Rhodobacter capsulatus gains a competitive advantage from respiratory nitrate reduction during light–dark transitions

M. J. K. Ellington1, D. J. Richardson2 and S. J. Ferguson1

1 Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK
2 Centre for Metalloprotein Spectroscopy and Biology, School of Biological Sciences, University of East Anglia, Norwich Research Park, Norwich NR4 7TJ, UK

Correspondence
S. J. Ferguson
stuart.ferguson{at}bioch.ox.ac.uk


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Rhodobacter capsulatus N22DNAR+ possesses a periplasmic nitrate reductase and is capable of reducing nitrate to nitrite under anaerobic conditions. In the absence of light this ability cannot support chemoheterotrophic growth in batch cultures. This study investigated the effect of nitrate reduction on the growth of R. capsulatus N22DNAR+ during multiple light–dark cycles of anaerobic photoheterotrophic/dark chemoheterotrophic growth conditions in carbon-limited continuous cultures. The reduction of nitrate did not affect the photoheterotrophic growth yield of R. capsulatus N22DNAR+. After a transition from photoheterotrophic to dark chemoheterotrophic growth conditions, the reduction of nitrate slowed the initial washout of a R. capsulatus N22DNAR+ culture. Towards the end of a period of darkness nitrate-reducing cultures maintained higher viable cell counts than non-nitrate-reducing cultures. During light–dark cycling of a mixed culture, the strain able to reduce nitrate (N22DNAR+) outcompeted the strain which was unable to reduce nitrate (N22). The evidence indicates that the periplasmic nitrate reductase activity supports slow growth that retards the washout of a culture during anaerobic chemoheterotrophic conditions, and provides a protonmotive force for cell maintenance during the dark period before reillumination. This translates into a selective advantage during repeated light–dark cycles, such that in mixed culture N22DNAR+ outcompetes N22. Exposure to light–dark cycles will be a common feature for R. capsulatus in its natural habitats, and this study shows that nitrate respiration may provide a selective advantage under such conditions.


Abbreviations: TMAO, trimethylamine-N-oxide; UQ/UQH2, ubiquinone/ubiquinol; {Delta}p, protonmotive force


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The photosynthetic {alpha}-proteobacterium Rhodobacter capsulatus N22DNAR+ is capable of the dissimilatory reduction of nitrate to nitrite, but is not capable of the complete reduction of nitrate to dinitrogen (McEwan et al., 1982). R. capsulatus N22, which was originally isolated from the St Louis strain, is the parent strain of N22DNAR+. It is a green mutant which was used in studies of light-driven electron transport because optical absorption bands overlapping those from the cytochromes were absent. Strain N22 takes up nitrate from the medium in a characteristically assimilatory, ammonium-sensitive and light-dependent manner. N22 can grow with nitrate as the nitrogen source but cannot respire nitrate. Relatively long-term phototrophic culturing of N22 with nitrate as nitrogen source resulted in a change of phenotype. Nitrate uptake from the medium became light-inhibited and ammonia-insensitive. Subsequently, a periplasmic nitrate reductase (Nap) was purified from this gain-of-function mutant, termed N22DNAR+ (McEwan et al., 1987), and some of the electron-transport chain to Nap was elucidated (Richardson et al., 1990). Nap is known to be present in some strains of R. capsulatus, but absent from others (McEwan et al., 1984; Alef et al., 1984). We assume that strain N22 had lost the ability to respire nitrate via Nap during prolonged phototrophic culturing in the laboratory on medium lacking nitrate. In phototrophic batch culture there must have been some selective advantage for either a reversion, or a compensating mutation, to permit the expression of Nap. By analogy with spontaneous DNA point mutations in Paracoccus pantotrophus (Sears et al., 2000) the simplest basis for this is that the upstream DNA regulatory region was the site of an inactivating mutation in N22, but other possibilities cannot be excluded.

It has long been recognized that R. capsulatus can grow under anaerobic heterotrophic (dark) conditions, in the presence of an appropriate exogenous N oxide terminal electron acceptor such as nitrous oxide, or other acceptors such as TMAO and DMSO (Shultz & Weaver, 1982; McEwan et al., 1985, 1987). However, the reduction of nitrate to nitrite by Nap is unable to support growth under anaerobic chemoheterotrophic conditions in batch culture despite catalysing the generation of protonmotive force {Delta}p (McEwan et al., 1982, 1983, 1984. One reason for this observation could be that nitrate-respiration-dependent {Delta}p may be too low to support a detectable growth rate in batch culture, as a threshold dependency of growth rate on {Delta}p in bacteria is recognized (Taylor & Jackson, 1985).

