Present addresses: Department of Microbiology and Immunology, University of British Columbia, 300-6174 University Blvd., Vancouver, BC, Canada V6T 1Z3. ‡Institut für Molekularbiologie und Tumorforschung, Philipps-Universität Marburg, Emil-Mannkopff-Str. 2, D-35037 Marburg, Germany.
Light-dependent regulation of photosynthesis genes in Rhodobacter sphaeroides 2.4.1 is coordinately controlled by photosynthetic electron transport via the PrrBA two-component system and the photoreceptor AppA
Article first published online: 29 SEP 2005
Volume 58, Issue 3, pages 903–914, November 2005
How to Cite
Happ, H. N., Braatsch, S., Broschek, V., Osterloh, L. and Klug, G. (2005), Light-dependent regulation of photosynthesis genes in Rhodobacter sphaeroides 2.4.1 is coordinately controlled by photosynthetic electron transport via the PrrBA two-component system and the photoreceptor AppA. Molecular Microbiology, 58: 903–914. doi: 10.1111/j.1365-2958.2005.04882.x
- Issue published online: 29 SEP 2005
- Article first published online: 29 SEP 2005
- Accepted 23 August, 2005.
Formation of the photosynthetic apparatus in Rhodobacter is regulated by oxygen tension and light intensity. Here we show that in anaerobically grown Rhodobacter cells a light-dependent increase in expression of the puc and puf operons encoding structural proteins of the photosynthetic complexes requires an active photosynthetic electron transport. The redox-sensitive CrtJ/PpsR repressor of photosynthesis genes, which was suggested to mediate electron transport-dependent signals, is not involved in this light-dependent signal chain. Our data reveal that the signal initiated in the photosynthetic reaction centre is transmitted via components of the electron transport chain and the PrrB/PrrA two-component system in Rhodobacter sphaeroides. Under blue light illumination in the absence of oxygen this signal leads to activation of photosynthesis genes and interferes with a blue-light repression mediated by the AppA photoreceptor and the PpsR transcriptional repressor in R. sphaeroides. Thus, light either sensed by a photoreceptor or initiating photosynthetic electron transport has opposite effects on the transcription of photosynthesis genes. Both signalling pathways involve redox-dependent steps that finally determine the effect of light on gene expression.
Responses of bacteria to light have been known for many years and it was widely anticipated that bacteria react to photosynthetic electron flow (Clayton, 1963; Weaver, 1971; Grishanin et al., 1997; Romagnoli and Armitage, 1999; El Bissati and Kirilovsky, 2001) or to reactive oxygen species (Yang et al., 1995; 1996; Browning et al., 2003). However, in recent years it was demonstrated that bacteria contain different types of photoreceptors that were previously believed to occur exclusively in plants [reviewed in the study by Braatsch and Klug (2004)] or represent new types of photoreceptors (Braatsch et al., 2002; Gomelsky and Klug, 2002; Masuda and Bauer, 2002).
Purple bacteria of the genus Rhodobacter are extremely metabolically versatile and contain branched electron transfer pathways (Fig. 1). Rhodobacter performs respiration by using oxygen or alternative electron acceptors like dimethyl sulphoxide (DMSO). Under anaerobic conditions in the light Rhodobacter cells gain ATP by anoxygenic photophosphorylation. The photosynthetic cyclic electron transport shares components with the respiratoric electron transport chain (ETC) (Fig. 1). Electron transfer between the two transmembrane complexes reaction centre and cyt bc1 is mediated by the mobile electron carrier ubiquinone, located in the hydrophobic domain of the cytoplasmic membrane, and by (a) c-type cytochrome(s) [reviewed in the study by McEwan (1994)]. This cyclic electron transport is linked to the generation of a proton motive force.
Oxygen and light are critical environmental signals that regulate the formation of the photosynthetic apparatus in Rhodobacter. Under high oxygen tension, production of the photosynthetic complexes is completely shut off in Rhodobacter sphaeroides, whereas it persists at low levels in Rhodobacter capsulatus. A drop in oxygen tension significantly increases formation of the photosynthetic complexes in both species even in the absence of light [reviewed in the study by Gregor and Klug (1999)]. High light intensity causes a decrease in the amount of photosynthetic complexes in phototrophically grown Rhodobacter cultures (Drews, 1986; Pemberton et al., 1998).
