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E-dependent manner
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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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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E factor and the anti-E factor ChrR affecting phrA expression, while superoxide anions do not stimulate phrA expression. Thus, the E regulon is involved not only in the response to singlet oxygen but also in the hydrogen peroxide response.
Present address: Department of Microbiology and Immunology, University of British Columbia Life Sciences Centre, 4557-2350 Health Sciences Mall, Vancouver, BC V6T 1Z3, Canada.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). Their accumulation blocks DNA replication and transcription and can therefore be lethal to cells. DNA photolyases repair these photoproducts in a blue/near-UV light dependent reaction referred to as photoreactivation (Rupert et al., 1958). Photolyases are closely related to cryptochromes, which belong to the same protein family (Cashmore et al., 1999).
Cryptochromes lack the ability for photoreactivation, but are instead known to function as blue light photoreceptors in diverse organisms by interacting with proteins such as COP1 and clock proteins (Yang et al., 2001; Lin & Shalitin, 2003; Sancar, 2003). Plant cryptochromes mediate e.g. stem elongation and photoperiodic flowering in response to light (reviewed by Lin & Shalitin, 2003). Cryptochromes in animals are light dependent and light independent regulators of the circadian clock (reviewed by Sancar, 2003). Almost all eukaryotic cryptochromes possess C-terminal domains beyond the photolyase-homology region while prokaryotic cryptochromes lack these C-terminal extensions (reviewed by Partch & Sancar, 2005; Fig. 1). The recently identified cryptochrome Drosophila, Arabidopsis, Synechocystis, Human (cryptochrome DASH) proteins lack the C-terminal extension. This group was found to specifically repair CPDs in single-stranded DNA (Selby & Sancar, 2006) and its members are therefore considered to be photolyases.
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; Ueda et al., 2005; Fujihashi et al., 2007).
Anoxygenic photosynthetic proteobacteria of the genus Rhodobacter are metabolically extremely versatile. R. sphaeroides uses light for anoxygenic photosynthesis but can also perform aerobic or anaerobic respiration. As a free-living aquatic bacterium, R. sphaeroides is exposed to sunlight in its natural habitat (Page et al., 2004). However, solar radiation of high intensity is harmful due to the formation of DNA photoproducts. In the presence of oxygen and bacteriochlorophyll, it generates highly reactive singlet oxygen, which causes photooxidative damage (Borland et al., 1987). To minimize the risk of photooxidative damage, Rhodobacter has evolved complex regulatory circuits that control the expression of photosynthesis genes according to environmental conditions. Under high oxygen tension, in either the light or dark, the bacterium is virtually unpigmented and performs aerobic respiration. Only when the oxygen tension decreases is the photosynthetic apparatus formed (Zeilstra-Ryalls et al., 1998). When R. sphaeroides grows in the absence of oxygen by anaerobic respiration, light has a stimulating effect on the expression of photosynthesis genes (Braatsch et al., 2002). Recent investigations on the protection of R. sphaeroides against photooxidative stress showed the involvement of E (RSP1092) and the anti-E factor ChrR (RSP1093) in this response (Anthony et al., 2005; Glaeser & Klug, 2005). E is a member of the extracytoplasmatic function (ECF) family of factors. ChrR belongs to a new family of zinc-containing anti- factors (Newman et al., 2001) and inhibits the ability of E to form a stable complex with core RNA polymerase by the formation of a E : ChrR complex (Anthony et al., 2004).
The genome of R. sphaeroides encodes three proteins, the products of RSP1981, RSP2143 and RSP3077, with similarity to photolyases and cryptochromes. Here we show that RSP2143, putatively named phrA (Braatsch et al., 2004), encodes a functional photolyase. RSP3077 also seems to act as a photolyase, while RSP1981 has no function in photoreactivation. Furthermore, analysis of the expression of phrA under different growth conditions revealed its regulation by the E/ChrR system.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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. Escherichia coli strains were cultured in Luria–Bertani broth at 37 °C with continuous shaking at 180 r.p.m. Rhodobacter sphaeroides strains were cultivated at 32 °C in a malate minimal salt medium (Drews, 1983) with continuous shaking at 140 r.p.m.
