Control of dimethylsulfoxide reductase expression in Rhodobacter capsulatus: the role of carbon metabolites and the response regulators DorR and RegA

The ability to use dimethylsulfoxide (DMSO) and trimethylamine N-oxide (TMAO) as electron acceptors is widespread among purple non-sulfur phototrophic bacteria (Zannoni, 1995 ). In Rhodobacter capsulatus DMSO reductase (DorA) is located in the periplasm and electron transfer to this terminal reductase has been shown to involve the cytochrome DorC (Shaw et al., 1999a , b ). The DMSO respiratory pathway has a role in redox homeostasis in Rhodobacter cells growing photoheterotrophically, especially when reduced carbon sources are present (Richardson et al., 1988 ). It also enables the cells to utilize a variety of carbon sources during dark anaerobic growth (Cox et al., 1980 ; Schultz &Weaver, 1982 ).

Recently, it has been shown that the dorCDA gene cluster is located upstream of a gene designated dorB (Shaw et al., 1999a ) and genes involved in the early steps of synthesis of the molybdenum cofactor of DMSO reductase (Solomon et al., 1999 ). The dorD gene encodes a protein that is required for the biogenesis of DorA but does not have a direct role in DMSO respiration (Shaw et al., 1999a ). Upstream of dorCDA are two additional genes, dorR and dorS. Mutational analysis of the dor genes upstream of dorCDA in Rhodobacter sphaeroides (Mouncey et al., 1997 ; Mouncey & Kaplan, 1998a ) has identified the sensor histidine kinase DorS and the response regulator DorR as a two-component regulatory system that is required for the DMSO-dependent induction of the dorCDA operon. A similar conclusion has been made from the analysis of a dorR mutant of R. capsulatus (Shaw et al., 1999a ). The dorR and dorC genes are divergently transcribed while the dorS gene is transcribed from a distinct promoter in the same direction as dorC (Mouncey et al., 1997 ). The DorR protein is a member of the OmpR family of response regulators. In R. sphaeroides it has been shown that DorR binds to the dorRdorC intergenic region that separates these two genes in both Rhodobacter species and contains the dorCDA and dorR promoters (Ujiiye et al., 1997 ; Yamamoto et al., 2001 ).

A variety of environmental and nutritional signals appear to affect DMSO reductase expression. Under aerobic conditions expression of the dor operon is very low. In R. sphaeroides (Mouncey & Kaplan, 1998b ) and R. capsulatus (Shaw, 1998 ) anaerobic induction appears to involve the activation of dorS expression by the transcriptional regulator FNR. An FNR-binding site has been identified upstream of dorS in R. sphaeroides (Mouncey & Kaplan, 1998b ) while no such binding site has been found in the dorRdorCDA intergenic region of either Rhodobacter species. In addition, Mouncey & Kaplan (1998a ) have shown that negative aerobic regulation of dor expression in R. sphaeroides is under redox control via the cytochrome cbb₃ oxidase, although the details of this signal transduction pathway are not entirely clear. Recently, we showed that the ModE orthologue in R. capsulatus, MopB, was also required for high levels of dor operon expression in this organism (Solomon et al., 2000 ). MopB activates dor expression in the presence of molybdate and is also required for molybdate-dependent repression of the alternative nitrogenase (Wang et al., 1993 ). Clearly, regulation of the dor anaerobic respiratory system is complex, and appears to be linked to other major bioenergetic and metabolic processes in phototrophic bacteria.

