Department of Bacteriology, University of Wisconsin Madison, 420 Henry Mall, Madison, WI 53706, USA
Correspondence
Timothy J. Donohue
tdonohue{at}bact.wisc.edu
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ABSTRACT |
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INTRODUCTION |
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We are studying transcriptional regulatory circuits that control respiratory pathways in the facultative phototroph Rhodobacter sphaeroides. This -proteobacterium has a choice of anaerobic energetic lifestyles: it can grow via photosynthesis if light is present or it can use DMSO as an alternative electron acceptor in the absence or presence of light. The decision of which anaerobic energy production strategy to use adds complexity to the regulation of the metabolic lifestyle of this bacterium. In fact, there appears to be a hierarchy of regulation since the addition of alternative electron acceptors to photosynthetic cultures of R. sphaeroides decreases the expression of genes encoding the photosynthetic apparatus (Karls et al., 1999
; Oh & Kaplan, 1999
). This work was aimed at providing a molecular explanation for how the alternative electron acceptor, DMSO, decreases expression of these photosynthesis genes under anaerobic conditions.
To analyse how DMSO decreases photosynthesis gene expression, we used the P2 promoter for the cytochrome c2 gene, cycA, because its activity was previously found to be significantly lower under these conditions (Karls et al., 1999). Expression of cycA P2 has been shown to be directly activated by the response regulator PrrA, as are other genes encoding other components of the R. sphaeroides photosynthetic apparatus (Eraso & Kaplan, 1994
; Pemberton et al., 1998
). The mechanism by which cycA P2 expression is decreased by DMSO under photosynthetic conditions was unknown, but one possibility was that electron transport to DMSO decreased activation of this promoter by PrrA. PrrA activity responds to the oxidationreduction state of the electron-transport chain to increase expression of some photosynthesis genes under anaerobic conditions (Zeilstra-Ryalls et al., 1998
). Alternatively, we previously hypothesized that the DorR response regulator, which is required for expression of genes in the DMSO respiratory pathway, could be acting to repress cycA P2 transcription in response to DMSO under anaerobic conditions (Karls et al., 1999
).
Our results show that DorR is required for DMSO to decrease the expression of cycA P2 during photosynthetic growth in the presence of DMSO, even though active preparations of this protein failed to interact directly with cycA P2. In addition, we found that the DMSO reductase, DorA, is required for the decreased expression from cycA P2 during photosynthetic growth in the presence of DMSO. A model is presented to explain how DMSO reductase function can lead to a decrease in cycA P2 expression when DMSO is present under photosynthetic conditions.
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METHODS |
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To purify DorR, cultures of ER2566(pJC403) were shaken for 6 h at 25 °C after addition of 0·3 mM IPTG. Cells were harvested by centrifugation, resuspended in 15 ml column buffer (20 mM Tris/HCl, pH 8·0, 0·1 mM EDTA, 0·5 mM KCl, 0·1 % Triton X-100), and sonicated (eight times for 1 min each at 50 % duty cycle) to lyse the cells. The cell lysate was centrifuged at 12 000 g for 30 min at 4 °C, and the supernatant loaded onto a 13 ml chitin column that was equilibrated with column buffer. Next, the column was washed with 10 volumes of column buffer, followed by a 2·5 volume wash with cleavage buffer (20 mM Tris/HCl, pH 8·0, 0·1 mM EDTA, 0·5 M KCl, 100 mM DTT) before storage overnight at 4 °C to permit intein self-cleavage. DorR was eluted with 3 volumes of column buffer, and fractions were analysed by SDS-PAGE. Fractions containing DorR (3 ml) were combined and dialysed against two changes of 1 l of 50 mM HEPES-Na, pH 7·8, 200 mM KCl, 10 mM MgCl2, 1 mM DTT, 50 µM PMSF, followed by dialysis into this buffer with 50 % (v/v) glycerol and storage at 80 °C.
Phosphorylation of DorR.