After the initial description of Nap in R. capsulatus, Nap systems have been identified and characterized biochemically and/or genetically in many bacteria (Berks et al., 1995b; Darwin et al., 1998; Flanagan et al., 1999; Grove et al., 1996; Reyes et al., 1996, 1998; Siddiqui et al., 1993). The physiological function of Nap varies between species (Richardson et al., 2001). These functions include anaerobic growth adaptation (Siddiqui et al., 1993), nitrate scavenging in nitrate-limited environments (Potter et al., 1999, 2001), denitrification (Bedzyk et al., 1999; Bell et al., 1990; Liu et al., 1999) and a redox-balancing role in the bacterium during chemoheterotrophic or photoheterotrophic growth conditions in the presence of a highly reduced carbon substrate (Richardson et al., 1988; Richardson & Ferguson, 1992; Roldan et al., 1994; Sears et al., 1997, 2000).

In Rhodobacter sphaeroides DSM158 the role of Nap is to provide a redox-balancing mechanism during photoheterotrophic growth (Reyes et al., 1996). The Nap system of R. sphaeroides DSM158 is encoded by the napKEFDABC gene cluster, and the regulation of the system has been studied (Castillo et al., 1996; Reyes et al., 1996, 1998). In vivo Nap activity is not responsive to the intracellular C/N ratio (Dobao et al., 1994), but is regulated by enzyme activation in response to the carbon substrate/electron supply and nitrate concentration (Gavira et al., 2002). Interestingly, in response to nitrate under anaerobic photoheterotrophic conditions with an oxidized carbon substrate, enzyme activation causes increased in vivo Nap activity (Gavira et al., 2002) (see below). Transcription of nap remains unchanged under all tested anaerobic conditions, but does increase under aerobic conditions. However, lower enzyme activities were observed, probably due to the preferred electron route to oxygen being available (Gavira et al., 2002). This has a superficial similarity to the situation in P. pantotrophus, where nap transcription is in part regulated in response to aerobicity (Sears et al., 2000; Ellington et al., 2002).

Although no molecular analyses have been carried out in R. capsulatus, the role for nitrate respiration as a redox-balancing pathway has been established experimentally (Richardson et al., 1988). The addition of exogenous terminal electron acceptors, such as nitrate, allowed growth on highly reduced carbon compounds to be supported (Richardson et al., 1988). Thus, growth on highly reduced substrates is only possible by combining the cyclic electron photosynthetic system with an anaerobic terminal electron-transport pathway to N oxides (Richardson et al., 1988). Conversely, with an oxidized substrate such as malate, photoheterotrophic growth occurs with no additional electron acceptors. Additions of terminal electron acceptors such as nitrate did not provide any extra support for growth (Richardson et al., 1988), although reduction of nitrate was observed. Given this, and the Nap enzyme activation seen in R. sphaeroides DSM158 under photoheterotrophic conditions in the presence of an oxidized carbon source and nitrate (see above) (Gavira et al., 2002), the question of the function of the Nap pathway during photoheterotrophic growth with a relatively oxidized carbon substrate such as malate still needs to be addressed.

The photosynthetic cyclic electron-transport chain of R. capsulatus includes a ubiquinone (UQ)-reducing reaction centre and a ubiquinol (UQH2)-oxidizing bc1 complex. At low {Delta}p electrons can enter the UQ/UQH2 pool via NADH dehydrogenase and it has been shown that during anaerobic dark incubation the cyclic electron-transport system becomes over-reduced (Jones et al., 1990). Nap interacts with the cyclic electron-transport system at the level of the UQH2 pool. Addition of nitrate to anaerobic dark cultures serves to repoise the electron-transport chain (Jones et al., 1990). This may aid culture survival and recovery from dark conditions.

Here we ask if the ability to respire nitrate during growth on relatively oxidized substrates is relevant during light–dark cycling of anaerobic cultures of R. capsulatus, conditions that the organism would encounter in its natural environment. We examine whether a selective advantage is conferred on a nitrate-respiring strain over a non-nitrate-respiring strain, during repeated light–dark cycling of an anaerobic mixed-strain continuous culture of R. capsulatus.


   METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains and batch culture.
The Rhodobacter capsulatus strains N22 and N22DNAR+ (McEwan et al., 1982), which are negative and positive, respectively, for the periplasmic nitrate reductase, were used. The rifampicin-resistant strain R. capsulatus N22 rifr was obtained from culture of N22 on peptone-yeast extract (PY) medium containing 100 µg rifampicin ml-1. Aerobic growth was achieved in 250 ml flasks containing 50 ml PY medium. Flasks were incubated at 30 °C in a rotary shaker operating at 200 r.p.m. For experimental growth, RCV medium (Weaver et al., 1975) was used, with ammonium as the nitrogen source; growth was thus deemed to be under nitrogenase-repressed conditions. Experimental starter cultures of R. capsulatus N22 and N22DNAR+ were grown aerobically at 30 °C in 5 ml minimal RCV medium with malate added to 30 mM final concentration.

Continuous culture.
For continuous culture, RCV medium with 10 mM (final concentration) malate was used. Prior to continuous culture the chemostat vessel was typically inoculated with 5 % (v/v) of overnight RCV starter culture. The vessel was operated in continuous-culture mode (D=0·1 h-1), aerobically under dark conditions until culture turbidity reached OD660 0·400±0·02. The vessel was switched to anaerobic conditions with high illumination, and the culture allowed to equilibrate for 4–5 h. Cultures were subjected to periods of light and dark to achieve culture growth and washout, respectively. Culture washout was monitored turbidimetrically at 660 nm and allowed to proceed to an OD660 of about 0·1. Upon re-illumination the culture was allowed to recover fully before the next period of darkness. Periods of 8–14 h were typically spent under each condition.

Illumination for photoheterotrophic growth under anaerobic conditions was supplied by 6x100 W domestic light bulbs placed 30 cm from the culture vessel. Anaerobic culture conditions were maintained by sparging oxygen-free nitrogen through the vessel at approximately 1·5 l min-1. The absence of dissolved oxygen was monitored with a polarographic pO2 electrode (Ingold). The vessel stirrer was operated at 200 r.p.m. A combined glass electrode (Ingold) was used to monitor pH; values between 6·5 and 8·0 were observed during operation.

Contamination of the culture was checked by enrichment agar plating of culture serial dilutions for colony morphology and c.f.u. determinations. To check for carbon limitation a 10 mM carbon substrate addition was made to the chemostat at the end of operation. Cultures rapidly increased their biomass, to double or more of that previously seen.

Analytical procedures.
Growth was followed turbidimetrically by measuring culture OD660. For determination of c.f.u. ml-1, 1/10 serial dilutions were made in a total volume of 1 ml. From single-strain culture, the strains were counted by plating dilutions from the vessel on PY and PYrif agar plates for R. capsulatus N22DNAR+ and R. capsulatus N22, respectively. For the mixed-strain culture, R. capsulatus N22 was counted directly from PYrif agar plates; R. capsulatus N22DNAR+ c.f.u. values were determined by subtracting the R. capsulatus N22 PYrif value from the c.f.u. count of the PY plates. Typically dilutions at 10-5, 10-6, 10-7 and 10-8 were plated in 100 µl amounts. Samples of 2 ml were withdrawn from the culture vessel through a small-bore tube attached to a sample bottle. After sampling, culture samples were placed on ice for immediate serial dilution and nitrite concentration determination. Nitrite concentrations in culture supernatants were determined by colorimetric change as described by Coleman et al. (1978). Each data point represents the mean of triplicate assays of nitrite concentration and culture turbidity.


   RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Effect of nitrate on growth of R. capsulatus N22DNAR+ in continuous culture, during light–dark cycles
The growth profile of R. capsulatus N22DNAR+ during light–dark cycles was determined under anaerobic heterotrophic growth conditions in continuous culture. At the onset of the experiment, under steady-state photoheterotrophic conditions at D=0·1 h-1 in the absence of nitrate, the maximum culture density was OD660 0·418 (Fig. 1a). The values for c.f.u. ml-1 (Fig. 1b) reflected the OD660 values (Fig. 1a, b). After transition from light to dark, culture washout began within 2 h of dark conditions commencing (Figs 1 and 3). Initial washout kinetics were -20x107 c.f.u. ml-1 h-1 (Fig. 1b, Table 1). Upon illumination, culture density began to increase within 2 h, with an initial growth rate of 14x107 c.f.u. ml-1 h-1 observed. Similar values for OD660, c.f.u. and washout/recovery were gained for all three of the light–dark cycles performed. It was established as a control that there was no nitrite accumulation in the absence of added nitrate (Fig. 1c).