Most of the genes required for the synthesis of the photosynthetic apparatus are clustered in a region of the Rhodobacter chromosome known as the photosynthesis (PS) gene cluster (Zsebo and Hearst, 1984; Choudhary and Kaplan, 2000). These genes include bch and crt operons (encoding enzymes involved in bacteriochlorophyll and carotenoid biosynthesis respectively), the puf operon and puhA gene [encoding the structural and assembly proteins of the light harvesting complex I (LHI) and reaction centre (RC)]. Unlinked to the PS gene cluster is the puc operon, encoding the structural and assembly proteins of the LHII complex.
Central roles in the oxygen-dependent gene regulation in Rhodobacter could be assigned to three systems, namely FnrL, Prr/Reg and AppA-PpsR/CrtJ (Gregor and Klug, 2002; Zeilstra-Ryalls and Kaplan, 2004). The FnrL regulator protein of Rhodobacter is supposed to directly sense molecular oxygen via an oxygen-labile 4Fe-4S cluster as reported for its Escherichia coli FNR homologue (Khoroshilova et al., 1997). The RegB/RegA and PrrB/PrrA two-component system were originally described to control the oxygen-dependent expression of PS genes in R. capsulatus (Sganga and Bauer, 1992; Mosley et al., 1994) and in R. sphaeroides respectively (Phillips-Jones and Hunter, 1994; Eraso and Kaplan, 1994; 1995). It is now well established that the RegB/RegA two-component system regulates a variety of energy-generating and energy-utilizing processes in Rhodobacter[for a review see the study by Elsen et al. (2004) and references therein]. The Prr/Reg two-component system consists of the membrane-associated PrrB/RegB histidine kinase and its cognate PrrA/RegA response regulator (Fig. 1). At low oxygen tension the sensor kinase undergoes autophosporylation and transfers the phospho group to the corresponding response regulator (Inoue et al., 1995; Oh and Kaplan, 2000). The phosphorylated DNA binding protein PrrA/RegA activates the transcription of several PS genes including those of the puf and puc operons (Eraso and Kaplan, 1996; Du et al., 1998; Bowman et al., 1999). Under aerobic growth reductant flow through the cbb3 oxidase enhances the phosphatase activity of PrrB relative to its kinase activity thus preventing activation of PS genes by phosphorylated PrrA (Oh and Kaplan, 2001). A direct interaction between cbb3 and PrrB was observed in vitro (Oh et al., 2004). By comparing the genome wide expression of a cbb3 mutant with that of the parental wild-type strain it was revealed recently that the cbb3 oxidase possesses additional regulatory activities under anaerobic conditions (Kaplan et al., 2005). It was suggested that under anaerobic conditions electrons flow through cbb3 to the 2-oxo donor for the conversion of spheroidene to spheroidenone and thereby affect the PrrB phosphatase activity.
Redox regulation occurs not only by activation of PS genes at low oxygen but also by gene repression at high oxygen tension. The PpsR protein of R. sphaeroides and its counterpart in R. capsulatus, CrtJ (53% identity), repress PS genes at high oxygen tension in a redox-dependent manner. Known targets of aerobic repression by PpsR/CrtJ are the bch, crt and puc genes, whose promoters reveal a consensus sequence of TGT-N12-ACA (Elsen et al., 1998; Choudhary and Kaplan, 2000). The CrtJ protein also affects oxygen-dependent puf expression (Abada et al., 2002), although binding sites for CrtJ in the puf promoter region have not been defined. Recent transcriptome studies revealed that all PS genes, whether they do or do not contain PpsR-binding sites, were regulated directly or indirectly via the AppA-PpsR pathway in R. sphaeroides (Moskvin et al., 2005).
PpsR/CrtJ binding to the puc promoter target sequence was shown to be stimulated directly by oxygen, which causes the formation of an intramolecular disulphide bond in PpsR/CrtJ (Masuda and Bauer, 2002; Masuda et al., 2002). The R. sphaeroides flavoprotein AppA is known to function as PpsR antagonist (Gomelsky and Kaplan, 1997), participating in the oxygen-dependent redox-control of PS gene expression by reducing the disulphide bond in PpsR (Masuda and Bauer, 2002). Oh and Kaplan (2001) proposed that AppA monitors the redox state of the quinone pool and thereby modulates PpsR repressor activity in response to changes in oxygen tension and light intensity. No AppA homologue exists in R. capsulatus.