In this study we have investigated in more detail the regulation of DMSO reductase expression via the response regulator DorR. An emerging phenomenon over the last few years has been the control of the phosphorylation state of response regulators by small metabolites such as acetyl phosphate (McCleary & Stock, 1994 ; Prüß, 1998 ). This metabolic control of response regulators provides an additional element in the signal transduction pathways of some sensor kinaseresponse regulator systems. In view of the key role of DMSO respiration in anaerobic dark growth of R. capsulatus we here consider the possibility that DorR may be under this form of metabolic control. A second aim was to determine whether the negative control of dor operon expression was dependent upon the global, redox-responsive RegBA histidine kinaseresponse regulator system (Swem et al., 2001 ; Elsen et al., 2000 ). Here we report that the regulation of dor operon expression is dependent upon the DorR protein, and is integrated into the RegBACco redox-regulatory system.

Strains, media and plasmids.
Strains and plasmids used are shown in Table 1. R. capsulatus cells were grown routinely at 30 °C on liquid or solid RCV minimal medium with malate as carbon source (Weaver et al., 1975 ) or on chemoautotrophic (CA) medium (Madigan & Gest, 1979 ) supplemented with a carbon source. When specified, DMSO was added to the growth medium in a final concentration of 0·27%. For growth on pyruvate, a 1 M pyruvate solution was prepared immediately prior to use, filter-sterilized and added to carbon-source-free CA medium to a final concentration of 40 mM. For growth on fructose or succinate, filter-sterilized, 1 M solutions were added to CA medium; final concentrations were 20 mM for fructose and 30 mM for succinate. For dark, anaerobic growth 30 mM bicarbonate was added to the growth medium from a sterile, 1 M stock solution when specified. Aerobic cultures of R. capsulatus for use in mating experiments were grown on TYS medium (Beringer, 1974 ) with vigorous shaking at 30 °C. Escherichia coli strains were routinely grown on liquid or solidified LuriaBertani or TYS medium (Sambrook et al., 1989 ). Where necessary, antibiotics were added to the media as follows: ampicillin, E. coli 100 µg ml^-1; gentamicin, E. coli 10 µg ml^-1, R. capsulatus 4 µg ml^-1; tetracycline, E. coli 10 µg ml^-1, R. capsulatus 1 µg ml^-1. After mating experiments, 50 µg tellurite ml^-1 was included in selective plates for R. capsulatus. In the case of the regA mutant MSO1 the existing kanamycin resistance was used for counter-selection.

Table 1. Strains and plasmids used in this study

Preparation of cell-free extracts.
Cells were harvested in the late exponential growth phase by low-speed centrifugation (3200 g) at 4 °C for 15 min, and pellets stored at -20 °C. For the preparation of cell-free extracts the pellets were resuspended in 20 mM potassium phosphate buffer pH 7·6, 1 mM EDTA (10 ml per g wet weight of cells), homogenized and passed twice through a French pressure cell (Aminco) at 1000 p.s.i. (6·9 MPa). Cell debris was removed by centrifugation at 32000 g at 4 °C for 30 min, and the resultant crude extract was subjected to ultracentrifugation at 145000 g at 4 °C for 90 min. The supernatant from this centrifugation step was used for DMSO reductase or ß-galactosidase assays.

Molecular biological methods.
Plasmids pSL111 and pALS69, harbouring dorA::lacZ and dorR::lacZ transcriptional fusions, respectively, have been described by Shaw et al. (1999a ) and Shaw (1998) . Plasmids were transferred into R. capsulatus by biparental matings (Simon et al., 1983 ) using E. coli S17-1 as donor strain.

DMSO reductase activity
This was measured in extracts of total soluble protein, using dithionite-reduced methylviologen as electron donor (McEwan et al., 1985 ). One unit of activity corresponds to 1 µmol methylviologen oxidized min^-1. All measurements were carried out at least in triplicate on each cell extract and are the means of three independent growths (four in the case of the reg mutant strain).

