regulation of apoptosis (Saitoh et al., 1998
). Thioredoxin is also involved in the regulation by light of photosynthetic enzymes in plant chloroplasts (Buchanan, 1984
; Buchanan et al., 1994
).
Because of its extremely low redox potential and free thiol in its reduced form, which can readily form a disulfide bridge, thioredoxin is considered to be involved in defence against oxidative stress not only by regeneration of oxidatively damaged proteins (Fernando et al., 1992; Natsuyama et al., 1992
), but also by its ability to reduce hydrogen peroxide (Mitsui et al., 1992
; Nakamura et al., 1994
; Spector et al., 1988
; Tomimoto et al., 1993
) or by acting as a hydrogen donor for peroxidase (Chae et al., 1994
). Thioredoxin also functions as a singlet oxygen quencher and hydroxyl radical scavenger independently of its redox state (Das & Das, 2000
).
Two thioredoxin genes, trxA and trxC, encoding thioredoxin 1 and 2, respectively, were identified in Escherichia coli (Laurent et al., 1964; Miranda-Vizuete et al., 1997
). Thioredoxin 1 and thioredoxin 2 have 29 % sequence identity, with the greatest difference being a 32 aa extension of thioredoxin 2 at its N terminus. Thioredoxin 2 possesses additional cysteine thiols apart from those of the active site, which, when oxidized, downregulate its activity in the reduction in insulin disulfides. Thioredoxin 2 is also less heat-stable than thioredoxin 1 and does not reduce ribonucleotide reductase as efficiently as thioredoxin 1. Thioredoxin 2 participates in the OxyR-orchestrated antioxidant response (Ritz et al., 2000
). Thioredoxin 1 expression is not controlled by OxyR but in the stationary phase is controlled by ppGpp (Lim et al., 2000
).
The facultatively phototrophic purple bacteria of the genus Rhodobacter can adapt rapidly to changes in their environment. As long as oxygen is available, they perform oxidative respiration. If the oxygen tension drops below a threshold value, the formation of pigment protein complexes is induced. When oxygen is no longer available, Rhodobacter gains energy by anoxygenic photosynthesis in the presence of light. Many different proteins are involved in the oxygen-dependent regulation of photosynthesis genes in Rhodobacter (e.g. Gregor & Klug, 2002). A central component of the redox control system of Rhodobacter capsulatus is the two-component system RegB/RegA (PrrB/PrrA in Rhodobacter sphaeroides) (Sganga & Bauer, 1992
). The phosphorylated response regulator RegA activates the expression of photosynthesis genes at a low oxygen tension (Masuda et al., 1999
; Sganga & Bauer, 1992
). Some photosynthesis genes are repressed at a high oxygen tension by the CrtJ protein (PpsR in R. sphaeroides). CrtJ binds to its target DNA sequences in a redox-dependent manner (Ponnampalam & Bauer, 1997
). It was shown recently that the CrtJ protein forms an intramolecular disulfide bond when exposed to oxygen, which is critical for binding to its target promoters (Masuda et al., 2002
). In addition to these DNA-binding proteins, thioredoxin 1 was shown to be involved in the redox-dependent expression of photosynthesis genes in R. sphaeroides by a yet unidentified mechanism. A mutant strain, which harbours reduced levels of thioredoxin 1, shows a lower degree of induction of the puf and puc genes encoding pigment-binding proteins after a decrease in oxygen tension than the wild-type strain (Pasternak et al., 1999
). In R. sphaeroides, the single thioredoxin 1 is essential for growth (Pasternak et al., 1997
). The expression of trxA increases during an increase in oxygen tension (Pasternak et al., 1999
).
The genome of R. capsulatus, a close relative of R. sphaeroides, harbours at least two genes encoding thioredoxins, trxA and trxC. Since thioredoxin 2 resembles the thioredoxin 1, it is possible that thioredoxin 2 of R. capsulatus is also involved in the redox-dependent regulation of photosynthesis genes. To learn more about the function of thioredoxin 2 in R. capsulatus, we constructed a trxC mutant of this strain and constructed plasmids with wild-type and mutated thioredoxin genes, which were expressed in E. coli. We demonstrate (1) that it can partially replace the thioredoxin 1 function as hydrogen donor for methionine sulfoxide reductase, but (2) that it cannot replace thioredoxin 1 as a subunit of phage T7 DNA polymerase independent on its redox potential, (3) that R. capsulatus thioredoxin 2 is involved in defence against oxidative stress and (4) that R. capsulatus thioredoxin 2 affects the oxygen-dependent expression of photosynthesis genes as thioredoxin 1 from R. sphaeroides, albeit in an opposite way. Thus, the trxA gene from R. sphaeroides and the trxC gene from R. capsulatus are reversely affected by oxygen, and the corresponding thioredoxins exert an opposite effect on the expression of photosynthesis genes.
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METHODS |
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Determination of survival rates.