In R. sphaeroides a blue light-dependent repression of PS genes including the puf and puc operons has been demonstrated at an intermediate oxygen concentration (104 ± 24 µM dissolved oxygen) (Shimada et al., 1992; Braatsch et al., 2002; 2004), and could be attributed to the interplay of the AppA and PpsR regulatory proteins (Masuda and Bauer, 2002; Braatsch et al., 2002; 2004; Fig. 1). No blue light-dependent puf and puc repression was observed at low (≤ 3 µM) oxygen tension (Braatsch et al., 2002). It is conceivable that this response represents an adaptation process, which prepares cells for photooxidative stress. FAD, non-covalently attached to the photoactive BLUF domain (Gomelsky and Klug, 2002) of AppA, was found to be essential for the blue light-dependent puc and puf repression. While the BLUF domain is required for transmission of the light signal, the C-terminal part of AppA is sufficient for normal redox regulation (Gomelsky and Kaplan, 1998; Braatsch et al., 2002; Han et al., 2004).
Our previous work (Braatsch et al., 2002) has shown that blue light does not repress PS genes at low oxygen tension and even leads to increased puf and puc expression under anaerobic growth conditions in R. sphaeroides. Evidence was provided that this gene activation in R. sphaeroides is not mediated by the FAD chromophore of AppA (Braatsch et al., 2002). By using mutant strains and different light qualities we show here that an increase in puf and puc expression caused by a dark-to-light transition of anaerobic grown R. capsulatus and R. sphaeroides cells is not restricted to blue light and requires an active photosynthetic electron transport. Our results lead to a model for light-dependent PS gene regulation that includes different signal chains.
Blue light affects puf and puc mRNA levels and the amount of photosynthetic complexes in Rhodobacter under anaerobic growth conditions
Rhodobacter sphaeroides shows a blue light-dependent repression of the puf and puc genes during semi-aerobic growth (Shimada et al., 1992), which is mediated by the AppA protein that functions as photoreceptor (Braatsch et al., 2002). An induction of puf and puc expression by blue light in the absence of oxygen was independent of AppA (Braatsch et al., 2002). R. capsulatus that does not harbour an AppA homologue does not show an effect of blue light on puf and puc expression during semi-aerobic growth (Braatsch et al., 2002). In order to test whether blue light affects puf and puc expression under anaerobic growth condition in R. capsulatus, cultures were grown in the dark by providing 60 mM DMSO as terminal electron acceptor for anaerobic respiration. Doubling times determined under these conditions in the dark (13–15 h) were much higher than during aerobic growth (2.7 h) or photosynthetic growth in white light (2.4 h). When these cultures were illuminated by blue light, doubling times decreased to 11–12 h indicating that blue light can support growth by initiating low rates of photosynthetic electron transport. As shown in Fig. 2, blue light (λmax 400 nm; band width 80 nm) passing through the BG12/KG1 filters can be absorbed by an bacteriochlorophyll absorption maximum at 375 nm (Soret band). We quantified puf, puc and rRNA levels in R. capsulatus wild-type cultures grown in the dark or illuminated with blue light by Northern blot analysis. The 23S rRNA (14S rRNA after in vivo processing; Kordes et al., 1994) was used as a quantitative control of total RNA. The signal intensities for rRNA were within 30% independently of light irradiation during the time-course of the experiment. Normalized puf and puc mRNA levels in R. capsulatus wild type increased by a factor of 1.8–2.3, 240 min after the onset of illumination, while there was no significant change in the dark controls (Fig. 3B). Although R. sphaeroides and R. capsulatus behave differently when illuminated by blue light at intermediate oxygen levels (Braatsch et al., 2002), they show similar increased puf and puc expression by blue light under anaerobic conditions (Figs. 3A and B and 4). This suggests that homologous proteins, which are present in both species, are involved in transmission of the blue light signal under anaerobic conditions.
In order to see whether the moderate increase of puf and puc expression observed for both Rhodobacter strains after illumination indeed causes a phenotypic effect, we performed spectral analysis on cell-free extracts of anaerobic R. sphaeroides cultures. As shown in Fig. 5 blue light illumination for 24 h resulted in significant higher amounts of photosynthetic complexes in the absence of oxygen compared with dark grown cultures. Calculation of the LHI and LHII specific peak areas (peaks at 800 and 855 nm) revealed 29.8 ± 4.5% higher amounts of photosynthetic complexes in the light.