ß-Galactosidase activity.
In time-course growth experiments and aerobic/anaerobic shift experiments the activity of chromosomal lacZ fusions was determined by the method of Miller (1972) , using chloroform and cetyltrimethylammonium bromide (CTAB). In all other experiments, ß-galactosidase activity was determined as nmol ONP (mg protein)^-1, using the method of Clark & Switzer (1977) . All measurements were carried out in triplicate on each cell extract and are the mean of at least two independent growth experiments. Protein was determined by the Lowry method or using the bicinchoninic acid assay (Sigma). Denaturing polyacrylamide gel electrophoresis and Western blotting were performed using standard procedures (Sambrook et al., 1989 ). Anti-DMSO reductase antibodies were used as described by Hatton et al. (1994) .

Growth experiments.
Measurements of bacterial growth were carried out under high-light (100 W m^-2) and low-light (16 W m^-2) conditions at 30 °C as described by Horne et al. (1996) for the dorR mutant, the regA mutant strain and their respective wild-type strains with either malate or pyruvate as carbon source. Experimental cultures were prepared by inoculating 100 ml medium with 2% (high light) or 5% (low light) of a pre-culture grown up under the conditions to be used for the main experiment and incubated as described above. Samples (1 ml) for optical density measurements (with a Hitachi U-3000 spectrophotometer) were removed at intervals suitable to the respective growth rate (between 1 h and 4 h). Cultures were topped up with the respective growth medium after sampling. All experiments were repeated at least twice. For experiments involving a shift from dark aerobic to dark anaerobic growth, 100 ml medium was shaken in 1 l flasks (30 °C, 200 r.p.m.) until an OD₆₈₀ of 0·30·5 was reached. Cultures were then transferred to sterile 100 ml bottles and incubated at 30 °C in the dark.

DorR is required for maximal induction and repression of dor operon expression
Previous experiments using a dorA::lacZ fusion showed that the response regulator DorR is required for a high level of dor operon expression (Shaw et al., 1999a ). This observation was confirmed here using DMSO reductase activity measurements to monitor dor operon expression. With malate as carbon source, DMSO reductase activity in wild-type cells (strain 37b4) grown phototrophically in the presence of DMSO was about 150-fold higher compared to cells grown without this electron acceptor under both high- and low-light conditions (Fig. 1). Under non-inducing conditions, DMSO reductase activity in strain 37b4 was 0·02 units (mg protein)^-1; expression levels were similar with both levels of light intensity. No DMSO reductase activity was detected in strain 37b4 grown aerobically.

(23K): Fig. 1. DMSO reductase activity in cell extracts of wild-type (37b4) or dorR mutant strain (DorR-R1) of R. capsulatus grown aerobically or photoheterotrophically in the presence of 30 mM malate. High-light conditions (HL) were 100 W m-2; low-light conditions (LL) were 16 W m-2. Enzyme assays were performed at least in triplicate for three independent growths experiments. Errors are given as 95% confidence intervals. Note the change in units on the y-axis after the break.

In the R. capsulatus dorR mutant strain, DorR-R1, no DMSO-dependent induction of DMSO reductase activity was observed in cells grown phototrophically on malate. However, a basal level of DMSO reductase activity could be detected in the dorR mutant under all growth conditions examined. The levels were between 5 and 10 times higher than in strain 37b4 under the same conditions (Fig. 1). This activity was independent of the presence of DMSO and, in contrast to the wild-type, low but detectable amounts of DMSO reductase were present in aerobically grown cells. Interestingly, in the absence of DMSO, there also appeared to be an increase in DMSO reductase activity with decreasing light intensity. The presence of DMSO reductase protein in DorR-R1 was confirmed by Western blotting (data not shown). This result contrasts with previous observations in R. sphaeroides (Mouncey et al., 1997 ), where an absence of DorA protein in a dorR mutant strain was reported. Taken together, these data indicate that DorR is required for induction of dor operon expression in the presence of DMSO and for repression in the absence of electron acceptor under photoheterotrophic conditions in R. capsulatus. In contrast, aerobic repression of the dor operon appears to be largely independent of DorR.

