Institut für Mikrobiologie und Molekularbiologie, University of Giessen, Heinrich-Buff-Ring 2632, D-35392 Giessen, Germany
Correspondence
Jens Glaeser
Jens.Glaeser{at}mikro.bio.uni-giessen.de
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ABSTRACT |
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INTRODUCTION |
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Only recently has the effect of the reactive oxygen species hydrogen peroxide and superoxide anion on growth, survival and gene regulation been investigated in Rhodobacter (Li et al., 2003a, b
, 2004
; Zeller & Klug, 2004
). In contrast, photo-oxidative stress and the protective role of carotenoids against the generation of singlet oxygen in R. sphaeroides were recognized 50 years ago (Griffiths et al., 1955
). In this study it was reported that a carotenoid-deficient mutant of R. sphaeroides is rapidly killed when light and oxygen are present simultaneously. Decades later, it was shown that singlet oxygen is generated by triplet bacteriochlorophyll a (BChl a) in the presence of molecular oxygen (Borland et al., 1987
). Evidence for the generation of singlet oxygen by BChl a in intact reaction centres (RCs) and light-harvesting (LH) complexes was obtained later. Protein subunits of carotenoid-free RCs are degraded quickly in the presence of light and oxygen (Tandori et al., 2001
) and carotenoid-free LH complexes generate triplet and cation-radical BChl a upon illumination, which give rise to singlet oxygen formation in the presence of oxygen (Cogdell et al., 2000
; Limantara et al., 1998
).
Carotenoids represent a structurally very diverse class of isoprenoid pigments, which occur in all photosynthetic organisms. In photosynthetic purple bacteria, they are essential constituents of LH antennae (Lang et al., 1995; Zurdo et al., 1993
), important in harvesting light energy and exciton transfer (Cogdell et al., 1999
), and essential for the protection of the photosynthetic apparatus under photo-oxidative stress (Cogdell & Frank, 1987
; Cogdell et al., 2000
). Carotenoids prevent the harmful effects of singlet oxygen by quenching (Foote & Denny, 1968
), reacting with singlet oxygen to form oxidized forms of carotenoids (Fiedor et al., 2001
, 2002
) or quenching the triplet state of BChl a (Borland et al., 1989
). In vivo, quenching of triplet BChl a by carotenoids is assumed to be the major process by which the generation of singlet oxygen is prevented in photosynthetic purple bacteria (Cogdell & Frank, 1987
; Cogdell et al., 2000
; Limantara et al., 1998
).
Singlet oxygen has been shown to rapidly kill cells in many biological systems (Foote, 1976; Krinsky, 1978
). Possible targets of damage by singlet oxygen include a large variety of biological molecules, such as DNA, proteins and lipids (Briviba et al., 1997
). Singlet oxygen has been proven to be genotoxic (Epe, 1991
) and mutagenic (Ouchane et al., 1997
). Its half-life ranges from 4 µs in water (Foote & Clennan, 1995
) to 200 ns in living cells (Gorman & Rodgers, 1992
). Diffusion of singlet oxygen is limited to a range of 10 nm in vivo (Sies & Menck, 1992
) and 100 nm in aqueous solutions (Kochevar & Redmond, 2000
). The efficiency of singlet oxygen quenching varies between structurally different carotenoids and depends in vitro mainly on their concentration (Foote & Denny, 1968
), the length of the conjugated system (Foote et al., 1970
) and functional groups (Di-Mascio et al., 1989
; Foote & Denny, 1968
; Hirayama et al., 1994
).
Singlet oxygen may elicit stress responses by affecting gene regulation. It has been shown that in response to singlet oxygen the expression of several genes is up-regulated in Chlamydomonas and Arabidopsis species (Leisinger et al., 2001; op-den-Camp et al., 2003
). A light-dependent induction of carotenoid synthesis was observed in Myxococcus xanthus, which presumably is mediated by singlet oxygen (Hodgson & Murillo, 1993
). The induction of carotenoid biosynthesis by singlet oxygen has also been shown in Pfaffia rhodozyma (Schroeder & Johnson, 1995
). The genetic basis of the response to stress induced by singlet oxygen has been recently unravelled in Arabidopsis (Wagner et al., 2004
). However, the mechanisms underlying gene regulation and signal transduction induced by singlet oxygen are so far mostly unclear (Kochevar, 2004
; Krieger-Liszkay, 2004
).
In the present study, we have investigated the capacity of different carotenoids to protect R. sphaeroides under conditions of photo-oxidative stress and address the question whether genes involved in the defence against photo-oxidative and oxidative stress are induced by singlet oxygen.
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METHODS |
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I=(I1I2)/ln(I1/I2) (Van Liere & Walsby, 1982).
Light intensity was adjusted throughout the experiments to compensate for changes in turbidity due to bacterial growth. Conditions of photo-oxidative stress were generated by the addition of methylene blue (SigmaAldrich) at a final concentration of 0·2 µM to aerobically growing cultures incubated under high light. Methylene blue specifically generates singlet oxygen in the presence of light and oxygen. Bleaching of methylene blue was checked by recording UV/VIS absorption spectra between 400 and 600 nm (Lambda12; Perkin Elmer) in culture medium without cells. Maximum absorption of methylene blue decreased during incubation under high light to 74 and 69 % after 90 and 150 min, respectively.