Blue light-dependent increase of puf and puc expression in anaerobic Rhodobacter cultures is a consequence of photosynthetic electron flow
As mentioned above, blue light illumination increased the growth rates of R. capsulatus and R. sphaeroides cultures grown anaerobically in the presence of DMSO. To test whether altered puf and puc expression upon blue light illumination was attributed to this photosynthetic electron flow, we studied the blue light response of Rhodobacter strains with deletions in the puhA gene that encodes a non-pigment binding protein of the reaction centre. The R. capsulatus strain DW5 harbours a non-polar translational in frame puhA deletion (Table 1). The R. sphaeroides strain PUHA1 (Table 1) has part of the puhA gene deleted and replaced by a resistance cartridge. Both strains are unable to form RC or LHI complexes and as a consequence fail to initiate photosynthetic electron flow. However, absorbency scans of cell-free extracts from anaerobically dark grown cultures of DW5 and parental strain SB1003 show that the total amounts of pigments in both strains are similar (Fig. 2). As shown in Fig. 3 blue light illumination has no significant effects on puf and puc expression in strains DW5 and PUHA1, implying a regulatory coupling between photosynthetic electron flow and the transcription of PS genes. The doubling time of PS-deficient mutants DW5 and PUHA1 did not change because of blue light irradiation.
|R. s. 2.4.1.||Wild-type||van Neil (1944)|
|R. s. PUHA1||ΔpuhA::Kmr cassette||Sockett et al. (1989)|
|R. s. PPS1||PpsR::Kmr cassette||Gomelsky and Kaplan (1997)|
|R. s. CBB3Δ||Deletion in ccoNOQP||Oh and Kaplan (2002)|
|R. s. Gadc2||crt Δ(cycA::Sp) Cyt c2–||Caffrey et al. (1992)|
|R. s. Gadcy||crt Δ(cycY::Km) Cyt cy–||Jenney and Daldal (1993)|
|R. s. BC17||crt Δ(petABC(fbcFBC):: Km) Cyt bc1–||Yun et al. (1990)|
|R. s. PRRB1||prrBΔNruI-RsrII::Ω Smr Spr||Eraso and Kaplan (1995)|
|R. c. SB1003||Wild-type||Yen and Marrs (1976)|
|R. c. DB469||crtJ::Kmr cassette||Bollivar et al. (1994)|
|R. c. DW5||ΔpuhA||Wong et al. (1996)|
|R. c. ΔRC6(pTX35)||Δpuf operon::Km; puf operon on plasmid||Klug and Cohen (1988)|
|R. c. ΔRC6(pTXΔRB6)||Δpuf operon::Km; puf operon on plasmid Δpuf LM||Klug and Cohen (1988)|
|R. c. MS01||regA:: Kmr cassette||Sganga and Bauer (1992)|
|R. c. CSM01||regB:: Kmr cassette||Mosley et al. (1994)|
In addition, the effect of blue light on puf and puc expression under anaerobic conditions was quantified in the R. capsulatus strains ΔRC6(pTX35) and ΔRC6(pTXΔRB6) (Klug and Cohen, 1988). Strain ΔRC6 has a large part of the puf operon deleted and is complemented to wild-type phenotype by the low copy number plasmid pTX35, which comprises the complete puf operon. Plasmid pTXΔRB6 contains the complete pufQBA and pufX genes, but lacks the complete pufLM genes that encode the pigment binding proteins of the reaction centre. As the other Rhodobacter strains lacking a functional reaction centre strain ΔRC6(pTXΔRB6) did not show increased puf and puc expression after illumination. Strain ΔRC6(pTX35) showed significant induction of puf and puc mRNA levels by blue light (Fig. 3B), although no significant differences in growth and content of photosynthetic complexes between strains ΔRC6(pTXΔRB6) and ΔRC6(pTX35) were observed during anaerobic growth in blue light (data not shown).