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80 nm and no detectable transmission at <317 or >515 nm. Photon flux density of each light quality measured at culture level was 10 µmol photons m–2 s–1 (3 W m–2) or at least 60 µmol photons m–2 s–1 (60 W m–2), respectively. For aerobic growth, overnight cultures with an OD660 of 0.2 were transferred to a flask and gassed with air, resulting in an oxygen concentration of approximately 180 µM. To test for the effect of illumination, cultures grown under dark aerobic conditions to an OD660 of 0.4 were illuminated with a 500 W halogen lamp with a light intensity of 800 W m–2 (750 µmol photons m–2 s–1; high intensity white light), or 800 W m–2 (380 µmol m–2 s–1; high intensity red light) or 30 W m–2 (18 µmol photons m–2 s–1; low intensity white light). A filter transmitting wavelength greater 600 nm was used for red light illuminations. Light energy in W m–2 was measured with a LI-250 light metre equipped with a LI-200SA pyranometer sensor (spectral response of 400–1100 nm, LI-Cor) and photon flux in µmol photons m–2 s–1 was measured with an LI-189 light metre equipped with a LI-190SA quantum sensor (spectral response of 400–700 nm, LI-Cor).
When required, antibiotics were used at the following concentrations: kanamycin 25 µg ml–1; tetracycline 20 µg ml–1 (E. coli) or 2 µg ml–1 (R. sphaeroides); trimethoprim 50 µg ml–1 (R. sphaeroides). In the presence of light, no tetracycline was added. For the generation of singlet oxygen, methylene blue (Sigma-Aldrich) was added to a final concentration of 0.2 µM. To test the effect of hydrogen peroxide (H2O2) and superoxide anions on phrA expression, H2O2 and paraquat were added to a final concentration of 1 mM to R. sphaeroides cultures with an OD660 of 0.5.
Photoreactivation activity.
Overnight cultures of Rhodobacter strains grown to an OD660 of 0.8 were diluted 1 : 100 in malate minimal salt medium. After exposure to various doses of UV light at 254 nm (UVcross-linker 1800, Stratagene), the cells were plated on agar plates in a dark room with dim red light, and afterwards exposed to the light of a 58 W fluorescent lamp (TLD 58W/25, Philips). For photoreactivation, the plates were covered with the plastic cover of the Petri dish. Photon flux density measured at culture level was 35–40 µmol photons m–2 s–1 (LI-190SA quantum Sensor, LI-Cor; spectral response 400–700 nm). Control plates were wrapped in aluminium foil for growth in the dark. After 3 days, the colonies were counted and survivals relative to UV-unirradiated controls were calculated.
Genetic techniques.
DNA cloning was performed according to standard protocols (Sambrook & Russell, 2001). All plasmids used are listed in Table 1
. Oligonucleotides generating suitable recognition sites for cloning or used as primers for RT-PCR analyses were synthesized by Roth. DNA sequencing was performed with an ABI-Prism 310 genetic analyser (Applied Biosystems).
Construction of R. sphaeroides 
phrA, 1981 and 3077.