Cultures were grown under semi-aerobic conditions until the optical density reached 0·40·5; then, oxidative stress agents were added at indicated concentrations for 1 h and dilutions were plated. Survival of 100 % corresponds to the viable cell number determined immediately before the addition of the oxidative agents. The percentage of colonies grown from the treated cultures is given as the percentage survival in Table 2. The values are the mean of three experiments. Hydrogen peroxide, tertiary-butyl hydroperoxide (t-BOOH), diamide and methyl viologen (Paraquat) were purchased from Sigma.
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Phage assay.
To determine whether TrxC of R. capsulatus can substitute for E. coli TrxA as an essential subunit of T7 DNA polymerase, T7 infection tests were performed as described by Sambrook et al. (1989). Bacteria were grown overnight in LB broth supplemented with the corresponding antibiotics. Appropriate culture dilutions were mixed with corresponding-titre T7, incubated for 20 min at room temperature for attachment then added to 0·7 % agar LB broth and spread on to 1 % LB plates, which were incubated at 37 °C for 4 h to monitor T7 development.
Cloning of R. capsulatus and R. sphaeroides trx genes.
The trxC sequence (RRC00979) was used to design the specific mutagenic primers RctrxCexp>A, 5'-GGATCCCGGAATCGCTGCGACTGA-3' (forward, introduction of BamHI site), and RctrxCexp>B, 5'-AAGCTTCTTGGGCGCGGGTTCTC-3' (reverse, introduction of HindIII site). The PCR product produced with the above primers was cloned into pGEM T-vector (Promega) and recloned into pQE32 His-tag vector (Qiagen) digested with HindIII and BamHI. The recombinant plasmid designated pQERctrxC was transformed into E. coli strain JM109. The correct construct as confirmed by sequencing was transformed into E. coli strain M15 (pREP4) for overexpression of thioredoxin 2 induced by the addition of 1 mM IPTG at 32 °C for 4 h.
For mutagenesis of the first cysteine of thioredoxin 1 and/or 2 active site -C-X-X-C-, the following primers were designed: RsC29A>A (forward), 5'-TGGGCCGGCCCCTGCCGGCAGAT-3', and RsC29A>B (reverse, complementary to RsC29A>A), 5'-ATCTGCCGGCAGGGGCCGGCCCA-3', for trxA of R. sphaeroides (RstrxA) and trxC of R. capsulatus (RctrxC); RcC29A>A (forward), 5'-GAATGGGCTGGCCCCTGCAAGATG-3', and RcC29A>B (reverse, complementary to RcC29A>A), 5'-CATCTTGCAGGGGCCAGCCCATTC-3', for trxA of R. capsulatus (RctrxA). The upstream fragments of RstrxA from primers EH12 and RsC29A>B, RctrxA from RctrxAstart and RcC29A>B, RctrxC from KH8 and RsC29A>B and the downstream fragments of RstrxA from primers EH7 and RsC29A>A, RctrxA from RctrxAendHind and RcC29A>A, RctrxC from EH26 and RsC29A>A were amplified by PCR. The diluted overlapped PCR products of upstream and downstream fragments, which acted as primers and templates, were amplified to produce the full-length thioredoxin fragments with the mutation of cysteine into alanine. The full-length trx fragments were cloned into plasmid pUTC5 (Assemat et al., 1995) replacing wild-type trxA of R. sphaeroides.
Mutant construction.
R. capsulatus strain SB1003trxC- was generated by transferring the suicide plasmid pPHRctrxC : : Km into wild-type SB1003 and screening for insertion of the kanamycin cassette into the chromosome by double crossover. To this end, parts of the trxC gene together with upstream and downstream sequences were amplified by PCR using the primers P1 and P2 and primers P3 and P4, respectively. The primer sequences were as follows: P1, 5'-GGGGTACCGCAGGCCTCCTACA-3'; P2, 5'-CGGGATCCGACGGCACCTTGTT-3'; P3, 5'-CGGGATCCTTGRCGGCTTCGTC-3'; P4, 5'-CCCAAGCTTATCGGCCATGCTCAG-3'. The PCR products were digested with appropriate restriction enzymes (KpnI and BamHI for the upstream fragment, BamHI and HindIII for the downstream fragment) and cloned into pPHU281 (Hübner et al., 1993) to give plasmid pPHRCtrxC and generating a BamHI site at the junction. Then, a 1·3 kb BamHI fragment containing the kanamycin cassette from pUC4KSAC (Barany, 1985
) was inserted into the BamHI site of pPHRctrxC to generate pPHRctrxC : : Km, which was transformed into E. coli strain SM10. For the purpose of generating strain SB1003trxC-, diparental conjugation was carried out with R. capsulatus SB1003 as a recipient strain. A Southern hybridization was performed to confirm the correct insertion of the kanamycin cassette into the chromosome.
RT-PCR.