If the blue light-dependent induction of puf and puc genes in Rhodobacter is not mediated by a specific light receptor but rather by photosynthetic electron flow, other light qualities that are absorbed by photopigments (Crt, Bchl) should have a similar effect on gene expression, while light qualitities not absorbed by the photosynthetic apparatus should not affect puf and puc expression. To further confirm our conclusion on the involvement of photosynthetic electron transport, we monitored puf and puc expression in anaerobically grown cultures of R. capsulatus or R. sphaeroides after far red (λmax 800 nm) or orange (λmax 630 nm) light illumination. The far red light but not the orange light can be efficiently absorbed by the photosynthetic apparatus of Rhodobacter (Fig. 2). In agreement with our hypothesis, both Rhodobacter wild-type strains responded to far red light illumination by a significant increase of puf and puc expression, while the reaction centre mutant DW5 did not (Figs 3 and 4). puf and puc expression of both wild-type strains did not change significantly in response to orange light (Figs 3 and 4).
The CrtJ/PpsR repressor protein is not involved in blue light-dependent puf and puc expression
Oh and Kaplan (2000; 2001) suggested that the redox status of the ubiquinone pool is sensed by the AppA protein, which acts as an anti-repressor of the PpsR protein. PpsR (CrtJ in R. capsulatus) is a soluble protein that exhibits a helix–turn–helix DNA-binding domain (Penfold and Pemberton, 1991), two critical cysteine residues (Masuda and Bauer, 2002; Masuda et al., 2002) and two Per-Arnt-Sim (PAS) domains (Penfold and Pemberton, 1991; 1994; Gomelsky and Kaplan, 1995).
As photosynthetic electron flow affects the redox state of ubiquinone, the suggested pathway would also lead to light-dependent gene expression. Blue light-dependent repression of the puf and puc genes under semi-aerobic conditions is indeed mediated by the PpsR and AppA proteins in R. sphaeroides 2.4.1, and the BLUF domain of AppA was shown to function as blue light photoreceptor (Braatsch et al., 2002; Masuda and Bauer, 2002). R. capsulatus SB1003 does not show this light response and does not harbour an AppA homologue. Based on previously published data (Braatsch et al., 2002) we can exclude a role of the AppA BLUF domain in blue light-dependent increase of puf and puc expression under anaerobic conditions in R. sphaeroides. It is however, conceivable that the PpsR homologue CrtJ is involved in the blue light response of R. capsulatus under anaerobic conditions and that PpsR can influence puf and puc expression independently of AppA. To test this hypothesis, we studied the blue light response in strains R. capsulatus DB496 and R. sphaeroides PPS1, which both have inactivated the crtJ/ppsR gene by partial deletion and insertion of a kanamycin cartridge (Table 1). Northern blot analysis demonstrated that puf and puc mRNA levels in anaerobically grown R. capsulatus DB469 or R. sphaeroides PPS1 are still induced by blue light (Fig. 3). We conclude that the CrtJ/PpsR protein is not required for this blue light response of Rhodobacter.
The anaerobic light signal is transmitted via components of the ETC
Interaction between photosynthetic and respiratory ETCs in Rhodobacter has been shown for many years. PS inhibits all the other bioenergetic processes. The light-induced proton motive force on the proton translocating complexes and changes in the redox state of electron transfer components common to the different ETCs have been proposed to explain the inhibition of respiration by light (Verméglio, 1995 and references therein).
During aerobic respiration of Rhodobacter ubiquinol oxidase and cytochrome cbb3 oxidase (Fig. 1) catalyse final electron transfer to oxygen (Baccarini Melandri et al., 1973; Marrs and Gest, 1973; La Monica and Marrs, 1976; Zannoni et al., 1976; Oh and Kaplan, 2002). It was suggested that electron flow through the cbb3 complex of R. sphaeroides can also take place anaerobically, resulting in the conversion of the carotenoid spheroidene to spheroidenone (Oh and Kaplan, 1999; Kaplan et al., 2005). In contrast to R. capsulatus, R. sphaeroides contains an additional cytochrome c oxidase of the aa3-type, which is the major cytochrome c oxidase under highly aerobic conditions (Oh and Kaplan, 1999). The aa3 oxidase is not expressed under anaerobic conditions (Kaplan et al., 2005) and therefore omitted from Fig. 1. To test whether the cbb3 oxidase is part of the light signal chain under anaerobic conditions we tested the response of puf and puc expression to blue light in an anaerobic culture of the cbb3 mutant strain R. sphaeroides CBB3Δ (Table 1). As shown in Figs 3 and 4puf and puc expression did not increase after blue light illumination supporting the role of cbb3 in transmission of the light signal.