Detection of singlet oxygen.
Singlet oxygen was detected by the reaction with DanePy [3-(N-diethylaminoethyl)-N-dansyl-aminomethyl-2,5-dihydro-2,2,5,5-tetramethyl-1H-pyrrole], yielding the nitroxide radical DanePyO [3-(N-diethyl-aminoethyl)-N-dansyl-aminomethyl-2,5-dihydro-2,2,5,5-tetramethyl-1H-pyrrol-1-yloxyl], which exhibits decreased fluorescence (Kálai et al., 1998). The change in relative DanePy fluorescence (
F/F545) indicates the relative amount of singlet oxygen generated and was calculated as described by Hideg et al. (2000)
. Fluorescence of DanePy in Rhodobacter cultures was measured by excitation of samples at 337 nm and recording the emission at 545 nm with a Kontron SFM-25 spectrophotofluorimeter. For fluorescence measurements, a slit width of 10 nm and a quartz cuvette with a path length of 10 mm were used. To obtain data on singlet oxygen generation, a final concentration of 20 µM DanePy was added to exponentially growing cultures and then incubated under conditions of high light. For normalization of raw fluorescence data in time series experiments, initial fluorescence values at 0 min were used. Prior to normalization, fluorescence values obtained from dark-incubated control cultures were subtracted from values obtained from cultures incubated under high light.
Survival assays under photo-oxidative stress.
The survival of Rhodobacter was determined after exposure to photo-oxidative stress for 90 min by plating aliquots of serial dilutions obtained from liquid cultures. Control cultures were incubated in the presence of high light (800 W m2) or were kept in the dark.
Analysis of bacterial pigments.
To gain absorption spectra, samples of cultures from directly before the shift to high light or photo-oxidative stress conditions were used. The optical density of all cultures was 0·4 at 660 nm and 30 ml was harvested at 10 000 g for 10 min. Pigments were extracted from cell pellets with a mixture of acetone and methanol (7/2, v/v) and analysed by UV/VIS spectroscopy (Lambda12). BChl a concentrations were calculated from the absorption at 772 nm with an extinction coefficient of 76 mM1 cm1 (Clayton, 1966). Carotenoids were analysed from the same extracts by HPLC (HP 1100 Series; Hewlett Packard) on a C18 silica column (Multophyp ODS 5 µm, 250x4·6 mm; CS-Chromatographie Service, Langerwehe, Germany) with a mixture of methanol and acetone (9/1, v/v) and a flow rate of 1 ml min1 (Permentier et al., 2001
). Identification of carotenoids was performed by their retention time and the absorption spectra were recorded between 300 and 800 nm with a diode array spectrophotometer (HP 1100 Series, model G1324A). Extinction coefficients used for quantification of carotenoids were
=2500 for sphaeroidenone, hydroxysphaeroidenone, sphaeroidene and hydroxysphaeroidene and
=2700 for neurosporene (Züllig, 1985
). For the preparation of carotenoid standards, acetone extracts of R. sphaeroides strains were separated on silica gel plates (Polygram Sil G; MachereyNagel) with a mixture of petroleum ether and acetone (8/2, v/v). Coloured bands containing carotenoid bands were cut out and resolved in acetone, and concentrations were determined by UV/VIS spectroscopy (Lambda12).
RNA extraction and quantitative real-time RT-PCR.
Samples from growth experiments with Rhodobacter cultures were obtained at 0, 5, 10, 20, 40 and 90 min after the shift of dark-incubated cultures to high light or to photo-oxidative stress. Samples were rapidly cooled in ice and pelleted by centrifugation at 10 000 g. In all cases, shift experiments for RNA isolation were started at an optical density of 0·4 at 660 nm. Total RNA was isolated by the hot phenol method and quantified by photometric analysis at 260 nm. Samples were treated with RQ1 RNase-free DNase I (Promega) to remove contaminating DNA. Absence of genomic DNA contamination was checked by PCR amplification of RNA samples. A final concentration of 4 ng total RNA µl1 was used and the following primers were synthesized to quantify relative gene expression by quantitative real-time RT-PCR: crtA, crtA-A (5'-GAATCGCCGATCTACCAG-3') and crtA-B (5'-GGCCTTCCAGAACTTGAC-3'); crtI, crtI-A (5'-CAACGTGACCTCGATGTA-3') and crtI-B (5'-GAAGCCGCATGTAGGTAT-3'); katE, katE-A (5'-CTATCCGCTGATCGAGGT-3') and katE-B (5'-GTCGGCATAGGAGAAGAC-3') (Zeller & Klug, 2004); rpoZ, 2.4.1rpoZ-A (5'-ATCGCGGAAGAGACCCAGAG-3') and 2.4.1rpoZ-B (5'-GAGCAGCGCCATCTGATCCT-3') (Zeller & Klug, 2004
); RSP0799, RSP0799-A (5'-GAACAATTACGCCTTCTC-3') and RSP0799-B (5'-CATCAGCTGGTAGCTCTC-3'); RSP2389, RSP2389-A (5'-CCGCAATACGACGATCTT-3') and RSP2389-B (5'-CGGAGTGATGGTGGTCAT-3'). Normalization of mRNA levels was performed with the rpoZ gene, which encodes the
-subunit of the R. sphaeroides RNA polymerase (Pappas et al., 2004
). The one-step RT-PCR kit (Qiagen) was used for reverse transcription followed by PCR as described in the manufacturer's manual, except SYBR Green (SigmaAldrich) was added at a final dilution of 1 : 50 000 to the final master mix. Master mix and RNA solution were mixed in a final volume of 10 µl for relative quantification of mRNA transcripts in a Rotor-Gene 3000 real-time PCR cycler (Corbett Research). For analysis slope correction and dynamic tube normalization options were applied in the rotor-gene software version 6.0 (Corbett Research). Crossing point (Cp) values representing the number of cycles where fluorescence signals started to increase in real-time RT-PCR was determined for all genes with a fluorescence threshold of 0·002 and relative expression of crtA, crtI, katE, RSP0799 and RSP2389 mRNA was calculated relative to the expression of untreated samples and relative to rpoZ, according to the method of Pfaffl (2001)
. Real-time PCR efficiencies were determined by applying serial dilutions of mRNA between final concentrations of 8 and 0·2 ng µl1 as 1·89 for crtA, 1·709 for crtI, 1·96 for katE, 2·02 for rpoZ, 2·31 for RSP0799 and 2·04 for RSP2389.