For RT-PCR analysis, the reverse transcription reaction was carried out in a final volume of 25 µl containing 0·125 µg random primer (Promega), 5 µg total RNA, 1 mM each of four deoxyribonucleoside triphosphates and 1·5 units reverse transcriptase (Promega) at 42 °C for 1 h and 99 °C for 2 min to inactivate the reverse transcriptase. PCR was carried out in a final volume of 50 µl containing 5 µl reverse-transcribed cDNA solution, 1 unit Taq polymerase (Qiagen) and 2 pmol each of the oligonucleotide primers 5'-GCGCCGCAGTTCCAAGCC-3' and 5'-CTTGCCGCGGACGAAGCC-3' for trxC, and 5'-GAAGTGCGCCAATCCGAC-3' and 5'-AGAGCGAGGCTTCGATCC-3' for trxA. Amplification was carried out by an initial denaturation step at 96 °C for 3 min followed by 38 cycles for trxC or 23 cycles for trxA at 96 °C for 1 min, 62 °C (for trxC) or 57 °C (for trxA) for 40 s and 72 °C for 30 s. A sample lacking reverse transcriptase was included for each reaction as a control for DNA contamination. Reaction products were subjected to electrophoresis on 4 % agarose gels (Biozym).
Southern, Northern and colony hybridization analysis.
Southern and Northern blot hybridizations were performed as described previously (Engler-Blum et al., 1993; Pasternak et al., 1997
). Colony hybridization was carried out according to the manufacturer's recommendation (PALL).
Production of antibodies and Western blot analysis.
For immunological detection of thioredoxin 1 or 2, 20 µg crude cell extracts as determined by Bradford (1976) were separated by SDSPAGE (Laemmli, 1970
) on 15 % polyacrylamide gels and transferred to Immobilon-P membrane (Millipore). The R. sphaeroides thioredoxin 1 and R. capsulatus thioredoxin 2 proteins were expressed as His-tag fusion proteins from pQE32-derived plasmids (Qiagen) in E. coli M15(pREP4) and the antibodies against the gel-purified proteins were raised in rabbits (Clontech). The antibodies were purified from the serum by protein A Sepharose. Western blotting was performed according to the Western Exposure Chemiluminescent Detection system (Clontech).
Bacteriochlorophyll measurements.
A sample (0·5 ml) of culture was sedimented and resuspended in 0·5 ml of acetonemethanol (7 : 2, v/v). The absorbance of the supernatant at 770 nm was determined after spinning in a microcentrifuge for 3 min. The relative bacteriochlorophyll content of the cells is given as the absorbance at 770 nm divided by the optical density at 660 nm.
Construction of the reporter genes for luciferase assays.
The luciferase reporter plasmids were constructed by cloning the KpnIStuI PCR fragments coding the 5'-flanking region of the trxC gene of R. capsulatus into restriction sites KpnI and SmaI of vector pBBR1MCS-5 (Kovach et al., 1994) to generate pBBR502. To generate pBBR502lux, the luxAB XbaI fragment from plasmid pILA (Kunert et al., 2000
) was cloned into the appropriate restriction sites of pBBR502. Plasmid pBBR502lux was transferred into R. capsulatus SB1003 by diparental conjugation to obtain SB1003(pBBR502lux) (Table 1
).
Luciferase assays were performed as described by Kunert et al. (2000) at room temperature with some modifications. In brief, luminescence of the luciferase reaction was induced by the addition of decanal (Sigma) to 1 ml culture (final concentration of decanal 0·5 mM). Light emission was monitored in a photomultiplier-based luminometer (BioOrbit; Labsystems). The mean value of four to five data near the maximum of the peak was used as the luminescence output. All the readings were related to the optical density of the culture at 660 nm, as determined with a UV/visible spectrophotometer. All measurements were made in duplicate and experiments were performed at least twice using independent cultures.
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RESULTS |
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Affinity-purified polyclonal antibodies were used to determine the presence of thioredoxin of R. sphaeroides or R. capsulatus in E. coli strains, which harbour the plasmids with trx genes of R. sphaeroides or R. capsulatus. Fig. 1 shows that the anti-thioredoxin 1 or anti-thioredoxin 2 antibodies reacted with one band of the expected size of thioredoxin 1 (1112 kDa) or thioredoxin 2 (15·3 kDa) in a total crude extract of the E. coli transformants. Thus, thioredoxin 1 of R. sphaeroides or R. capsulatus and thioredoxin 2 of R. capsulatus are expressed in E. coli strain BH216. Thioredoxin 1 proteins of E. coli, R. sphaeroides and R. capsulatus were not detectable with anti-thioredoxin 2 antibody of R. capsulatus: only one clear band was observed in BH216(pUTRctrxC) cell extracts (Fig. 1b
). In contrast, antibodies raised against E. coli thioredoxin 2 could react with E. coli thioredoxin 1, while E. coli thioredoxin 1 antibodies did not cross-react with thioredoxin 2 (Miranda-Vizuete et al., 1997
).
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Reduced thioredoxin complexes with bacteriophage T7 DNA polymerase, resulting in an enzyme with a high processivity (Huber et al., 1987). T7 phage cannot be propagated in E. coli strain BH216, which is unable to express thioredoxin 1. T7 phage can be propagated in E. coli strain BH216 containing a plasmid habouring the E. coli trxA gene, the R. sphaeroides trxA gene or the R. capsulatus trxA gene (data not shown). Thus, thioredoxins 1 from R. sphaeroides and R. capsulatus can function as subunits of T7 DNA polymerase in E. coli. However, we observed no propagation of phage T7 in BH216 containing a plasmid, which allows expression of the R. capsulatus thioredoxin 2 (data not shown). We conclude that R. capsulatus thioredoxin 2 cannot function as a subunit of T7 DNA polymerase.