We also tested the involvement of other components of the ETC in transmission of the light signal. Mutants of R. sphaeroides lacking the cyt bc1 complex, the membrane-anchored cyt cy or the mobile cyt c2 failed to increase puf and puc expression after illumination of anaerobic cultures to similar amounts as the wild type. These data indicate that the redox state of the ETC is involved in light-dependent signal transmission in the absence of oxygen.
The AppA/PpsR and the PrrB/PrrA signal chains co-ordinately regulate light-dependent gene expression in R. sphaeroides
In order to prove the transmission of the anaerobic light signal in Rhodobacter from the reaction centre via the ETC to PrrB/PrrA, RegB/RegA we also tested the response of blue light in R. capsulatus strains MS01 (lacks RegA), CSM01 (lacks RegB) and R. sphaeroides strain PRRB1 that lacks PrrB. Northern blot analysis revealed that puf and puc expression in the R. capsulatus mutants does no longer respond to blue light (Fig. 3). Our data demonstrate that the RegB/RegA system is not only involved in the transmission of oxygen-dependent redox signals but also in the transmission of light-dependent signals that are mediated by the ETC.
Similar to the Reg mutants of R. capsulatus blue light did not lead to increased expression of the puf and puc operons in R. sphaeroides strain PRRB1. In contrast to the R. capsulatus mutant we observed, however, a blue light-dependent decrease of puf and puc mRNA levels (Figs 3 and 4). puf expression was inhibited by 59%, puc expression by 68%, which is comparable to the levels of blue light inhibition we observed in semi-aerobically growing wild-type cultures of R. sphaeroides (Braatsch et al., 2002). These data reveal that both, the PrrB/PrrA and the AppA/PpsR signal chain, are involved in blue light regulation of PS genes under anaerobic conditions and lead to a model that is discussed below.
Although a regulating effect of light on the formation of photosynthetic complexes in Rhodobacter has been known for many years (Klug et al., 1985; 1991; Drews, 1986; Shimada et al., 1992) the factors involved in light sensing and the transmission of light-dependent signals were unknown. Our previous work demonstrated that two light-dependent signal chains affect the expression of PS genes in R. sphaeroides (Braatsch et al., 2002): blue light-dependent repression of PS genes under intermediate oxygen, which is mediated by the AppA/PpsR system and blue light-dependent induction of puf and puc expression in the absence of oxygen, which is independent of AppA (Braatsch et al., 2002). This blue light repression is not found in R. capsulatus, which lacks the AppA protein (Braatsch et al., 2002). Here we show that R. capsulatus, like R. sphaeroides responds to blue light under anaerobic conditions by increasing puf and puc mRNA levels, suggesting that similar light-dependent signal chains exist in the two species.
Cohen-Bazire et al. (1956) first proposed that the redox state of the ETC might ultimately control the levels of spectral complexes in the facultatively phototrophic purple bacterium R. sphaeroides in response to changes in oxygen tension and light intensity. Our data reveal that puf and puc genes of mutants unable to initiate photosynthetic electron transport fail to respond to blue light under anaerobic conditions and that only light qualities absorbed by the photosynthetic apparatus cause elevated puf and puc expression in wild-type strains. These findings are in agreement with the view that photosynthetic electron transport is required for light-induced expression of PS genes in Rhodobacter.
Oh and Kaplan (2000; 2001) presented a general model describing the interrelationship between the regulation of the PS genes and the activity of the ETC in R. sphaeroides. According to this model the redox status of the ubiquinone pool is sensed by the AppA protein, which acts as an anti-repressor of the PpsR protein. PpsR and its homologue CrtJ bind to the puc promoter region and repress transcription at high oxygen tension (Ponnampalam and Bauer, 1997; Gomelsky and Kaplan, 1998; Masuda and Bauer, 2002). Recent in vitro studies demonstrated that direct interaction with oxygen promotes the formation of an intramolecular disulphide bond in PpsR/CrtJ that stimulates DNA binding activity (Ponnampalam and Bauer, 1997; Masuda and Bauer, 2002; Masuda et al., 2002). Cho et al. (2004) reported that the binding activity of PpsR to target promoters was maintained even under anaerobic conditions. Apparently, not all of the repressor activity of PpsR/CrtJ disappeared under anaerobic conditions in vivo. Therefore, we had to consider that a blue light-dependent change in the redox state of an electron transport component affects the DNA-binding affinity of CrtJ/PpsR in anaerobically dark grown cells. This hypothesis was, however, falsified by our data: as well the R. sphaeroides ppsR mutant PPS1 as the R. capsulatus crtJ mutant DB469 show normal increase of puf and puc mRNA levels when anaerobic cultures are illuminated by blue light. Our findings also argue against signal transmission from the ubiquinone pool to AppA as postulated by Oh and Kaplan (2001) or to PpsR/CtrJ.