To assay autophosphorylation of DorR, the reaction was initiated by the addition of 30 mM 32P-labelled acetyl phosphate to 10 µM DorR in 50 mM Tris/HCl, pH 7·0, 5 mM MgCl2, 0·1 mM DTT, 0·1 mg BSA ml1 and incubated at 30 °C (McCleary & Stock, 1994). For the time-course experiment, the reaction volume was 90 µl; a 10 µl aliquot was removed at each time point and added to 5 µl of 3x SDS sample buffer (150 mM Tris/HCl, pH 6·8, 30 mM DTT, 6 % SDS, 0·3 % bromophenol blue, 30 % glycerol) on ice. Samples were analysed on a 12 % SDS-polyacrylamide gel, followed by drying the gel and overnight exposure to a phosphorscreen.
Isolation of promoter fragments.
The 215 bp dorCBA promoter fragment was generated by PCR using the DorC-1 (5'-GCGAGGAAGCTTGCGGCCGGTTGGACCTAATGC-3') and DorC-2 (5'-CCCGGATCCGCAGGAATGCGGCGGGCG-3') primers, using pNMT16 as a template (Mouncey et al., 1997). The PCR product was purified from an agarose gel using a Qiagen Gel Extraction Kit. The cycA P2 promoter fragments were obtained by restriction digestion of pRKK148 (with XbaI and KpnI for 73 to +22 of P2) and pRKK128 (with HindIII and NcoI for 228 to +22 of P2), followed by agarose gel purification.
Gel shift assays.
For gel shift assays, DNA fragments were end-labelled with [-32P]ATP (PerkinElmer Life Sciences) using T4 polynucleotide kinase (Promega), following the manufacturer's instructions. Free [
-32P]ATP was removed using a Qiaquick Nucleotide Removal Kit (Qiagen).
DNA binding reactions were performed in binding buffer (50 mM Tris/HCl, pH 8·0, 1 mM EDTA, 0·25 M sucrose, 0·025 % bromophenol blue) in the presence of 50-fold excess non-specific competitor ( DNA). Labelled promoter fragment DNA (8·8 nM) was added to binding buffer, followed by the addition of DorR or DorR that had been previously incubated with 25 mM acetyl phosphate at 30 °C for 1 h. In control reactions, DorR was also incubated at 30 °C for 1 h without addition of acetyl phosphate. Binding reactions were incubated at room temperature for 30 min and analysed at 250 V, 4 °C on a 6 % TBE polyacrylamide gel that had been pre-run for 30 min.
Analysis of cycA P2 reporter activity.
Levels of -galactosidase activity were measured from independent cultures of exponential-phase R. sphaeroides(pRKK232) cells using established protocols (Schilke & Donohue, 1995
).
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RESULTS |
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DorR accepted a phosphoryl group from 32P-labelled acetyl phosphate to generate phosphorylated DorR (PDorR). This incubation resulted in a time-dependent increase in the level of 32P-radioactivity associated with DorR (Fig. 2
). Under the tested conditions, maximal autophosphorylation of DorR occurred after between 20 and 30 min of incubation (Fig. 2b
). These data suggest that purified R. sphaeroides DorR has biological activity typical of other response regulators and can be phosphorylated using acetyl phosphate as a phosphodonor in vitro.
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DorR does not interact with the cycA P2 promoter
Since the cycA P2 promoter contains a sequence similar to a DorR binding site (sequence CTAGTCACATC) centred around 88 nt upstream of the start of transcription (Karls et al., 1999), we also tested if the negative effect of DMSO on this promoter resulted from a direct interaction with this protein. Two cycA P2 promoter fragments were tested: one including the sequence from 73 to +22 (data not shown) and the other from 228 to +22 relative to the start of transcription, both of which had demonstrated a DMSO-dependent decrease in promoter activity (Karls et al., 1999
). At concentrations of up to 2 µM protein (
40 times higher than the amount necessary to detect binding to dorCBA), neither DorR nor DorR treated with acetyl phosphate caused a shift in the mobility of either cycA P2 promoter fragment (Fig. 3b
and data not shown). From this analysis, we conclude that neither DorR nor its phosphorylated counterpart interacts with cycA P2 under conditions where this protein can bind to the dorRdorCBA intergenic region.