The ability of T7 to grow on the E. coli trxA mutant strains in which one or both active site cysteine residues of thioredoxin had been changed to serine or alanine definitively demonstrated that the redox capacity of thioredoxin is not required for stimulation of DNA polymerase activity (Huber et al., 1986). In contrast, neither thioredoxin-S2 nor modified thioredoxin in which a single cysteine had been methylated formed a complex with T7 DNA polymerase (Adler & Modrich, 1983
), suggesting that the conformation of the active site is very important for this interaction. To test the effect of thioredoxin 1 mutation C29A, the identical constructs as described above for the in vivo methionine sulfoxide assay were used for T7 infection. Interestingly, clear plaques were observed in the plate with strain BH216(pUTRctrxC29A), but not in the plate with strain BH216(pUTRstrxC29A). Thus, the mutated thioredoxin 1 (C29A) from R. capsulatus still acts as a subunit of T7 DNA polymerase to allow T7 phage to amplify, while mutated thioredoxin 1 of R. sphaeroides does not. Both thioredoxins 1 of the closely related species, R. sphaeroides and R. capsulatus, share an amino acid sequence identity of 82 %. However, the two active sites have different amino acids surrounding the cysteines. The mutation in the active site may lead to different conformational changes in the two thioredoxins (see Discussion).
Construction of a trxC mutant of R. capsulatus
To learn more about the role of thioredoxin 2 in R. capsulatus and to test for a function in redox-dependent regulation of photosynthesis genes, we inactivated the trxC gene of R. capsulatus. The trxC gene is positioned between the genes for thymidine kinase and Ala-tRNA on the R. capsulatus chromosome (Fig. 3). All these genes are oriented in the same direction as the trxC gene. To inactivate the trxC gene in R. capsulatus, we cloned the trxC upstream and downstream regions including the N-terminal and C-terminal segments of the coding regions into plasmid pPHU281 (Hübner et al., 1993
), which is unable to replicate in Rhodobacter. We then inserted the kanamycin cassette gene lacking transcriptional terminators between the trx upstream and downstream segments. After conjugational transfer into R. capsulatus, about 400 colonies were screened for insertion of the resistance gene into the chromosome via a double crossover by testing for resistance to kanamycin but a lack of the plasmid encoded tetracycline resistance. The fact that all of the kanamycin-resistant colonies were tetracycline-sensitive indicated that insertion of the tetracycline resistance gene into the chromosome by a single crossover may not lead to expression of tetracycline resistance. We therefore analysed those kanamycin-resistant clones by colony hybridization. Twenty-nine clones did not show hybridization to plasmid pPHU281. Isolated chromosomal DNA from these clones was used for Southern hybridization to confirm the presence of the resistance cassette and to test for the interruption of trxC gene. Two trxC mutant clones were identified, which had the kanamycin cassette integrated into the chromosomal trxC gene by a double crossover.
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The addition of 1 mM H2O2 inhibited growth of the trxC mutant to a larger extent than growth of the wild-type strain. One hour after addition of 1 mM H2O2 to wild-type cells, the number of viable cells was increased to a mean of 134 % of the number before the treatment, while the number of viable cells increased to a mean of 157 % in the untreated control. In the culture of the trxC mutant, the number of viable cells was decreased to 73 % after 1 h of H2O2 treatment. There were no significant differences in the survival rates of wild-type and mutant strain after treatment with lower concentrations of H2O2. The trxC mutant was very sensitive against treatment with 1 mM Paraquat. The survival rate after 1 h of treatment with Paraquat was 70 % in a wild-type culture, but only 20 % in a culture of the trxC mutant. The trxC mutant also showed a higher sensitivity against 0·5 mM Paraquat, but survival of both the mutant and the wild-type strain was affected to the same extent by 0·1 mM Paraquat. One hour after the addition of 1·5 mM diamide, 62 % of the cells of a wild-type culture were viable, but only 46 % of the cells of the trxC mutant were viable. The trxC mutant also showed lower survival rates after treatment with 1 or 0·5 mM diamide. One hour after the addition of 0·6 mM t-BOOH, the wild-type and the mutant strain showed survival rates of about 87 and 53 %, respectively, while the survival rates of the mutant and wild-type were similar at lower concentrations of t-BOOH. These results show that trxC mutant cells are more sensitive to the agents generating oxidative stress than the wild-type SB1003.
The observation from the determination of survival rate was verified by the zone of growth inhibition (Fig. 4b). The trxC mutant showed a decreased resistance to all oxidative agents applied. While clear zones of growth inhibition were observed when the disks were treated with H2O2 or diamide, turbid inhibition zones formed around the disks treated with t-BOOH or Paraquat.
Our results imply a function of thioredoxin 2 of R. capsulatus in the defence against oxidative stress.