Statistical analysis.
Statistical analysis for comparison of Cp values for the above-mentioned genes under different physiological conditions was performed with Student's t-test using Microsoft Excel. In all cases, significance was assumed if P<0·05.
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RESULTS |
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To investigate in more detail the role of carotenoid composition and abundance, R. sphaeroides wild-type cultures and mutants impaired in carotenoid synthesis were tested for their survival after incubation under high light and photo-oxidative stress conditions. The survival rate of R. sphaeroides wild-type cultures obtained under photo-oxidative stress was not significantly different from high-light- and dark-incubated controls (Fig. 2). As determined by HPLC, wild-type cultures contained 76·6 % sphaeroidenone, 20·5 % neurosporene and 2·6 % hydroxysphaeroidenone as major carotenoids (Table 2
). The molar ratio indicated that four times more BChl a was present than carotenoids (Table 2
). These results support the earlier finding that the carotenoid content and composition of R. sphaeroides efficiently prevent the generation of singlet oxygen in the light.
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In cultures of strain TC40 only 81 % of the cells survived incubation under photo-oxidative stress, but under high light no change was observed compared to dark controls (Fig. 2). Neurosporene is the most abundant carotenoid in strain TC40 and accounts for 97 % of total carotenoids (Fig. 3
, Table 2
), whereas sphaeroidene and hydroxysphaeroidene comprise only 3 % and sphaeroidenone was absent. The total amount of carotenoids was only 11-fold higher in strain TC40 compared to strain TC23 (Table 2
), which explains its higher survival rate (Fig. 2
). The results suggest that neurosporene is less efficient in quenching singlet oxygen than sphaeroidenone in vivo, which is mainly present in the wild-type and in strain TC23, because survival decreased despite a higher carotenoid to BChl a ratio compared to the wild-type. However, the decrease in survival rate of strain TC40 under photo-oxidative stress was not statistically significant when compared to high-light- and dark-incubated cultures as indicated by the standard deviations (Fig. 2
).
The survival rate of strain TC52, which contained mainly sphaeroidene (93·3 %), small amounts of hydroxysphaeroidene (5 %) and neurosporene (1·7 %) decreased to 76 and 63 % under high light conditions and photo-oxidative stress, respectively (Figs 2 and 3). Compared to strain TC40, survival rates under both high light and photo-oxidative stress were lower in strain TC52, although similar amounts of total carotenoids are present in both strains and the ratio of carotenoids to BChl a is larger in strain TC52 (Fig. 3
, Table 2
). Therefore, it is very likely that sphaeroidene is less efficient in quenching singlet oxygen than neurosporene in vivo.
In conclusion, the abundance of total carotenoids and the type of carotenoid present in vivo are important factors for the survival of R. sphaeroides under high light and photo-oxidative stress.
Detection of singlet oxygen generated in vivo
To investigate if singlet oxygen is generated in R. sphaeroides cultures under physiological conditions, we measured singlet oxygen during exponential growth under high light conditions. Relative amounts of singlet oxygen generated were determined by the decrease in fluorescence of DanePy, a trap molecule that specifically reacts with singlet oxygen (Kálai et al., 1998). Only small amounts of singlet oxygen were generated by R. sphaeroides wild-type cultures incubated at 800 W m2 as indicated by the small decrease in relative DanePy fluorescence (Fig. 4
). However, singlet oxygen was clearly detected after 120 min incubation. A much larger decrease in DanePy fluorescence was observed when strain TC67 was incubated at light intensities as low as 20 W m2. In comparison, the relative DanePy fluorescence in strain TC67 was fourfold lower than in wild-type cultures (Fig. 4
) even though TC67 cultures were incubated under a 40-fold lower light intensity and contained fivefold lower levels of BChl a (Table 2
). Obviously, low amounts of BChl a are sufficient to generate toxic levels of singlet oxygen under low light intensities. Interestingly, singlet oxygen was also generated under high light in wild-type cultures. However, cellular damage was not observed by a decrease in growth or survival rate (Figs 1 and 2
).