DMSO reductase activity is required for DMSO to decrease cycA P2 expression
Given the apparent inability of DorR to interact with cycA P2, it was likely that the effect of DMSO on cycA expression was indirect. One possibility is that this effect depends on the function of a protein that requires DorR for its synthesis or activity: for instance, one of the dorCBA gene products (Mouncey & Kaplan, 1998). To determine if any dorCBA gene products are required for the DMSO-dependent decrease in expression from cycA P2 : : lacZ under photosynthetic conditions, we tested expression of this promoter in cells containing an insertion in dorC that has been shown to be polar onto dorBA (Mouncey et al., 1997
). In the DorC strain, the presence of DMSO did not decrease activity of cycA P2 under photosynthetic conditions (Fig. 1
), suggesting that one or more dorCBA gene products were required for this effect. DMSO also did not alter the activity of the cycA P2 reporter in DorA cultures under photosynthetic conditions. In addition, cycA P2 expression levels in the DorA strain were comparable to those of the DorR strain and to those seen when wild-type cells are grown photosynthetically without DMSO (Fig. 1
). Thus, the negative effect of DMSO during photosynthetic growth on cycA P2 : : lacZ appeared to require the presence of DorA. Considering these results together with the lack of DorR binding to this promoter, we conclude that the loss of DorR indirectly affects this promoter through the inability to activate dorCBA expression. A model to explain the role of DorA in this response is presented below.
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DISCUSSION |
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Is DorR necessary for DMSO to decrease cycA P2 expression in R. sphaeroides?
Consistent with the hypothesis that DorR was both an activator of dorCBA and a repressor of cycA P2 expression, a DorR strain does not show a DMSO-dependent decrease in cycA P2 expression during photosynthetic growth. Also, DorR expression is low during aerobic growth (Mouncey & Kaplan, 1998), when cycA P2 expression has been seen to be unaffected by DMSO (J. C. Comolli & T. J. Donohue, unpublished). However, DorR failed to interact with cycA P2, though it did interact with another putative target sequence, the dorRdorCBA intergenic region, suggesting that DorR is not repressing transcription at this photosynthesis promoter.
If DorR does not act directly at cycA P2, why is the DorR strain unable to decrease expression of this promoter in response to DMSO? Cells lacking the DMSO reductase (because of a mutation in dorA) also do not show a detectable DMSO-dependent decrease in cycA P2 expression, which is not significantly different from the results seen in the DorR mutant. Thus, the inability of the DorR mutant to activate dorCBA transcription in the presence of DMSO is probably the reason why cycA P2 activity is not decreased in this strain (Mouncey & Kaplan, 1998).
How might the function of DorA, the DMSO reductase, alter cycA P2 expression anaerobically in the presence of DMSO?
When an alternative electron acceptor, such as DMSO, is present, electron flow through the photosynthetic apparatus should be altered, according to what is known about anaerobic electron transport in this bacterium. During photosynthesis in R. sphaeroides, electrons are carried by quinones from the photochemical reaction centre to the cytochrome bc1 complex to generate a proton gradient. These electrons are then used by cytochrome c2 to reduce the photo-oxidized bacteriochlorophyll molecules in the reaction centre. It is likely that the reduced quinone generated during photosynthetic growth can be oxidized either by the cytochrome bc1 complex or by DorC, a membrane-bound c-type cytochrome that is proposed to act as an electron donor to DMSO reductase, DorA (Shaw et al., 1999). Then, shuttling of reductant to DorA under photosynthetic growth conditions could alter the activity of PrrA, a global response regulator known to increase cycA P2 activity under these conditions (Karls et al., 1999
). PrrA is required for expression from cycA P2 and for photosynthetic growth (Karls et al., 1999
). It is believed that the PrrBA two-component system (homologous to the RegBA, RegSR and RoxRS systems in other bacteria) monitors changes in the oxidationreduction state of the electron-transport chain, to control photosynthesis gene expression (Comolli & Donohue, 2002
; Emmerich et al., 2000
; Eraso & Kaplan, 1994
; Oh & Kaplan, 2000
). Consequently, our model predicts that when electrons are diverted from the electron-transport chain by the reduction of alternative electron acceptors like DMSO, PrrB/PrrA activity may be modulated to decrease the expression of target genes like cycA P2.