Role of thioredoxin 2 in expression of genes for pigment-binding proteins and formation of photosynthetic complexes in R. capsulatus
We have previously characterized the trxA mutant TK1 of R. sphaeroides, which produces less thioredoxin than a wild-type strain. Strain TK1 accumulates less bacteriochlorophyll and less puf and puc mRNAs encoding pigment-binding proteins after a transition from growth under high oxygen tension to growth under low oxygen tension compared with its parental wild-type strain (Pasternak et al., 1999). When the trxC mutant of R. capsulatus was subjected to the same change in growth conditions, it accumulated similar levels of bacteriochlorophyll to that of the parental wild-type strain, indicating that the same amounts of photosynthetic complexes are formed in both strains. However, the bacteriochlorophyll content increased more rapidly in the trxC mutant than in the isogenic wild-type strain (Fig. 5
). We also analysed the levels of puf and puc mRNA in the trxC mutant. puf and puc are both polycistronic operons, which harbour genes required for the formation of pigment protein complexes (Alberti et al., 1995
; Choudhary & Kaplan, 2000
; Zsebo & Hearst, 1984
). Using Northern blot analysis, we determined the level of the 0·5 kb pucBA mRNA that encodes the two pigment-binding proteins of the light-harvesting II complex. This mRNA species is a stable processing product of the 2·3 kb primary puc transcript. We also determined the level of the 0·5 kb pufBA mRNA, which is a processing product of the pufQBALMX primary transcript and encodes the proteins of the light-harvesting I complex.
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Effects of oxygen on trxC expression
Our data show that thioredoxin 2 of R. capsulatus affects the oxygen-dependent expression of photosynthesis genes. To test how trxC expression reacts to changes in oxygen tension in the environment, we monitored trxC expression under different growth conditions. Since the trxC mRNA does not show up on Northern blots as a distinct band (unpublished), we used a semiquantitative RT-PCR approach for directly quantifying trxC transcript levels. In addition, we used a trxC'luxAB fusion harbouring 450 nt of sequence upstream of trxC for following the expression of trxC.
As shown in Fig. 7(b), the trxC mRNA level strongly increased after a shift of the cultures from high to low oxygen tension. It has been reported that the trxA mRNA level in R. sphaeroides shows a slight decrease under these conditions (Pasternak et al., 1996
). Likewise, we observed a significant decrease for trxA expression in R. capsulatus in the Northern blot analysis (Fig. 7a
) and by semiquantitative RT-PCR (Fig. 7b
). Thus, trxA and trxC expression are regulated by oxygen tension in an opposite way. When we expressed the trxC'luxAB fusion in the wild-type strain, we were able to confirm this effect of oxygen on trxC expression. The luciferase activity increased by a factor of about eight to nine 1·5 h after the transition and then dropped again. When the cultures were shifted from low to high oxygen tension, we observed a strong decrease in luciferase activity (Fig. 7c
).
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DISCUSSION |
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R. capsulatus, a close relative of R. sphaeroides, contains at least two thioredoxin genes, trxA and trxC. To determine whether thioredoxin 2 from R. capsulatus functionally resembles thioredoxin 2 of E. coli and to test its involvement in the oxygen-dependent expression of photosynthesis genes, we inactivated the trxC gene of R. capsulatus and expressed it in the E. coli strain BH216. Since strain BH216, which lacks a functional trxA gene but harbours the intact trxC gene, cannot grow on minimal medium with methionine sulfoxide and cannot propagate phage T7, E. coli TrxC is not able to replace TrxA for its functions as hydrogen donor for methionine sulfoxide reductase and as a subunit of T7 DNA polymerase when expressed at a normal cellular level. When the trxC gene is overexpressed in an E. coli metEtrxA background, it confers weak growth to this strain on minimal medium with methionine sulfoxide (Ritz et al., 2000; Stewart et al., 1998
), indicating that it can complement the defect poorly. We made the identical observation after expressing the trxC gene from R. capsulatus in an E. coli metEtrxA background. This strain was unable to allow propagation of phage T7, indicating that thioredoxin 2 is unable to function as a subunit for T7 DNA polymerase, even when the trxC gene is overexpressed.
The inactivation of the trxC gene had little effect on the growth of R. capsulatus, even under a high oxygen tension. An extended lag phase of the mutant strain after increasing the oxygen tension in the culture indicates the involvement of thioredoxin 2 in adaptation to high oxygen tension. This assumption is supported by the fact that no growth of the mutant was observed 1 h after a transition from low to high oxygen tension. More pronounced differences between the wild-type strain and the trxC mutant of R. capsulatus were observed in the presence of oxidative-stress-generating agents (Table 2). The mutant strain showed a higher sensitivity against diamide, Paraquat, t-BOOH and H2O2 than the parental wild-type strain. An E. coli trxC mutant showed a sensitivity similar to that of the isogenic wild-type after exposure to high concentrations of H2O2 (5 mM) or 0·8 mM diamide (Ritz et al., 2000
). Our data suggest that thioredoxin 2 of R. capsulatus, like its E. coli counterpart (Ritz et al., 2000
), is involved in the oxidative stress response but is more important in the resistance against diamide and H2O2 than its E. coli counterpart. The way in which thioredoxin 2 is regulated by oxidative stress and the molecular mechanisms of oxidative stress defence in R. sphaeroides or R. capsulatus are under investigation.