Signal sensing by the sensor kinase PrrB of R. sphaeroides (Eraso and Kaplan, 1995) and its counterpart RegB (58% identity) of R. capsulatus (Sganga and Bauer, 1992; Mosley et al., 1994) may occur by two mechanisms. In R. sphaeroides the cbb3 oxidase was suggested to generate an inhibitory signal that shifts the relative equilibrium of PrrB from the kinase mode to the phosphatase mode. The strength of the inhibitory signal is supposed to be proportionally related to the extent of electron flow through the cbb3 oxidase under aerobic conditions (Oh and Kaplan, 2000) and also under anaerobic conditions (Kaplan et al., 2005). Exposure of RegB to oxidizing conditions results in the formation of an intermolecular disulphide bond, converting the kinase from an active dimer into an inactive tetrameric state (Swem et al., 2003). Our results are in agreement with a signal chain that links photosynthetic electron flow to the ETC and the PrrB/PrrA (RegB/RegA) system as proposed by Oh and Kaplan and as outlined in Fig. 1. As well Rhodobacter mutants lacking components of the ETC as mutants in the RegB/RegA, PrrB/PrrA system are unable to transmit the signal generated by photosynthetic electron transport to puf and puc expression. The lack of an inhibitory signal from cbb3 on the PrrB/RegB kinase activity would result in stronger induction of the puf and puc genes even in the dark. In agreement with this assumption we found an increased basal puf (1.7-fold) and puc (2.5-fold) mRNA level in the dark grown R. sphaeroides cbb3 mutant compared with the isogenic wild-type strain (data not shown, average from two independent experiments). In accordance with our model cyt bc1 and cyt c2 mutants failed to show blue light-dependent puf and puc expression, because of the lack of electron transfer to cbb3 or lack of cyclic photosynthetic electron transport respectively. Similarly, the cyt cy mutant did not show significant light-dependent puf and puc gene expression. Increased amounts of photosynthetic complexes and increased puf and puc levels in cy mutants under aerobic conditions have been reported previously (Daldal et al., 2001; Oh and Kaplan, 2002). This suggests that the presence of c2 does not fully compensate the lack of cy and that the lack of cy influences the rate of electron flow through cbb3. It is currently unknown, which proportion of electrons flows to cbb3 via cyt c2 or cyt cy under anaerobic conditions. The mutations of components of the ETC will also influence the redox state of the ubiquinone pool, which was suggested to constitute the signal for regulatory proteins (Oh and Kaplan, 2002). Based on our data we cannot exclude the possibility of signal transfer directly from the ubiquinone pool. However, our data proof that such light-dependent signal is not transmitted via AppA or Ppsr/CrtJ but by the PrrB/PrrA, RegB/RegA two-component system.
To our surprise the PpsR mutant of R. sphaeroides showed similar blue light-dependent repression of the puf and puc operons under anaerobic conditions as the wild-type strain under intermediate oxygen concentration. We have reported previously that blue light has no repressing effect on PS genes in R. sphaeroides when oxygen tension is low (≤ 3 µM). We concluded that AppA is in a certain redox state under low oxygen tension that prevents PpsR from binding to its DNA targets, independently of light (Braatsch et al., 2002). The repressing effect of blue light under anaerobic condition in the PrrB mutant reveals that the AppA/PpsR system responds to blue light even under anaerobic conditions. Even in the absence of oxygen AppA can release the repressing effect of PpsR at least partially when it senses blue light. This is in agreement with a model presented by Masuda and Bauer (2002) based on in vitro studies. The repressing effect under anaerobic conditions cannot be observed in a wild-type strain, as moderate activation of gene expression by PrrA counteracts the repression by PpsR. This is in line with our observation that a decrease in oxygen tension under continuous blue light illumination leads to increasing puc and puf mRNA levels (not shown). A connection between the PrrB/PrrA system and the AppA/PpsR-dependent regulation of PS gene expression was observed recently (Moskvin et al., 2005). The phototrophic incompetence of the prrA mutant was attributed to the inability to counteract PpsR, as a prrA/ppsR double mutant does grow phototrophically under anaerobic conditions. As the puf promoter contains no PpsR binding sites, an indirect effect of PpsR was suggested, i.e. PpsR-dependent expression of a regulatory factor(s) that controls puf expression (Moskvin et al., 2005). To our knowledge no experimental data are available for the exact PrrA binding sequence within the puc and puf promoter regions of R. sphaeroides. However, binding of the PrrA homologue RegA to the puc promoter of R. capsulatus is centred at around 60 bp upstream of the transcription initiation site as located by DNase I footprint protection analysis (Du et al., 1998; Kirndörfer et al., 1998). This interferes with the DNA-sequence known to be protected from DNase I digestion by CrtJ (Elsen et al., 1998) and covers partly the CrtJ recognition site (TGTN12TGT) spanning the −35 promoter region (Elsen et al., 1998). In vitro experiments revealed a competition of CrtJ and RegA for DNA binding at the puc promoter (Bowman et al., 1999). It will be interesting to evaluate the interdependence of PrrA and PpsR DNA-binding characteristics to the puc promoter, i.e. co-ordinated binding or competition and displacement under different redox or light conditions in vivo.