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In a time-course experiment, the relative expression of RSP2389 increased 16-fold after 40 min incubation under photo-oxidative stress when compared to the non-treated control at 0 min (Fig. 6). Mean mRNA levels from three independent experiments also showed an increase after 20 min exposure to photo-oxidative stress when raw data were normalized to rpoZ and dark controls (Fig. 7
). The statistical significance of this increase was demonstrated by the comparison of raw Cp values (Table 3
). In contrast to photo-oxidative stress, relative levels of RSP2389 were not increased under high light (Fig. 7
, Table 3
). Glutathione peroxidase very likely has a similar function in R. sphaeroides as in Chlamydomonas, because its expression is induced by the same stress factor, singlet oxygen.
A putative Zn-dependent hydrolase encoded by gene RSP0799 increased in relative abundance under photo-oxidative stress, determined by the comparison of protein patterns generated by 2D gel electrophoresis (unpublished data). Similar to glutathione peroxidase, the expression of RSP0799 was highest after 40 min exposure to photo-oxidative stress (Fig. 6). Mean mRNA levels of RSP0799 increased 15-fold after 20 min exposure to photo-oxidative stress in three independent experiments when normalized to rpoZ and dark controls (Fig. 7
). The increase of RSP0799 expression was statistically significant under photo-oxidative stress when raw Cp values were compared to dark controls (Table 3
). The same analysis showed that relative expression of RSP0799 was not significantly increased under high light (Fig. 7
, Table 3
).
Relative mRNA levels of RSP0799 and RSP2389 did not increase after the addition of 1 mM hydrogen peroxide using the same time points as depicted in Fig. 6 (data not shown). Both genes showed very similar expression in the carotenoid-deficient strain TC67 under a light intensity of 15 W m2 in comparison to photo-oxidative stress in wild-type cultures (data not shown). In conclusion, RSP0799 and RSP2389 were induced in wild-type cultures only if singlet oxygen was generated by methylene blue. Taking into account that glutathione peroxidase degrades protein peroxides generated by singlet oxygen, it is very likely that singlet oxygen or damage caused by singlet oxygen is a specific signal for gene regulation in R. sphaeroides.
Relative expression of catalase E is not induced by singlet oxygen
Genes involved in the detoxification of other reactive oxygen species such as hydrogen peroxide may also be induced by singlet oxygen or damage generated by singlet oxygen. Catalase E (katE) mRNA levels were strongly increased by the addition of hydrogen peroxide (Zeller & Klug, 2004). Therefore, we elucidated katE expression under photo-oxidative stress in a time-course experiment over 90 min. Relative katE expression was slightly increased after 20 min incubation under photo-oxidative stress when data were normalized to a non-treated control at 0 min (Fig. 6
). Three independent experiments showed that relative katE expression was not significantly increased after 20 min exposure to photo-oxidative stress when raw Cp values were compared (Table 3
) or when relative expression of katE was normalized to rpoZ and dark controls (Fig. 7
). Surprisingly, cultures exposed to high light showed a significant decrease in katE mRNA levels (Table 2
). The data clearly show that relative katE expression was not induced by singlet oxygen. Together with the data obtained on mRNA levels of crtA, crtI, RSP0799 and RSP2389, it can be concluded that singlet oxygen and hydrogen peroxide provide different signals for gene regulation. Therefore, a regulative cascade specific for stress inferred by singlet oxygen can be proposed based on our data.
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DISCUSSION |
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Protective role of carotenoids: high light conditions
Experiments providing evidence for a fast triplet energy transfer in from BChl a to carotenoids in RC and LHII suggested that singlet oxygen could only be generated in R. sphaeroides when carotenoids are absent (Cogdell et al., 2000; Limantara et al., 1998
). Since mostly isolated RC and LHII complexes have been investigated so far to study the protection against singlet oxygen formation by carotenoids, the question remains, which carotenoids prevent the formation of singlet oxygen in vivo most efficiently? R. sphaeroides strains containing mainly neurosporene (TC40) or low amounts of sphaeroidenone and hydroxysphaeroidenone (TC23) survived 90 min of high light at the same rate as the wild-type, but a strain containing mainly sphaeroidene and traces of hydroxysphaeroidene (TC52) showed a lower survival rate (Fig. 2
). In contrast, neurosporene was least efficient in singlet oxygen quenching among R. sphaeroides carotenoids as determined by the oxidation of linolic acid in a mixture of n-hexane and methanol (Hirayama et al., 1994
). According to this study, carotenoids found in R. sphaeroides can be sorted by decreasing efficiency in singlet oxygen quenching in the following order: hydroxysphaeroidenone, sphaeroidenone, hydroxysphaeroidene, sphaeroidene, neurosporene. Despite this finding, a neurosporene-containing strain (TC40) was not harmed, but a sphaeroidene-containing strain was harmed by high light conditions. This result cannot be explained by different amounts of carotenoids, which are similar in both strains (Table 2
). Presumably, sphaeroidene is less efficient in quenching triplet BChl a in vivo than neurosporene. However, the lack of sphaeroidenone monooxygenase, which is missing in strain TC52, could also explain a decreased rate of survival (Table 1
). It has been suggested that sphaeroidene monooxygenase is involved in photoprotection (Yeliseev & Kaplan, 1997
). In this photoprotection mechanism sphaeroidene acts as a sink for singlet oxygen via chemical quenching as mediated by sphaeroidenone monooxygenase. The results on relative mRNA levels of crtA were not increased under photo-oxidative stress and, hence, do not support a role for crtA in protection against singlet-oxygen-generated damage, because an increase in relative mRNA levels should then be expected. Since post-transcriptional regulation has been observed for crtA expression (O'Gara & Kaplan, 1997
), its role in photoprotection under photo-oxidative stress remains an open question.