R. sphaeroides phrA, 1981 and 3077 were generated by transferring plasmid pPHUphrA, pPHU1981 or pPHU3077 into R. sphaeroides. Fragments (500 bp) of the 5' and 3' ends of the genes were amplified by PCR using the oligonucleotides phrAHindIIIup (5'-GGGAAGCTTCAGGCCGATCTCCCCCC-3'), phrAEcoRIup (5'-GGAATTCGAAGATCGTATGGCCC-3'), phrAEcoRIdown (5'GAATTCGTGACCGGCACCATG-3') and phrAXbaIdown (5'-GCTCTAGAGCCGTCATTCGTGTCC-3') for phrA; 1981HindIIIup (5'-GGGAAGCTTCGCGACCTGCCTCG-3'), 1981ScaIup (5'-CCGGCCCAGTACTGCAGG-3'), 1981ScaIdown (5'-GTGGATGCAGTACTGCGCG-3') and 1981XbaIdown (5'-CGTCTAGAAACATGACCAATCCGCC-3') for RSP1981 and 3077HindIIIup (5'-CCCAAGCTTGGGCTCGACTGGAGCCGA-3'), 3077ScaIup (5'-AAAAGTACTTTTCGATCAGCCGCCAGTC-3'), 3077ScaIdown (5'-AAAAGTACTTTTCGCGCTGCTGGCGGG-3') and 3077XbaIdown (5'-GCTCTAGAGCTTCCGCCTCGGTCAGA-3') for RSP3077. The Kmr gene (Vieira & Messing, 1982), flanked by the corresponding fragments, was inserted in the suicide plasmid pPHU281 (Hübner et al., 1993). The plasmids were transferred into E. coli S17-1 and each was then transferred into R. sphaeroides 2.4.1 by biparental conjugation. Southern blotting was performed of kanamycin-resistant, tetracycline-sensitive clones, to confirm that the target genes had been replaced by the inactive gene through double crossover.
Complementation of R. sphaeoides phrA and 3077.
For complementation of R. sphaeroides phrA or 3077, the genes phrA or RSP3077 with around 100 bp of the flanking sequences on each end were generated with primers phrAHindIIIup and phrAXbaIdown or 3077HindIIIup and 3077XbaIdown, respectively. The fragments were ligated into plasmid pRK415, and the resulting plasmids pRKphrA and pRK3077 were transferred into the recipient strain by triparental mating as described earlier (Klug & Drews, 1984).
Construction of the phrA : lacZ reporter plasmid.
The putative promoter region of phrA (–108 to+130 bp) was amplified with the oligonucleotides phrAlacZup (5'-GCGAATTCAGGCCGATCTCCCCC-3') and phrAlacZdown (5'-CGGTCTCGGGATCCAGAATGAAC-3') and ligated in the vector pPHU234, carrying the promoterless lacZ gene. The construct was conjugated into R. sphaeroides strains, as well as the plasmid pPHU234, as a control.
β-Galactosidase assay.
This was carried out as described previously (Hübner et al., 1991).
RNA extraction and real-time RT-PCR.
Total RNA of R. sphaeroides cells grown under different conditions was isolated as described previously (Nieuwlandt et al., 1995) and quantified by spectrophotometric analysis (absorbance at 260 nm). For quantitative real-time RT-PCR, 40 ng total RNA was used and the One-Step RT-PCR kit (Qiagen) and Reverse-iT One-Step RT-PCR Kit (ABgene), following the manufacturer's instructions. The amplification of PCR products [oligonucleotides phrA-A (5'-GTTCGAGGACTGCCTGAT-3') and phrA-B (5'-AGGAACCTCTGGCGATAG-3') for phrA, lacZ-A (5'-CATTCAGGCTGCGCAACT-3') and lacZ-B (5'-TTAATCGCCTTGCAGCAC-3') for lacZ and rpoZ-A (5'-ATCGCGGAAGCCAGAG-3') and rpoZ-B (5'-GAGCAGCGCCGATCCT-3') for rpoZ] was monitored with SYBR Green using a Rotor-Gene 3000 real-time PCR cycler (LTF). Relative expression of phrA and rpoZ (a housekeeping -factor) mRNA were calculated by the method of Pfaffl (2001).