In summary, the following model for regulation of PS genes under anaerobic conditions emerged. When Rhodobacter is growing anaerobically with DMSO as terminal electron acceptor PS genes are transcribed because of activation by PrrB/RegB, but not at maximal level. As some electrons flow through cbb3 even under anaerobic conditions a small inhibitory signal of the PrrB kinase activity is generated. Light that is absorbed by the photosynthetic apparatus initiates photosynthetic electron flow that reduces the inhibitory signal of cbb3 cytochrome oxidase (Oh and Kaplan, 2000) on PrrB (RegB) and consequently transcription of PS genes is enhanced. In R. sphaeroides this activation dominates the blue light repression mediated by AppA/PpsR. As a consequence of this regulatory network the oxygen tension decides whether PS genes are activated or repressed by light and allows Rhodobacter to adequately respond to environmental changes.
Bacterial strains and growth conditions
Table 1 summarizes the characteristics of the bacterial strains used in this study. Rhodobacter strains were grown chemoheterotrophically at 32°C on a malate minimal salt medium (Drews, 1983). Growth under semi-aerobic conditions was performed by incubating 40 ml of culture in 50 ml flasks under gentle agitation in the dark. For photoheterotrophic growth, cell cultures were illuminated with a bank of 35 W fluorescent lamps. For anaerobic growth, DMSO (final concentration 60 mM) was added to the medium as terminal electron acceptor and cells were grown in the dark in screw-cap flasks filled to the top. When required, antibiotics were added to the growth medium at the following concentrations: kanamycin, streptomycin 25 µg ml−1; spectinomycin 10 µg ml−1.
Light shift experiments
Light was obtained from a lamp of a slide projector (250 W, 24 V; General Electric) and filtered through a combination of heat-absorbing filter KG1 and filters BG12, SFK15 and RG780 (Schott) respectively. The resulting light had maxima at 400, 630 and 800 nm with a half-band width ≤ 80 nm. Photon flux density of each light quality measured at culture level was approximately 27 µmol of photons m−2 s−1 (Pyranometer Sensor, Li-200SA). For light shift experiments, cultures grown in the dark to an optical density (OD) at 660 nm of 0.1–0.2 were transferred into the light at time zero. Flasks containing cultures grown in the dark were wrapped in aluminium foil.
Gene expression analysis
Expression of puf, puc and rRNA genes was monitored by RNA gel-blot analysis as described in the study by Braatsch et al. (2002).
Absorbance spectroscopy was performed on a spectrophotometer (Lambda 12, Perkin Elmer). Rhodobacter cell extracts were obtained by sonication of cultures and removing cell debris by centrifugation. Spectral analyses were performed on crude cell-free lysates. Samples contained 400–600 µg of protein per millilitre as determined according to the Bradford method (Bradford, 1976).
We thank J. T. Beatty, F. Daldal, M. Gomelsky and S. Kaplan for providing Rhodobacter strains, P. Galland for sharing his equipment and S. Kaplan for valuable discussions. This work was supported by Deutsche Forschungsgemeinschaft DFG Kl563/15-2/15-3 and Fonds der Chemischen Industrie.
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