E. coli thioredoxin 1 is active in reconstituting T7 DNA polymerase activity by forming a complex with the gene 5 protein in its reduced form; thioredoxin-S2 shows no activity or competition (Adler & Modrich, 1983). The trxA mutant strains in which one or both active-site cysteine residues of E. coli thioredoxin had been changed to serine or alanine can be lysed by T7 phage (Huber et al., 1986
), indicating that conformation of the reduced thioredoxin at the active site is more important than whether or not the active site can provide the redox potential. Interestingly, the C29A mutant of R. sphaeroides thioredoxin 1 could not act as a subunit of T7 DNA polymerase, whereas the C29A mutant of R. capsulatus thioredoxin 1 could. R. capsulatus thioredoxin 1 has the same amino acids KM directly behind the second cysteine of the active site as E. coli thioredoxin 1. However, R. sphaeroides thioredoxin 1 has the amino acids RQ following the second cysteine of the active site, as R. capsulatus and E. coli thioredoxin 2, which were found not to act as a subunit of T7 DNA polymerase. These two residues KM are important in protein interactions and may stabilize thiolate in the active site (Eklund et al., 1991
). This suggests that the thioredoxin 1 mutation C29A of R. capsulatus might not change the conformation at the active site, probably partly contributed by the following residues KM. The R. sphaeroides thioredoxin 1 C29A mutation might change it. It is conceivable that the flexibility of the active site region to allow the reduced form of the protein to take up functionally significant conformations of a slightly higher energy than the oxidized form is more subtle in R. sphaeroides thioredoxin 1 than in R. capsulatus thioredoxin 1.
Although thioredoxin 1 and 2 have similar functions in some regards, the two genes respond to changes in oxygen tension in an opposite way. While a reduction in oxygen tension in cultures of R. capsulatus resulted in a significant increase in trxC mRNA level, the trxA mRNA level decreased. Interestingly, the opposite response of the two genes to oxygen tension correlates with an opposite effect on the oxygen-dependent expression of photosynthesis genes. A trxA mutant of R. sphaeroides harbouring lower levels of thioredoxin 1 than the isogenic wild-type showed a diminished increase in puf and puc mRNA levels after such a transition when compared with the parental wild-type strain (Pasternak et al., 1999). Until now we were unable to construct a trxA mutant of R. capsulatus even by applying exactly the same strategy as that used for constructing the R. sphaeroides strain with an altered thioredoxin 1 level. The trxC mutant of R. capsulatus showed a faster increase and a higher accumulation of puf and puc mRNA levels after a reduction in oxygen tension than the isogenic wild-type strain. We conclude that the two thioredoxins have opposite effects on some cellular functions. At present, it is unknown how the effect of thioredoxin on the expression of photosynthesis genes is exerted. Thioredoxins may influence the activity of some of the proteins (RegB/RegA, CrtJ; see introduction), which have been identified as regulators of photosynthesis gene expression in R. capsulatus. We are in the process of unravelling the molecular basis for the effect of thioredoxin on the expression of photosynthesis genes to learn more about such differing functions of thioredoxin 1 and thioredoxin 2, which may also be of importance for other cellular functions.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Alberti, M., Burke, D. H. & Hearst, J. E. (1995). Structure and sequence of the photosynthesis gene cluster. In Anoxygenic Photosynthetic Bacteria, pp. 10831106. Edited by R. E. Blankenship, M. T. Madigan & C. E. Bauer. Dordrecht: Kluwer.
Assemat, K., Alzari, P. M. & Clement-Metral, J. D. (1995). Conservative substitutions in the hydrophobic core of Rhodobacter sphaeroides thioredoxin produce distinct functional effects. Protein Sci 4, 25102516.
Barany, F. (1985). Two-codon insertion mutagenesis of plasmid genes by using single-stranded hexameric oligonucleotides. Proc Natl Acad Sci U S A 82, 42024206.[Abstract]
Boschi-Muller, S., Azza, S. & Branlant, G. (2001). E. coli methionine sulfoxide reductase with a truncated N terminus or C terminus, or both, retains the ability to reduce methionine sulfoxide. Protein Sci 10, 22722279.
Boschi-Muller, S., Azza, S., Sanglier-Cianferani, S., Talfournier, F., Van Dorsselear, A. & Branlant, G. (2000). A sulfenic acid enzyme intermediate is involved in the catalytic mechanism of peptide methionine sulfoxide reductase from Escherichia coli. J Biol Chem 275, 3590835913.
Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of proteindye binding. Anal Biochem 72, 248254.[CrossRef][Medline]
Buchanan, B. B. (1984). The ferredoxin/thioredoxin system: a key element in the regulatory function of light in photosynthesis. Bioscience 34, 378383.[Medline]
Buchanan, B. B., Schurmann, P. & Jacquot, J. P. (1994). Thioredoxin and metabolic regulation. Semin Cell Biol 5, 285293.[CrossRef][Medline]
Chae, H. Z., Chung, S. J. & Rhee, S. G. (1994). Thioredoxin-dependent peroxide reductase from yeast. J Biol Chem 269, 2767027678.