Pyruvate activates dor operon expression via DorR at low light intensity and under aerobic conditions
Since DMSO respiration is a key activity associated with anaerobic dark growth using fermentable carbon sources in R. capsulatus we determined whether a carbon source that produced phosphorylated intermediates such as acetyl phosphate might exert an effect on dor operon expression. Fig. 2 shows that DMSO reductase activity in DMSO-induced R. capsulatus 37b4 cells grown at high light intensity was reduced by 50% when pyruvate replaced malate as carbon source. Time-course induction experiments confirmed that this result was not dependent on the growth phase of the culture (data not shown). Under low-light conditions DMSO reductase activity in DMSO-induced wild-type cells was similar in cells grown on either carbon source. However, in the absence of DMSO, the activity level in wild-type cells grown at low light intensity was about 10-fold higher in cells grown on pyruvate and was similar to the level found in the dorR mutant grown on malate. In this mutant the DMSO reductase activity levels observed were comparable to those found in cells grown on malate, and there also appeared to be a slight effect of light intensity on enzyme expression. It is worth noting that the aerobic repression of the dor operon also seemed to be less stringent with pyruvate as carbon source (Fig. 2).

(21K): Fig. 2. DMSO reductase activity in cell extracts of wild-type (37b4) or dorR mutant strain (DorR-R1) of R. capsulatus grown aerobically or photoheterotrophically in the presence of 40 mM pyruvate. High light conditions (HL) were 100 W m-2; low light conditions (LL) were 16 W m-2. Enzyme assays were performed at least in triplicate for three independent growths experiments. Errors are given as 95% confidence intervals. Note the change in units on the y-axis after the break.

Influence of a dorR mutation on cell growth
Growth of R. capsulatus wild-type 37b4 and the dorR mutant under phototrophic conditions using either malate or pyruvate showed that the dorR mutant grew at a slightly slower rate (∼15% slower) than the wild-type, while there was no difference in growth rates during aerobic growth. Replacement of malate with pyruvate as a carbon source resulted in a 50% stimulation of the phototrophic growth rate of both wild-type strain 37b4 and the dorR mutant. Likewise, aerobic growth rates on pyruvate were higher than on malate (Table 2). Under low-light conditions the presence of DMSO led to an increase (30%) in the growth rate of the wild-type strain when malate was the carbon source. However, the dorR mutant showed reduced growth rates (about 50%) in the presence of DMSO under all conditions used except for low-light conditions with pyruvate as carbon source (Table 2). This reduced growth rate might be related to DMSO-induced damage to the cells.

Table 2. Growth rates of the wild-type (37b4) and dorR mutant strain (DorR-R1) of R. capsulatus with 30 mM malate or 40 mM pyruvate as carbon source under photoheterotrophic or aerobic conditions

The limited repertoire of redox-balancing reactions associated with fermentation pathways in R. capsulatus means that this bacterium requires an external electron acceptor to facilitate anaerobic dark growth. Most studies describing anaerobic dark growth of R. capsulatus have been carried out in the presence of TMAO or DMSO (Madigan & Gest, 1978 ; Cox et al., 1980 ; Madigan et al., 1980 ). During experiments in which R. capsulatus cultures containing either dorR::lacZ or dorA::lacZ chromosomal fusions (Shaw et al., 1999a ) were shifted from aerobic conditions to anaerobic dark growth conditions in the absence of DMSO, an approximately twofold induction of dorR and dorA expression was observed (data not shown). This induction was independent of the carbon source (fructose, malate or pyruvate). In order to investigate a possible role of DorR for the dark, anaerobic growth mode, fructose, succinate, malate and pyruvate were used as carbon sources in media containing either DMSO or 30 mM bicarbonate. It was found that both the dorR mutant and the wild-type were able to grow with fructose and pyruvate in the presence of bicarbonate, while neither showed significant growth with either of the two other carbon sources and bicarbonate. These data indicate that bicarbonate-mediated dark anaerobic growth is independent of the DorR regulatory system and requires carbon sources other than TCA cycle intermediates. As expected, the dorR mutant was unable to grow with DMSO as electron acceptor, while the wild-type was able to grow with all four carbon sources in the presence of DMSO.