In comparison to the carotenoid-deficient strain TC67, all other strains tested had much higher survival rates under high light and photo-oxidative stress (Fig. 2). These results were supported by an earlier study performed with Rubrivivax gelatinosus. Although the carotenoid composition is different in Rubrivivax gelatinosus, a similar general conclusion was drawn: mutants containing carotenoids survived under high light conditions, whereas survival decreased drastically in carotenoid-free mutants (Ouchane et al., 1997
).
Protective role of carotenoids: photo-oxidative stress
Not only does the prevention of singlet oxygen generation play a pivotal role in organisms, but so does the quenching of singlet oxygen (Di-Mascio et al., 1989; Foote & Denny, 1968
). Prosthetic groups and the extension of the conjugated electron system, i.e. the structural features of carotenoids, largely influence their efficiency to quench singlet oxygen (Foote et al., 1970
; Hirayama et al., 1994
). Taking into account this information, the question arose whether the carotenoids found in R. sphaeroides are able to prevent damage from externally generated singlet oxygen. Water-soluble photosensitizers have been frequently used to increase the amount of singlet oxygen generated in biological systems (Kochevar & Redmond, 2000
). The survival rate of all strains impaired in carotenoid biosynthesis decreased under conditions of photo-oxidative stress (Fig. 2
), indicating that quenching of singlet oxygen generated by methylene blue depends on the specific amount of carotenoid present in vivo (Foote et al., 1970
). This theory is supported by the amount of carotenoids found and the survival rate observed in strains impaired in carotenoid synthesis and wild-type cultures. Clearly, survival and carotenoid content increased in the order TC67, TC23, TC40/TC52 and wild-type cultures (Fig. 3
, Table 2
). The importance of the type of carotenoid present in vivo becomes clear from the comparison of strains TC40 and TC52. Although similar amounts of carotenoids were observed, the neurosporene-containing strain (TC40) exhibited higher survival rates than the sphaeroidene-containing strain (TC52). Hence, our in vivo data are not in agreement with results obtained from in vitro systems where it was clearly observed that neurosporene is less efficient in singlet oxygen quenching than sphaeroidene (Hirayama et al., 1994
).
Evidence for the cellular adaptation to photo-oxidative stress
In our growth experiments, cultures of strain TC67 and wild-type cultures recovered from low growth rates during prolonged incubation under low light and photo-oxidative stress, respectively (Fig. 1). This suggests that an adaptation to the stress conditions has occurred. However, the spontaneous generation of mutants more resistant to photo-oxidative stress may also explain this phenomenon. In a previous study with carotenoid-deficient mutants of Rubrivivax gelatinosus, a frequency of 103 mutants with a higher resistance to photo-oxidative stress compared to carotenoid-deficient mutants was observed (Ouchane et al., 1997
). Therefore, generation of mutations can be ruled out as a reason for increased resistance, but a frequency of 103 mutations would only have generated a higher resistance in 0·1 % of the cellular population.
Singlet oxygen induces carotenoid biosynthesis in Pfaffia rhodozyma (Schroeder & Johnson, 1995) and very likely mediates the light-dependent induction of carotenoid biosynthesis in Myxococcus xanthus (Hodgson & Murillo, 1993
). In plants, zeaxanthin accumulates only under photo-oxidative stress and plays an essential role in protection of the photosynthetic apparatus via the xanthophyll cycle (Demmig-Adams et al., 1999
). Therefore, we expected an increase of relative carotenoid content in R. sphaeroides under photo-oxidative stress. However, neither an increase in carotenoid contents nor increased expression of carotenoid synthesis genes was observed in R. sphaeroides (Figs 4 and 7
). Therefore, other factors must exist in R. sphaeroides which are expressed in response to photo-oxidative stress to allow adaptation to higher singlet oxygen levels.
Induction of putative cellular defence systems
Induction of gene expression by singlet oxygen has been observed in bacteria, yeast and plants (Hodgson & Murillo, 1993; Leisinger et al., 2001
; op-den-Camp et al., 2003
). As an example, glutathione peroxidase significantly increased upon incubation under photo-oxidative stress in Chlamydomonas (Leisinger et al., 2001
) and is also involved in the degradation of peptide peroxides generated by singlet oxygen exposure (Morgan et al., 2004
). As in Chlamydomonas the increase of glutathione peroxidase in R. sphaeroides was specifically induced under photo-oxidative stress. Assuming the same function for glutathione peroxidase in R. sphaeroides, its induction indicates a specific response to stress caused by singlet oxygen. Further evidence for the induction of gene expression by singlet oxygen in R. sphaeroides was obtained by the increase of mRNA levels for a putative Zn-dependent hydrolase in response to photo-oxidative stress. These findings and the lack of katE expression by photo-oxidative stress clearly suggested that putative transcriptional regulators for the induction of genes in response to singlet oxygen must exist in R. sphaeroides. More than likely, these factors are different from those involved in the regulation of genes detoxifying hydrogen peroxide, such as katE. The identification of the regulatory factors that mediate the singlet-oxygen-specific response in R. sphaeroides will be the subject of future investigations. Only recently has the genetic basis for the response to singlet oxygen been investigated in Arabidopsis and regulation factors responding specifically to singlet oxygen have been found (Wagner et al., 2004
).