Choudhary, M. & Kaplan, S. (2000). DNA sequence analysis of the photosynthesis region of Rhodobacter sphaeroides 2.4.1. Nucleic Acids Res 28, 862867.
Das, K. C. & Das, C. K. (2000). Thioredoxin, a singlet oxygen quencher and hydroxyl radical scavenger: redox independent functions. Biochem Biophys Res Commun 277, 443447.[CrossRef][Medline]
Drews, G. (1983). Mikrobiologisches Praktikum. Berlin: Springer.
Eklund, H., Gleason, F. K. & Holmgren, A. (1991). Structural and functional relations among thioredoxins of different species. Proteins 11, 1328.[Medline]
Engler-Blum, G., Meier, M., Frank, J. & Müller, G. A. (1993). Reduction of background problems in nonradioactive Northern and Southern Blot analyses enables higher sensitivity than 32P-based hybridizations. Anal Biochem 210, 235244.[CrossRef][Medline]
Fernando, M. R., Nanri, H., Yoshitake, S., Nagata-Kuno, K. & Minakami, S. (1992). Thioredoxin regenerates proteins inactivated by oxidative stress in endothelial cells. Eur J Biochem 209, 917922.[Abstract]
Gregor, J. & Klug, G. (2002). Oxygen-regulated expression of genes for pigment binding proteins in Rhodobacter capsulatus. J Mol Microbiol Biotechnol 4, 249253.[CrossRef][Medline]
Huber, H. E., Russel, M., Model, P. & Richardson, C. C. (1986). Interaction of mutant thioredoxins of Escherichia coli with the gene 5 protein of phage T7. The redox capacity of thioredoxin is not required for stimulation of DNA polymerase activity. J Biol Chem 261, 1500615012.
Huber, H. E., Tabor, S. & Richardson, C. C. (1987). Escherichia coli thioredoxin stabilizes complexes of bacteriophage T7 DNA polymerase and primed templates. J Biol Chem 262, 1622416232.
Hübner, P., Masepohl, B., Klipp, W. & Bickle, T. A. (1993). nif gene expression studies in Rhodobacter capsulatus: ntrC-independent repression by high ammonium concentrations. Mol Microbiol 10, 123132.[Medline]
Keen, N. T. & Tamaki, S. (1988). Improved broad-host-range plasmids for DNA cloning in Gram-negative bacteria. Gene 70, 191197.[CrossRef][Medline]
Kovach, M. E., Phillips, R. W., Elzer, P. H., Roop, R. M. & Peterson, K. M. (1994). pBBR1MCS: a broad-host-range cloning vector. Biotechniques 16, 800802.[Medline]
Kunert, A., Hagemann, M. & Erdmann, N. (2000). Construction of promoter probe vectors for Synechocystis sp. PCC 6803 using the light-emitting reporter systems Gfp and LuxAB. J Microbiol Methods 41, 185194.[CrossRef][Medline]
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680685.[Medline]
Laurent, T. C., Moore, E. C. & Reichard, P. (1964). Enzymatic synthesis of deoxyribonucleotides. IV. Isolation and characterization of thioredoxin, the hydrogen donor from Escherichia coli. J Biol Chem 239, 34363444.
Lillig, C. H., Prior, A., Schwenn, J. D., Aslund, F., Ritz, D., Vlamis-Gardikas, A. & Holmgren, A. (1999). New thioredoxins and glutaredoxins as electron donors of 3'-phosphoadenylylsulfate reductase. J Biol Chem 274, 76957698.
Lim, C. J., Daws, T., Gerami-Nejad, M. & Fuchs, J. A. (2000). Growth-phase regulation of the Escherichia coli thioredoxin gene. Biochim Biophys Acta 1491, 16.[Medline]
Mark, D. & Richardson, C. C. (1976). Escherichia coli thioredoxin: a subunit of bacteriophage T7 DNA polymerase. Proc Natl Acad Sci U S A 73, 780784.[Abstract]
Masuda, S., Dong, C., Swem, D., Setterdahl, A. T., Knaff, D. B. & Bauer, C. E. (2002). Repression of photosynthesis gene expression by formation of a disulfide bond in CrtJ. Proc Natl Acad Sci U S A 99, 70787083.
Masuda, S., Matsumoto, Y., Nagashima, K. V., Shimada, K., Inoue, K., Bauer, C. E. & Matsuura, K. (1999). Structural and functional analyses of photosynthetic regulatory genes regA and regB from Rhodovulum sulfidophilum, Roseobacter denitrificans, and Rhodobacter capsulatus. J Bacteriol 181, 42054215.
Miranda-Vizuete, A., Damdimopoulos, A. E., Gustafsson, J.-A. & Spyrou, G. (1997). Cloning, expression, and characterization of a novel Escherichia coli thioredoxin. J Biol Chem 272, 3084130847.