DMSO reductase expression is repressed by the response regulator RegA
The RegBA system is a global regulator of gene expression in Rhodobacter species. It has a key role in the induction of the photosynthetic apparatus (Sganga & Bauer, 1992 ; Du et al., 1998 ) and has also been shown to modulate expression of nitrogen and carbon dioxide fixation pathways, the uptake hydrogenase and proteins involved in respiratory processes (Elsen et al., 2000 ; Qian & Tabita, 1996 ; Swem et al., 2001 ). Recently it has also been demonstrated that wild-type RegA controls activity of the puf and puc promoters directly by binding to the relevant sequences (Hemschemeier et al., 2000 ). It had been previously observed that DMSO reductase activity in R. sphaeroides is influenced by mutations in the rdx and cco operons (Mouncey & Kaplan, 1998a ), and a direct interaction between components of these operons and the sensor kinase DorS had been suggested by the authors. As recent studies have established an involvement of the RegBA regulatory system (known as PrrBA in R. sphaeroides) in the transmission of signals generated by components of the cco and rdx operons (OGara & Kaplan, 1997 ; Roh & Kaplan, 2000 ), the effect of a mutation in the regA gene on DMSO reductase expression was investigated. Measurements of DMSO reductase activity of R. capsulatus wild-type (strain SB1003) and regA mutant strain MSO1 (Sganga & Bauer, 1992 ), grown under phototrophic (high light) conditions in the absence of DMSO showed that activity was increased in the regA mutant, reaching levels comparable to that found in DMSO-induced wild-type cells (Fig. 3a). In DMSO-induced cultures a further twofold increase of activity was observed in the mutant (Fig. 3a). The presence of DMSO reductase in the regA mutant strain grown in the absence of DMSO was confirmed by Western blotting (Fig. 3a). In a corresponding set of experiments the activity of a dorA::lacZ gene fusion in the regA mutant background was determined and was also found to be higher than that of a wild-type genetic background (Fig. 4a). This result confirmed that regA was exerting an effect on the transcription of the dor operon. These data suggest that the RegBA system is involved in repression of dor operon expression and that RegA might be acting as a repressor.

(35K): Fig. 3. DMSO reductase activities in wild-type (SB1003) and regA mutant strain MSO1 of R. capsulatus when grown under high-light conditions in the presence of either 30 mM malate (a) or 40 mM pyruvate (b). The results obtained by the spectrophotometric assays were confirmed by Western blot analysis as shown in the lower half of each panel. In each lane 10 µg protein was loaded. Lanes: 1, SB1003; 2, SB1003+DMSO; 3, MSO1; 4, MSO1+DMSO. The DMSO reductase band is arrowed. Four independent experiments were performed for each growth condition, and enzyme activities for each determined at least in triplicate. Errors are given as 95% confidence intervals.

(17K): Fig. 4. Levels of Φ[dorA::lacZ] activity in the wild-type strain SB1003 and the regA mutant strain MSO1 when grown under high-light conditions in the presence of either 30 mM malate (a) or 40 mM pyruvate (b). Three independent experiments were performed for each growth condition; errors are given as 95% confidence intervals.

Metabolic control of DMSO reductase expression by pyruvate occurs in a regA mutant
Experiments similar to those presented in Fig. 3(a) were conducted with pyruvate instead of malate as carbon source. Surprisingly, this change of carbon source restored the control of DMSO reductase expression to normal levels in the reg mutant (Fig. 3b). Under inducing conditions the levels of DMSO reductase activity were found to be similar in both the regA mutant and the wild-type strain. The results were confirmed by Western blotting (Fig. 3b) and using the dorA::lacZ gene fusion (Fig. 4b). The fact that control of dor expression was restored in the regA mutant indicates that RegA is not directly responsible for control of DMSO reductase expression by pyruvate. By the same token, regulation cannot have been restored by phosphorylation of DorR via a phosphate donor, as such a phosphorylation should have led to increased dor expression.