Although R. sphaeroides wild-type cultures do not seem to encounter stress by BChl a-mediated generation of singlet oxygen, the presence of a defence system against singlet oxygen generated by an extracellular photosensitizer would potentially be very important under environmental conditions. Extracellular production of singlet oxygen by humic acids has been reported recently (Paul et al., 2004). Humic acids are widespread in environments with high nutrient loads in aquatic and terrestrial habitats. Different efficiencies of singlet oxygen generation by humic acids have been reported to be dependent on an aquatic or terrestrial origin. In the case of aquatic origin, the amounts of singlet oxygen generated depend on the depth of the water column and the season of sampling (Paul et al., 2004
). Extracellular sources of singlet oxygen might be important under natural conditions. Carotenoids are potentially insufficient to prevent damage by singlet oxygen generated through humic acids or other extracellular photosensitizers under natural conditions. Therefore, singlet oxygen generated in the environment may affect growth and survival of Rhodobacter under natural conditions, which shows the potentially pivotal role of a defence system expressed in response to high levels of singlet oxygen.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Borland, C. F., McGarvey, D. J., Truscott, T. G., Cogdell, R. J. & Land, E. J. (1987). Photophysical studies of bacteriochlorophyll a and bacteriopheophytin a singlet oxygen generation. J Photochem Photobiol B Biol 1, 93101.[CrossRef]
Borland, C. F., Cogdell, R. J., Land, E. J. & Truscott, T. G. (1989). Bacteriochlorophyll a triplet state and its interactions with bacterial carotenoids and oxygen. J Photochem Photobiol B Biol 3, 227245.[CrossRef]
Briviba, K., Klotz, L. O. & Sies, H. (1997). Toxic and signaling effects of photochemically or chemically generated singlet oxygen in biological systems. Biol Chem 378, 12591265.[Medline]
Clayton, R. K. (1966). Spectroscopic analysis of bacteriochlorophylls in vitro and in vivo. Photochem Photobiol 5, 669677.
Cogdell, R. J. & Frank, H. A. (1987). How carotenoids function in photosynthetic bacteria. Biochim Biophys Acta 895, 6379.[Medline]
Cogdell, R. J., Isaacs, N. W., Howard, T. D., McLuskey, K., Fraser, N. J. & Prince, S. M. (1999). How photosynthetic bacteria harvest solar energy. J Bacteriol 181, 38693879.
Cogdell, R. J., Howard, T. D., Bittl, R., Schlodder, E., Geisenheimer, I. & Lubitz, W. (2000). How carotenoids protect bacterial photosynthesis. Philos Trans R Soc Lond B Biol Sci 355, 13451349.[CrossRef][Medline]
Demmig-Adams, B., Adams, W. W., Ebbert, V. & Logan, B. A. (1999). Ecophysiology of the Xanthophyll Cycle. In The Photochemistry of Carotenoids. Edited by H. A. Frank, A. Young, G. Britton & R. J. Cogdell. Dordrecht: Kluwer.
Di-Mascio, P., Kaiser, S. & Sies, H. (1989). Lycopene as the most efficient biological carotenoid singlet oxygen quencher. Arch Biochem Biophys 274, 532538.[CrossRef][Medline]
Drews, G. (1983). Mikrobiologisches Praktikum. Heidelberg: Springer.
Edge, R. & Truscott, T. G. (1999). Carotenoid radicals and the interaction of carotenoids with active oxygen species. In The Photochemistry of Carotenoids, pp. 223234. Edited by H. A. Frank, A. J. Young, G. Britton & R. J. Cogdell. Dordrecht: Kluwer.
Epe, B. (1991). Genotoxicity of singlet oxygen. Chem Biol Interact 80, 239260.[CrossRef][Medline]
Fiedor, J., Fiedor, L., Winkler, J., Scherz, A. & Scheer, H. (2001). Photodynamics of the bacteriochlorophyll-carotenoid system. 1. Bacteriochlorophyll-photosensitized oxygenation of beta-carotene in acetone. Photochem Photobiol 74, 6471.[CrossRef][Medline]
Fiedor, J., Fiedor, L., Kammhuber, N., Scherz, A. & Scheer, H. (2002). Photodynamics of the bacteriochlorophyll-carotenoid system. 2. Influence of central metal, solvent and beta-carotene on photobleaching of bacteriochlorophyll derivatives. Photochem Photobiol 76, 145152.[CrossRef][Medline]
Foote, C. S. (1976). Photosensitised oxidation and singlet oxygen: consequences in biological systems. In Free Radicals and Biological Systems, pp. 85133. Edited by W. A. Pryor. New York: Academic Press.
Foote, C. S. & Clennan, E. L. (1995). Properties and reactions of singlet oxygen. In Active Oxygen in Chemistry, pp. 105141. Edited by C. S. Foote, J. S. Valentine, A. Greenberg & J. F. Liebmann. London: Blackie Academic and Professional.