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). In contrast to R. sphaeroides this protein appears to be the only member of the photolyase/cryptochrome family in this organism. PhrA, RSP1981 and RSP3077 exhibit 61 %, 31 % and 24 % sequence identity, respectively, to the protein encoded by ORF90. Primary sequence comparison with the CPD-photolyase derived from Synechococcus sp. 6301 (Tamada et al., 1997) revealed several conserved residues shown to be involved in cofactor binding (Tamada et al., 1997; Fig. 1). Photolyases provide three conserved tryptophan residues, which were supposed to be involved in electron transport to reduce the FADH2 (Byrdin et al., 2003, Fig. 1). These residues are present in PhrA, but are notably absent in RSP1981 and RSP3077. However, since blocking photoreduction through this tryptophan chain by point mutations has little or no physiological significance for the in vivo activity of the E. coli photolyase (Kavakli & Sancar, 2004), the absence of the tryptophan triad does not necessarily correlate with a loss of photolyase function in RSP1981 and RSP3077.
phrA and RSP3077, but not RSP1981, are responsible for photoreactivation in R. sphaeroides
A quantitative analysis of UV light challenge revealed that R. sphaeroides 2.4.1 indeed shows photoreactivation (Fig. 2). While only 8 % of R. sphaeroides wild-type cells survived 100 J m–2 UV light exposure without photoreactivation, subsequent illumination increased the survival rate by a factor of 6.6 to 53 %. To test for a photolyase function, the gene phrA, RSP1981 or RSP3077 was inactivated by insertion of a kanamycin resistance cassette, and the resulting mutants were tested for photoreactivation. All mutants showed a comparable survival after UV light as the wild-type when light was not available (Fig. 2). When R. sphaeroides phrA was illuminated after exposure to UV light, we observed a significantly decreased survival rate (3 %) in comparison to the wild-type (53 %, Fig. 2). Complementation of R. sphaeroides phrA by plasmid-borne phrA restored photoreactivation properties and increased the survival rate of the deletion mutant 14-fold. The complementation of the photolyase mutant by the phrA gene is a clear proof for its function as photolyase.
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R. sphaeroides lacking RSP3077 showed photoreactivation; survival increased about 3.5-fold after illumination. Interestingly, the RSP3077 mutant showed 2.5-fold reduced survival (21 %) in comparison to the wild-type (53 %). When this strain was complemented, illumination increased the survival to 67 %, similar to that of the complemented phrA mutant. Selby & Sancar (2006) reported recently that members of the previously found cryptochrome DASH family (Brudler et al., 2003; Daiyasu et al., 2004) repair single stranded DNA. Although RSP3077 shows only minor (up to 9 %) sequence identity to members of this family, we can not exclude a function of RSP3077 in this regard and a role as putative second photolyase.
Expression of phrA is regulated by E and the anti-E factor ChrR
To date, there has been no evidence of a specific regulatory mechanism for the photolyase gene in any organism studied in this regard. Previous analysis of the promoter region of phrA revealed the presence of a putative E DNA-binding site, which is identical to the rpoE consensus sequence (Braatsch et al., 2004). Additional transcriptome analysis and in vitro transcription studies support the view that phrA is under control of E (Anthony et al., 2005).
In order to confirm these data and to test for the involvement of the anti-E factor ChrR, we compared the expression level of phrA in the wild-type strain R. sphaeroides 2.4.1 and in the derived mutant strains TF18 and ChrR grown at high oxygen tension in the dark. Mutant strain TF18 has the rpoE and the chrR genes deleted (Schilke & Donohue, 1995), while mutant strain ChrR has only chrR inactivated (Newman et al., 1999). When a phrA : lacZ reporter plasmid (pPHUphrAlacZ) was introduced into the wild-type strain, we observed a low β-galactosidase activity of 6 Miller units (Fig. 3a). In strain TF18, the activity was only 1 unit (Fig. 3a), which was the same as background (not shown). In contrast, we obtained about 200 Miller units in strain ChrR harbouring the reporter plasmid (Fig. 3a). These data suggest that E is required for transcription of phrA and that ChrR strongly counteracts E-mediated transcription, as was shown previously (Anthony et al., 2004).
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E or to ChrR of R. sphaeroides, nor an E-like binding region in the promoter of ORF90.