The cytochrome cbb₃ oxidase is a negative regulator of dor operon expression
Having shown that a mutation in the regA resulted in deregulation of dor expression, we investigated whether the observed effect is part of a redox-signalling pathway involving cytochrome cbb₃. It is already established that the cytochrome cbb₃ oxidase is a key regulator of anaerobic gene expression in R. sphaeroides (Oh & Kaplan, 1999 ; Mouncey & Kaplan, 1998a ; Oh & Kapla n, 2001 ). In R. capsulatus, dor operon expression, as indicated by the level of Φ[dorA::lacZ] activity, was about twofold higher in a cco mutant compared to the wild-type strain. However, the most obvious difference between the two strains was the sixfold amplification in Φ[dorA::lacZ] activity that was observed upon addition of DMSO. This was only observed in the cco mutant at high light intensity. At low light intensity Φ[dorA::lacZ] activity was much higher in both strains even under non-induced conditions, and no difference between induced and non-induced activity levels was observed for the cco mutant.

Previous analysis of the expression of the dor operon in R. capsulatus and R. sphaeroides has shown that regulation is complex and a number of regulatory proteins are involved in its control. These regulatory proteins appear to respond to a variety of environmental factors and this enables the control of dor operon expression to be deeply integrated into the metabolic circuits of these purple phototrophic bacteria. The response regulator DorR is central to the induction of dor operon expression; in the absence of this transcription factor dor operon expression was found to be low and induction by DMSO was not observed. These results are consistent with phosphorylated DorR acting as a positive regulator of transcription of both dorCDA and dorR as indicated by the measurements of DMSO reductase activity. In addition to this recognized role, DorR appears to be involved in repression of dorCDA expression under phototrophic growth conditions, as the regulatory mutant seemed to express DMSO reductase at a constant, low level which was well above (510-fold) the levels detected in non-induced wild-type cells. Thus, our findings indicate that the DorR protein may bind to the dorCdorR intergenic region in both phosphorylated (i.e. activated) and non-phosphorylated form. This would suggest a mechanism similar to the one described for the closely related TorR protein from E. coli (Simon et al., 1995 ; Ansaldi et al., 2000 ), which can bind to the tor promoter in both forms and also shows cooperative binding patterns. Our results are also consistent with observations made by Ujiiye et al. (1997) for the homologous DmsR protein from R. sphaeroides. These authors demonstrated that DmsR expressed in E. coli could bind DNA. From this observation they concluded that DmsR can bind to the dms promoter in its non-phosphorylated form, because the heterologously expressed protein was likely to be non-phosphorylated. Under non-inducing phototrophic conditions we suggest that non-phosphorylated DorR acts as a repressor of dor operon expression, and the data in Figs 1 and 2 are consistent with this view.

The low level of expression of DMSO reductase under aerobic conditions in both the wild-type and the dorR mutant strain may be partly due to the failure to express the DorS sensor, since the dorS gene is under FNR control (Mouncey & Kaplan, 1998a ). This would prevent a high level of phosphorylated DorR from being generated. Support for the suggestion that it is a limitation in the phosphorylated form of DorR that limits dor operon expression comes from the observation that in the presence of pyruvate elevated levels of DMSO reductase can be measured under aerobic conditions. The simplest explanation for this result is the phosphorylation of DorR by acetyl phosphate produced from pyruvate metabolism. Such a situation would allow the DorR protein to act as a positive regulator in the absence of the sensor DorS. It is established in E. coli that acetyl phosphate levels can vary greatly in cells, depending on carbon source, and under some circumstances can be as high as 1 mM (McCleary & Stock, 1994 ). Acetyl phosphate has also been shown to be involved in the regulation of OmpR-mediated processes in E. coli (Prüß, 1998 ). A similar mechanism might be involved in the effects observed with respect to DorR in our experiments.