Foote, C. S. & Denny, R. W. (1968). Chemistry of singlet oxygen. VII. Quenching by -carotene. J Am Chem Soc 90, 62336235.[CrossRef]
Foote, C. S., Chang, Y. C. & Denny, R. W. (1970). Chemistry of singlet oxygen. X. Carotenoid quenching parallels biological protection. J Am Chem Soc 92, 52165218.[CrossRef][Medline]
Foyer, C. H. & Jeremy, H. (1999). Relationships between antioxidant metabolism and carotenoids in the regulation of photosynthesis. In The Photochemistry of Carotenoids. Edited by H. A. Frank, A. J. Young, G. Britton & R. J. Cogdell. Dordrecht: Kluwer.
Fraser, N. J., Hashimoto, H. & Cogdell, R. J. (2001). Carotenoids and bacterial photosynthesis: the story so far. Photosynth Res 70, 249256.[CrossRef]
Gorman, A. A. & Rodgers, M. A. (1992). Current perspectives of singlet oxygen detection in biological environments. J Photochem Photobiol B Biol 14, 159176.[CrossRef][Medline]
Gregor, J. & Klug, G. (1999). Regulation of bacterial photosynthesis genes by oxygen and light. FEMS Microbiol Lett 179, 19.[CrossRef][Medline]
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]
Griffiths, M., Sistrom, W. R., Cohen-Bazire, G. & Stanier, R. Y. (1955). Function of carotenoids in photosynthesis. Nature 176, 12111214.[Medline]
Hideg, É., Kálai, T., Hideg, K. & Vass, I. (2000). Do oxidative stress conditions impairing photosynthesis in the light manifest as photoinhibition? Philos Trans R Soc Lond B Biol Sci 355, 15111516.[CrossRef][Medline]
Hirayama, O., Nakamura, K., Hamada, S. & Kobayasi, Y. (1994). Singlet oxygen quenching ability of naturally occurring carotenoids. Lipids 29, 149150.[Medline]
Hodgson, D. A. & Murillo, F. J. (1993). Genetics of regulation and pathway of synthesis of carotenoids. In Myxobacteria II, pp. 157181. Edited by M. Dworkin & D. Kaiser. Washington, DC: American Society for Microbiology.
Imhoff, J. F., Trüper, H. G. & Pfennig, N. (1984). Rearrangements of the species and genera of the phototrophic purple nonsulfur bacteria. Int J Syst Bacteriol 34, 340343.
Kálai, T., Hideg, É., Vass, I. & Hideg, K. (1998). Double (fluorescent and spin) sensors for detection of reactive oxygen species in the thylakoid membrane. Free Rad Biol Med 24, 649652.[CrossRef][Medline]
Kochevar, I. E. (2004). Singlet oxygen signaling: from intimate to global. In Science's STKE [Electronic Resource] Signal Transduction Knowledge Environment, pe7. doi:10.1126/stke.2212004pe7
Kochevar, I. E. & Redmond, R. W. (2000). Photosensitized production of singlet oxygen. In Methods of Enzymology: Singlet Oxygen, UV-A and Ozone, pp. 2028. Edited by L. Packer & H. Sies. London: Academic Press.
Krieger-Liszkay, A. (2004). Singlet oxygen production in photosynthesis. J Exp Bot, 110.
Krinsky, N. I. (1978). Non-photosynthetic functions of carotenoids. Philos Trans R Soc Lond B Biol Sci 284, 581590.
Lang, H. P. & Hunter, C. N. (1994). The relationship between carotenoid biosynthesis and the assembly of the light-harvesting LH2 complex in Rhodobacter sphaeroides. Biochem J 298, 197205.[Medline]
Lang, H. P., Cogdell, R. J., Gardiner, A. T. & Hunter, C. N. (1994). Early steps in carotenoid biosynthesis: sequences and transcriptional analysis of the crtI and crtB genes of Rhodobacter sphaeroides and overexpression and reactivation of crtI in Escherichia coli and R. sphaeroides. J Bacteriol 176, 38593869.[Abstract]
Lang, H. P., Cogdell, R. J., Takaichi, S. & Hunter, C. N. (1995). Complete DNA sequence, specific Tn5 insertion map, and gene assignment of the carotenoid biosynthesis pathway of Rhodobacter sphaeroides. J Bacteriol 177, 20642073.
Leisinger, U., Rufenacht, K., Fischer, B., Pesaro, M., Spengler, A., Zehnder, A. J. & Eggen, R. I. (2001). The glutathione peroxidase homologous gene from Chlamydomonas reinhardtii is transcriptionally up-regulated by singlet oxygen. Plant Mol Biol 46, 395408.[CrossRef][Medline]
Li, K., Hartig, E. & Klug, G. (2003a). Thioredoxin 2 is involved in oxidative stress defence and redox-dependent expression of photosynthesis genes in Rhodobacter capsulatus. Microbiology 149, 419430.[CrossRef][Medline]
Li, K., Pasternak, C. & Klug, G. (2003b). Expression of the trxA gene for thioredoxin 1 in Rhodobacter sphaeroides during oxidative stress. Arch Microbiol 180, 484489.[CrossRef][Medline]
Li, K., Hein, S., Zou, W. & Klug, G. (2004). The glutathione-glutaredoxin system in Rhodobacter capsulatus: part of a complex regulatory network controlling defense against oxidative stress. J Bacteriol 186, 68006808.