Upregulation of phrA expression depends on light intensity and can be enhanced by the photosensitizer methylene blue
Although photolyase activity is only required upon the formation of pyrimidine dimers, phrA seems to be expressed even in the absence of light (Fig. 3a and our unpublished microarray chip data). However, we have reported before that blue light stimulates phrA expression (Braatsch et al., 2004). In the fungus Trichoderma harzianum (Berrocal-Tito et al., 1999) and in the goldfish Carassius auratus (Mitani et al., 1996; Mitani & Shima, 1995; Yasuhira et al., 1991) blue light as well as H2O2 induces the transcription of the photolyase gene. Kihara et al. (2004) showed that near-UV light (300–400 nm) and sunlight enhance the expression of the photolyase in the fungus Bipolaris oryzae. Therefore, it is possible that phrA gene expression is upregulated in response to general stressors. To test this hypothesis, we used the phrA : lacZ reporter plasmid and performed real-time RT-PCR to monitor E/ChrR dependent phrA expression in cultures grown under different light conditions. We used methylene blue as a photosensitizer to generate singlet oxygen in irradiated cultures and tested whether phrA may also respond to other reactive oxygen species, i.e. H2O2 or superoxide anions, in a E/ChrR dependent manner.
R. sphaeroides 2.4.1 and mutant strains TF18 and ChrR carrying the reporter plasmid pPHUphrAlacZ were grown in the dark under high oxygen concentration and then shifted to high intensity white light (800 W m–2 or 750 µmol photons m–2 s–1) in the presence or absence of methylene blue. In all strains tested the addition of 0.2 µM methylene blue in the dark did not affect the β-galactosidase activity (data not shown). In the wild-type, the β-galactosidase activity increased from 5.5 to 19 Miller units after 120 min of white light illumination (Fig. 3a, b). An identical light shift experiment in the presence of methylene blue resulted in a slightly increased β-galactosidase activity (25 Miller units, Fig. 3a, b). No such light dependent increase in activity was observed in strains TF18 or ChrR (Fig. 3a, b). Here, methylene blue did not affect the β-galactosidase activity of the illuminated mutant strains (Fig. 3a, b).
To confirm the reporter gene studies, we used real-time RT-PCR for determination of the relative increase in phrA levels due to illumination in the presence or absence of methylene blue (Fig. 3c). For the wild-type, we observed a relative fourfold upregulation in phrA expression as early as 5 min of irradiation. A prolonged illumination of 15 min resulted in a fivefold increase in phrA transcript level compared to the dark aerobic control. No such significant upregulation was observed for the mutant strains TF18 and ChrR, which is in line with our reporter gene studies (Fig. 3a, b). Interestingly, illumination of wild-type cultures in the presence of methylene blue caused a further 1.8-fold upregulation of phrA expression at the given time points while no effect of the photosensitizer was detectable for the mutant strains (Fig. 3c). The higher phrA transcript level observed in the wild-type cultures treated with the photosensitizer suggests that singlet oxygen is a trigger for this response. It is conceivable that the β-galactosidase activity is impaired by the presence of singlet oxygen. However, our results strongly suggest that (i) white light illumination triggers a phrA upregulation in aerobically grown R. sphaeroides cells and that (ii) the E/ChrR pathway is responsible for signal transduction, resulting in a fast transcriptional response.
When we illuminated semi-aerobically grown wild-type cells with blue light of 10 µmol photons m–2 s–1 (3 W m–2), we did not see any significant change in phrA expression as determined by the phrA : lacZ reporter plasmid or by real-time RT-PCR (not shown). This is not in agreement with previously published data (Braatsch et al., 2004), where a 3.9-fold increase in phrA expression was described after 5 min blue light illumination with 10 µmol photons m–2 s–1 of the wild-type. In the latter case light intensities were monitored with a lux metre (DX-100), so it is conceivable that the light intensities used for the previous experiments were indeed higher than the 10 µmol photons m–2 s–1 used in the experiments reported here and monitored by a quantum sensor (LI-190SA). Indeed, when we increased the intensity of blue light to 60 µmol photons m–2 s–1, the β-galactosidase activity in wild-type cells increased twofold (not shown).