In addition to cycA, other photosynthesis genes have been shown to be negatively affected by the presence of alternative electron acceptors. For example, the levels of light-harvesting complexes and reaction centres are decreased when electron acceptors like DMSO, nitrate or carbon dioxide are present during photosynthetic growth of cultures that are supplied with a fixed carbon source (Michalski et al., 1985; Oh & Kaplan, 1999
). As expected, this decrease in the levels of light-harvesting complex was not detected in DorA or DorR strains under photosynthetic conditions in the presence of DMSO (data not shown). Since these external electron acceptors share the ability to siphon reducing power from the electron-transport chain, it is possible that they act by a common mechanism to decrease the activation of photosynthesis genes, potentially via PrrA.
Interactions of DorR with dorCBA
In R. sphaeroides, DorR is a member of the DorSR two-component regulatory system, whose activity is thought to be stimulated by the presence of DMSO (Mouncey & Kaplan, 1998). The properties of DorR mutants predict that DorR is required for activation of the dorCBA operon, which encodes a c-type cytochrome (DorC), a putative membrane protein of unknown function (DorB), and the DMSO reductase enzyme (DorA) (Mouncey & Kaplan, 1998
; Mouncey et al., 1997
). The
100 bp of DNA between the divergent dorCBA and dorR open reading frames contains four proposed DorR binding sites (Mouncey & Kaplan, 1998
) that share sequence similarity with elements bound by the related response regulator, TorR, in E. coli (Simon et al., 1994
). TorR directly controls expression of the genes for TMAO reductase (Ansaldi et al., 2000
). A similar DMSO-responsive system has been found in R. sphaeroides f. sp. denitrificans, which includes the DorR homologue, DmsR, and the dmsCBA operon, which encodes a DMSO reductase (Ujiiye et al., 1997
). Additionally, extracts of E. coli cells expressing DmsR contain a polypeptide that binds a dmsCBA promoter fragment in vitro, suggesting that this protein may be a DNA-binding protein (Ujiiye et al., 1997
).
In the course of these experiments, we purified DorR, phosphorylated it using the low-molecular-mass phosphodonor acetyl phosphate, and showed that it formed a stable complex with dorRdorCBA intergenic DNA. If DorR is binding to a series of elements in this DNA that are similar to sites recognized by the homologous E. coli TorR (Simon et al., 1995), it is possible that the different complexes observed with the dorRdorCBA fragment reflect the formation of higher-order complexes between DorR and the target DNA, as proposed for TorR (Simon et al., 1995
). While the treatment of DorR with acetyl phosphate may have increased its relative affinity for target DNA, additional experiments are needed to assess the functional significance of this observation.
Concluding remarks
In summary, we have demonstrated that both DorR and DMSO reductase (DorA) are required for the negative effect of DMSO on cycA P2 expression under photosynthetic conditions. Our data indicate that DMSO reductase activity is needed for the decline in expression of cycA P2 and possibly other photosynthesis genes under anaerobic conditions with DMSO. This effect may be due to changes in the activation state of the photosynthesis response regulator, PrrA, in response to alteration of electron flow through the electron-transport chain caused by the presence of DMSO. Further experiments are needed to determine if this sensing occurs via PrrA, and, if so, exactly how changes in electron flow are monitored in the absence of oxygen and what other genes involved in anaerobic energy generation in R. sphaeroides are regulated by the presence of alternative electron acceptors like DMSO.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 8 December 2003;
revised 27 January 2004;
accepted 3 February 2004.
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