Mitsui, A., Hirakawa, T. & Yodoi, J. (1992). Reactive oxygen-reducing and protein-refolding activities of adult T cell leukemia-derived factor/human thioredoxin. Biochem Biophys Res Commun 186, 12201226.[Medline]
Nakamura, H., Matsuda, M., Furuke, K. & 7 other authors (1994). Adult T cell leukemia-derived factor/human thioredoxin protects endothelial F-2 cell injury caused by activated neutrophils or hydrogen peroxide. Immunol Lett 42, 7580.[CrossRef][Medline]
Natsuyama, S., Noda, Y., Narimoto, K., Umaoka, Y. & Mori, T. (1992). Release of two-cell block by reduction of protein disulfide with thioredoxin from Escherichia coli in mice. J Reprod Fertil 95, 649656.[Abstract]
Orr, M. D. & Vitols, E. (1966). Thioredoxin from Lactobacillus leichmannii and its role as hydrogen donor for ribonucleoside triphosphate reductase. Biochem Biophys Res Commun 25, 109115.[Medline]
Pasternak, C., Assemat, K., Breton, A. M., Clement-Metral, J. D. & Klug, G. (1996). Expression of the thioredoxin gene (trxA) in Rhodobacter sphaeroides Y is regulated by oxygen. Mol Gen Genet 250, 189196.[CrossRef][Medline]
Pasternak, C., Assemat, K., Clement-Metral, J. D. & Klug, G. (1997). Thioredoxin is essential for Rhodobacter sphaeroides growth by aerobic and anaerobic respiration. Microbiology 143, 8391.[Abstract]
Pasternak, C., Haberzettl, K. & Klug, G. (1999). Thioredoxin is involved in oxygen-regulated formation of the photosynthetic apparatus of Rhodobacter sphaeroides. J Bacteriol 181, 100106.
Pille, S., Chuat, J.-C., Breton, A. M., Clement-Metral, J. D. & Galibert, F. (1990). Cloning, nucleotide sequence, and expression of the Rhodobacter sphaeroides Y thioredoxin gene. J Bacteriol 172, 15561561.[Medline]
Ponnampalam, S. N. & Bauer, C. E. (1997). DNA binding characteristics of CrtJ. A redox-responding repressor of bacteriochlorophyll, carotenoid, and light harvesting-II gene expression in Rhodobacter capsulatus. J Biol Chem 272, 1839118396.
Ritz, D. & Beckwith, J. (2001). Roles of thiol-redox pathways in bacteria. Annu Rev Microbiol 55, 2148.[CrossRef][Medline]
Ritz, D., Patel, H., Doan, B., Zheng, M., Aslund, F., Storz, G. & Beckwith, J. (2000). Thioredoxin 2 is involved in the oxidative stress response in Escherichia coli. J Biol Chem 275, 25052512.
Russel, M. & Model, P. (1985). Thioredoxin is required for filamentous phage assembly. Proc Natl Acad Sci U S A 82, 2933.[Abstract]
Saitoh, M., Nishitoh, H., Fujii, M., Takeda, K., Tobiume, K., Sawada, Y., Kawabata, M., Miyazono, K. & Ichijo, H. (1998). Mammalian thioredoxin is a direct inhibitor of apoptosis signal-regulating kinase (ASK) 1. EMBO J 17, 25962606.
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Schulze-Osthoff, K., Schenk, H. & Droge, W. (1995). Effects of thioredoxin on activation of transcription factor NF-kappa B. Methods Enzymol 252, 253264.[CrossRef][Medline]
Sganga, M. W. & Bauer, C. E. (1992). Regulatory factors controlling photosynthetic reaction center and light-harvesting gene expression in Rhodobacter capsulatus. Cell 68, 945954.[Medline]
Shi, J., Vlamis-Gardikas, A., Aslund, F., Holmgren, A. & Rosen, B. P. (1999). Reactivity of glutaredoxins 1, 2, and 3 from Escherichia coli shows that glutaredoxin 2 is the primary hydrogen donor to ArsC-catalyzed arsenate reduction. J Biol Chem 274, 3603936042.
Spector, A., Yan, G. Z., Huang, R. R., McDermott, M. J., Gascoyne, P. R. & Pigiet, V. (1988). The effect of H2O2 upon thioredoxin-enriched lens epithelial cells. J Biol Chem 263, 49844990.
Stewart, E. J., Aslund, F. & Beckwith, J. (1998). Disulfide bond formation in the Escherichia coli cytoplasm: an in vivo role reversal for the thioredoxins. EMBO J 17, 55435550.
Tomimoto, H., Akiguchi, I., Wakita, H., Kimura, J., Hori, K. & Yodoi, J. (1993). Astroglial expression of ATL-derived factor, a human thioredoxin homologue, in the gerbil brain after transient global ischemia. Brain Res 625, 18.[Medline]
Yen, H. C. & Marrs, B. (1976). Map of genes for carotenoid and bacteriochlorophyll biosynthesis in Rhodopseudomonas capsulata. J Bacteriol 126, 619929.[Medline]
Zsebo, K. M. & Hearst, J. E. (1984). Genetic-physical mapping of a photosynthetic gene cluster from Rhodobacter capsulata. Cell 37, 937947.[Medline]
Received 3 September 2002;
revised 4 November 2002;
accepted 8 November 2002.
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