When wild-type cells were grown under high oxygen tension and illuminated with red light of high intensity (800 W m–2 or 380 µmol photons m–2 s–1) we determined a 2.8-fold increase in phrA expression by the reporter assay and a 4.8-fold increase in the presence of methylene blue (Fig. 3a, b). Controls in the dark with or without methylene blue did not show any significant changes in β-galactosidase activity. These data reveal that phrA does not specifically respond to blue light and that no specific photoreceptor appears to be involved.
phrA expression of R. sphaeroides 2.4.1 responds to singlet oxygen
The amplifying effect of methylene blue on phrA expression of illuminated cells (Fig. 3a–c) suggests that singlet oxygen is a trigger for this response. To further support this assumption, we monitored phrA expression in strain TC67, which lacks carotenoids. Carotenoids are essential constituents of the photosynthetic apparatus and are assumed to prevent the formation of singlet oxygen by quenching of triplet bacteriochlorophyll a in vivo (Borland et al., 1989). It was shown previously that, in the presence of oxygen and high light, R. sphaeroides forms small amounts of singlet oxygen in vivo, which do not adversely affect cell growth (Glaeser & Klug, 2005). Higher amounts of singlet oxygen were generated by bacteriochlorophyll a in the carotenoid-deficient strain TC67, causing a decrease in growth and survival rates (Glaeser & Klug, 2005). When strain TC67(pPHUphrAlacZ) was illuminated with white light of low intensity (30 W m–2), the β-galactosidase activity increased about fivefold within 2 h (Fig. 3a, b). The induction of a comparable response in the wild-type required a 26-fold higher light intensity (800 W m–2; Fig. 3a, b). The more light sensitive phrA upregulation in strain TC67 supports our view that singlet oxygen can trigger this response.
We also monitored phrA expression by using the reporter plasmid in strain PUHA1, which lacks the H-subunit of the reaction centre (RC). In the RC all the cofactors are bound to the L and M subunits. A RC complex in which the H peptide has been stripped away, leaving an LM remnant, retains full primary photoactivity. However, in these LM complexes, the QA to QB reaction is impaired and the complexes are significantly less stable than LMH (Debus et al., 1985). In vivo, reaction centres lacking the H subunit are virtually absent as determined by spectroscopy (Tehrani et al., 2003) and as a consequence, strain PUHA1 can not grow phototrophically. Illumination of PUHA1 by high white light intensity resulted in about sixfold increase of phrA expression, high light and methylene blue in about sevenfold increase (Fig. 3a, b). This is significantly higher than in the wild-type (3.5- and 4.2-fold, respectively), suggesting that the lack of RC may amplify the signal acting on phrA. Most likely, energy transfer from excited bacteriochlorophyll to oxygen is enhanced when no photosynthetic electron transport can be initiated in the RC. Furthermore, we conclude that photosynthetic electron transport is not involved in light dependent regulation of phrA expression, as demonstrated for the upregulation of photosynthesis genes (Happ et al., 2005).
phrA expression of R. sphaeroides 2.4.1 responds to hydrogen peroxide but not to superoxide anions
In order to test whether phrA expression may also respond to other reactive oxygen species, we quantified its expression in the presence of H2O2 and the superoxide-generating agent paraquat. By using the phrA : lacZ reporter plasmid we could not observe any significant expression changes in response to H2O2 (Fig. 5) or paraquat (not shown). However, we had observed before that the lacZ gene is not suitable to monitor the response to H2O2 and superoxide anions (see below), presumably due to loss of activity of the β-galactosidase under oxidative stress.
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). As soon as 7 min after H2O2 exposure, we observed a 5.6-fold increase in phrA transcript level (Fig. 4). In strains TF18 and ChrR, which lack E and ChrR or ChrR, respectively, the phrA activity dropped about twofold upon 7 min exposure to H2O2 (Fig. 4). However, after 30 min of H2O2 treatment a relative 
1.5-fold increase in phrA transcript levels was observed in all three strains (Fig. 4). Based on gene expression dynamics we conclude that the E/ChrR pathway is responsible for the fast upregulation of phrA. For unknown reasons, expression of this gene was found to be slightly downregulated in mutant strains TF18 and ChrR after 7 min (Fig. 4). Since a prolonged exposure to H2O2 resulted in a similar 1.5-fold increase in phrA transcript levels, we suggest that other factors besides E and ChrR are involved in a retarded upregulation of phrA expression. A fast and transient response is expected, since the R. sphaeroides catalases detoxify H2O2 very quickly (Zeller & Klug, 2004).