Two modes of anaerobic dark growth have been described for R. capsulatus, namely anaerobic respiration using DMSO or TMAO, and fermentative growth in the presence of bicarbonate. Of these two modes, anaerobic respiration is the more energetically favourable one, allowing growth to higher cell densities. A twofold induction of dorR and dorA expression was observed when aerobically grown cells were shifted to anaerobic conditions, suggesting that the dor respiratory system might be involved in regulation of this growth mode. However, subsequent experiments using a dorR mutant showed clearly that the two dark anaerobic growth modes are unrelated, and DorR is not involved in general regulation of dark anaerobic growth. The observed increase in expression might be caused by a relaxation of the aerobic repression of dor operon expression.

DMSO respiration is a key energy generator under anaerobic dark conditions but the superiority of photophosphorylation and oxidative phosphorylation with oxygen as electron acceptor has led to the evolution of mechanisms that ensure that dor operon expression is negatively controlled by light and oxygen. A major factor in this control is the cytochrome cbb₃ oxidase since a cco mutant exhibits higher levels of dor operon expression under both aerobic and phototrophic conditions. A similar result has been reported by Mouncey & Kaplan (1998a ), and this group has recently suggested that redox signals are transduced via the CcoQ protein to the PrrBA two-component system (the homologue of the RegBA system in R. capsulatus). The pathway transducing the signal generated by the cytochrome cbb₃ oxidase has also been shown to influence the expression of photosynthesis and accessory pigments (Oh & Kaplan, 1999 ; OGara et al., 1998 ). Investigation of dor operon expression in a regA mutant showed a very significant deregulation of DMSO reductase expression, with the non-induced mutant strains reaching activity levels comparable to those found in induced wild-type cells. The pattern of DMSO-dependent induction of expression, however, remained the same. Together these results indicate that DMSO reductase expression is negatively regulated by a signal generated by the cytochrome cbb₃ oxidase, and that this signal is transmitted via the RegBA two-component system rather than by direct interaction of CcoQ with the DorS sensor kinase, as had been suggested for R. sphaeroides (Mouncey & Kaplan 1998a ). The way in which RegA influences dor operon expression remains to be determined; examination of the dorRdorC intergenic region for a RegA binding consensus sequence, recently described by two groups (Emmerich et al., 2000 ; Swem et al., 2001 ), failed to identify any such site.

An interesting finding with regard to dor operon regulation is that the deregulation of DMSO reductase observed in the malate-grown regA mutant strain can be reversed by a change of carbon source; when this strain was grown with pyruvate as carbon source, the familiar regulation pattern for DMSO reduction was restored. In the regA mutant, expression levels in the absence of DMSO were very low, while addition of DMSO led to a strong induction of activity. Both malate and pyruvate are substrates that are slightly more oxidized than the cell carbon, and the main difference in their utilization pathways is that pyruvate gives rise to enhanced levels of acetyl phosphate. Acetyl phosphate has been shown to be involved in metabolic control in enteric bacteria (Bouché et al., 1998 ; Nyström, 1994 ; Prüß, 1998 ), and is known to act as a phosphate donor to some response regulators. However, it is unlikely that the effect observed in the pyruvate-grown regA mutant is due to an increase in phosphorylated DorR, as that should result in increased DMSO reductase expression rather than a re-establishment of control mechanisms. We therefore suggest that another regulatory circuit is activated by acetyl phosphate and causes the observed repression of DMSO reductase expression.

This work was supported by a grant from the Australian Research Council to A. G. McEwan. We thank Carl Bauer and Fevzi Daldal for the provision of some of the strains used in this study, and T. J. Hurse for advice on statistical analyses.