Limantara, L., Fujii, R., Zhang, J. P., Kakuno, T., Hara, H., Kawamori, A., Yagura, T., Cogdell, R. J. & Koyama, Y. (1998). Generation of triplet and cation-radical bacteriochlorophyll a in carotenoidless LH1 and LH2 antenna complexes from Rhodobacter sphaeroides. Biochemistry 37, 1746917486.[CrossRef][Medline]
Morgan, P. E., Dean, R. T. & Davies, M. J. (2004). Protective mechanisms against peptide and protein peroxides generated by singlet oxygen. Free Rad Biol Med 36, 484496.[CrossRef][Medline]
O'Gara, J. P. & Kaplan, S. (1997). Evidence for the role of redox carriers in photosynthesis gene expression and carotenoid biosynthesis in Rhodobacter sphaeroides 2.4.1. J Bacteriol 179, 19511961.
op-den-Camp, R. G., Przybyla, D., Ochsenbein, C. & 9 other authors (2003). Rapid induction of distinct stress responses after the release of singlet oxygen in Arabidopsis. Plant Cell 15, 23202332.
Ouchane, S., Picaud, M., Vernotte, C. & Astier, C. (1997). Photooxidative stress stimulates illegitimate recombination and mutability in carotenoid-less mutants of Rubrivivax gelatinosus. EMBO J 16, 47774787.
Pappas, C. T., Sram, J., Moskvin, O. V. & 7 other authors (2004). Construction and validation of the Rhodobacter sphaeroides 2.4.1 DNA microarray: transcriptome flexibility at diverse growth modes. J Bacteriol 186, 47484758.
Paul, A., Hackbarth, S., Vogt, R. D., Roder, B., Burnison, B. K. & Steinberg, C. E. (2004). Photogeneration of singlet oxygen by humic substances: comparison of humic substances of aquatic and terrestrial origin. Photochem Photobiol Sci 3, 273280.[CrossRef][Medline]
Permentier, H. P., Neerken, S., Overmann, J. & Amesz, J. (2001). A bacteriochlorophyll a antenna complex from purple bacteria absorbing at 963 nm. Biochemistry 40, 55735578.[CrossRef][Medline]
Pfaffl, M. W. (2001). A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29, e45.
Schroeder, W. A. & Johnson, E. A. (1995). Singlet oxygen and peroxyl radicals regulate carotenoid biosynthesis in Pfaffia rhodozyma. J Biol Chem 270, 1837418379.
Sies, H. & Menck, C. F. (1992). Singlet oxygen induced DNA damage. Mutat Res 275, 367375.[CrossRef][Medline]
Takaichi, S. (1999). Carotenoids and carotenogenesis in anoxygenic photosynthetic bacteria. In The Photochemistry of Carotenoids, pp. 3969. Edited by H. A. Frank, A. Young, G. Britton & R. J. Cogdell. Dordrecht: Kluwer.
Tandori, J., Hideg, E., Nagy, L., Maroti, P. & Vass, I. (2001). Photoinhibition of carotenoidless reaction centers from Rhodobacter sphaeroides by visible light. Effects on protein structure and electron transport. Photosynth Res 70, 175184.[CrossRef]
Van Liere, E. & Walsby, A. (1982). Interaction of cyanobacteria with light. In The Biology of Cyanobacteria, pp. 945. Edited by N. G. Carr & B. A. Whitton. Oxford: Blackwell.
van Niel, C. B. (1941). The culture, general physiology, morphology, and classification of the non-sulfur purple and brown bacteria. Bacteriol Rev 8, 1118.
Wagner, D., Przybyla, D., op-den-Camp, R. G. & 8 other authors (2004). The genetic basis of singlet oxygen-induced stress responses of Arabidopsis thaliana. Science 306, 11831185.
Yeliseev, A. A. & Kaplan, S. (1997). Anaerobic carotenoid biosynthesis in Rhodobacter sphaeroides 2.4.1: H2O is a source of oxygen for the 1-methoxy group of spheroidene but not for the 2-oxo group of spheroidenone. FEBS Lett 403, 1014.[CrossRef][Medline]
Zeilstra-Ryalls, J. H. & Kaplan, S. (2004). Oxygen intervention in the regulation of gene expression: the photosynthetic bacterial paradigm. Cell Mol Life Sci 61, 417436.[CrossRef][Medline]
Zeller, T. & Klug, G. (2004). Detoxification of hydrogen peroxide and expression of catalase genes in Rhodobacter. Microbiology 150, 34513462.[CrossRef][Medline]
Züllig, H. (1985). Pigmente phototropher bakterien in seesedimenten und ihre bedeutung für die seenforschung. Schweiz Ztg Hydrol 47, 87126.
Zurdo, J., Fernandez-Cabrera, C. & Ramirez, J. M. (1993). A structural role of the carotenoid in the light-harvesting II protein of Rhodobacter capsulatus. Biochem J 290, 531537.[Medline]
Received 25 November 2004;
revised 1 February 2005;
accepted 23 February 2005.
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