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). Thus, superoxide anions generated by paraquat do not lead to E dependent stimulation of phrA.
Anthony et al. (2005) reported that E together with its anti- factor ChrR transmit singlet oxygen dependent signals but are not involved in the response to H2O2, diamide and superoxide anions. An increase of rpoE expression in the presence of H2O2 was not observed according to rpoE : lacZ reporter gene studies (Anthony et al., 2005). In contrast to this previous study, we found a significant upregulation of phrA (Fig. 4) and a similar upregulation in rpoE expression (not shown) due to H2O2 stress using real-time RT-PCR and DNA microarray analysis (Zeller et al., 2005).
In the non-phototrophic 
-proteobacterium Caulobacter crescentus cadmium sulphate (6 µM) caused a sixfold upregulation of the phrA homologue CC1428 (Hu et al., 2005). Cd2+ binds to glutathione in Gram-negative bacteria, which subsequently results in the formation of a considerable amount of H2O2 (reviewed by Nies, 1999). Interestingly, the upstream regions of CC1428 and rpoE/chrR (CCR0645/CCR0644) encode a putative R. sphaeroides E-dependent promoter sequence. Therefore it is likely that in C. crescentus (i) H2O2 formation due to Cd2+ stress results in a E/ChrR dependent upregulation of the phrA homologue CCR1428 and (ii) rpoE is transcribed by its own product as suggested for R. sphaeroides (Newman et al., 1999).
β-Galactosidase-activity in R. sphaeroides is affected by H2O2
We observed frequently that lacZ reporter assays in R. sphaeroides are not suitable to monitor responses to H2O2 and superoxide anions, indicating sensitivity of the β-galactosidase enzyme. The data presented in this paper suggest that this might also be the case for singlet oxygen.
We analysed by real-time RT-PCR whether the expression of lacZ in the phrA : lacZ reporter plasmid is enhanced in the wild-type after 7 min incubation with 1 mM H2O2. We measured a 2.6-fold increase in lacZ-mRNA level (Fig. 5), and a 5.6-fold increase of the mRNA transcribed from the chromosomal copy of phrA (5.6-fold, Figs 4 and 5). When the wild-type harbouring the reporter plasmid was cultured under high intensity white light conditions for 15 min, we observed a 7.4-fold increase of lacZ mRNA level (Fig. 5), which is similar to the increase of phrA mRNA level (Figs 3c and 5).
Based on the result that the lacZ gene expression but not the β-galactosidase enzymic activity increased, we suggest that this activity is somehow affected by reactive oxygen species. Toptchieva et al. (2003) found that intrinsic tellurite toxicity prevented the use of the lacZ fusion as a reporter for regulatory studies in Proteus mirabilis. Since tellurite oxidizes thiol groups, it contributes to oxidative stress and might affect the β-galactosidase enzyme in a similar way as ROS.
Conclusion
Our data reveal that the phrA gene of R. sphaeroides encodes a functional photolyase. A response of increased phrA expression caused by light is indeed triggered by singlet oxygen, which is generated by energy transfer from bacteriochlorophyll to molecular oxygen, and the response requires the E/ChrR system. Involvement of photoreceptors or photosynthetic electron transport can be excluded. In contrast to previous studies, we provide evidence that the E/ChrR system is also stimulated by H2O2. Future investigations will focus on determining by which mechanisms singlet oxygen or H2O2 affect E : ChrR complex dissociation.
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ACKNOWLEDGEMENTS |
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Edited by: J. Green
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Received 15 November 2006;
revised 1 February 2007;
accepted 5 February 2007.
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