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Fig. 1. Growth profile of R. capsulatus N22DNAR+ and time dependence of nitrite concentration in the absence of nitrate, during repeated light–dark cycling under anaerobic heterotrophic conditions. Open and filled bars above and below plots denote periods of light and dark, respectively. Bacterial growth was measured turbidimetrically at 660 nm (a) and serial dilution c.f.u. ml-1 counts made at 10-6 or 10-7 on PY agar plates (b). Nitrite was measured colorimetrically (c).

 


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Fig. 3. Response of the growth profile of R. capsulatus N22DNAR+ to nitrate under dark conditions. The data were taken from each of the three light–dark cycles shown in Figs 1(a) and 2(a). Panels (a), (b) and (c) show c.f.u. counts in the absence (+) and presence of 25 mM nitrate ({blacksquare}) during the first, second and third light–dark transitions, respectively. Broken lines indicate the theoretically predicted washout profiles for non-growing cultures in the presence (dashed lines) and absence (dotted lines) of nitrate, derived from application of equation 4 (see Discussion).

 

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Table 1. Initial rates of culture decrease for R. capsulatus N22DNAR+ and N22 during periods of darkness which were preceded and separated by periods of illumination

Rates were calculated for the 6 h after the onset of dark conditions. The decreases detailed occurred under chemoheterotrophic growth conditions in malate-limited continuous culture at D=0·1. Input nitrate concentrations are shown. Bold type denotes the reported strain in the mixed-strain culture.

 
In order to determine the effect of nitrate reduction on the growth profile of this nitrate-respiring strain, the above experiment was repeated with 25 mM nitrate added to the medium. The presence of nitrate did not affect the steady-state maximum culture density, with the OD660 values of <=0·4 observed being similar to those observed in the absence of nitrate. Comparison of the washout and growth kinetics revealed that they were similar for all three light–dark cycles (Fig. 2a, b). Furthermore the data show that nitrate respiration slowed the initial rate of washout (Fig. 2a, b and Fig. 3). A mean initial washout rate of -4x107 c.f.u. ml-1 h-1 was observed over the three light–dark cycles for the nitrate-respiring culture. This compares to a mean initial washout rate of -19x107 c.f.u. ml-1 h-1 for N22DNAR+ in the absence of nitrate (see Table 1). The mean growth rate observed was 17x107 c.f.u. ml-1 h-1 (Fig. 2a). As before, the c.f.u. ml-1 values (Fig. 2b) reflected the OD660 values (Fig. 2a), although in some cases OD660 values declined before c.f.u. ml-1 values decreased during culture washout. This suggests that cell size was decreasing with concomitant attenuation of the turbidity of the culture whilst the number of viable cells in solution remained higher. For this reason c.f.u. ml-1 values for initial washout kinetics were used for comparison between strains and conditions in the present study. In the presence of nitrate, nitrite accumulated to 1500±150 µM during photoheterotrophic growth (Fig. 2c). These values fluctuated during light–dark cycles. Decreases in nitrite accumulation coincided with culture washout under dark conditions. Consideration of nitrite production per c.f.u. revealed that during re-illumination after a dark period, the rate of nitrite production was approximately 2·5x10-7 µmol c.f.u.-1. The rate of nitrite production per c.f.u. increased twofold, to 5x10-7 µmol c.f.u.-1, as the culture density increased to OD660>0·15. The higher rate of nitrite production, 5x10-7 µmol c.f.u.-1, was observed throughout the remainder of the growth cycle. The lower rate of nitrite production is attributable to the previously observed inhibition of nitrate reduction by light in this organism (McEwan et al., 1982). This light-inhibition is more pronounced at low culture densities upon re-illumination as a consequence of less light-shading in a dilute culture.



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Fig. 2. Growth profile of R. capsulatus N22DNAR+ and time dependence of nitrite concentration in the presence of 25 mM nitrate, during repeated light–dark cycling under anaerobic heterotrophic conditions. Open and filled bars above and below plots denote periods of light and dark, respectively. Bacterial growth was measured turbidimetrically at 660 nm (a) and serial dilution c.f.u. ml-1 counts made at 10-6 or 10-7 on PY agar plates (b). Nitrite was measured colorimetrically (c).

 
Fig. 3 shows a comparison of the culture washout profiles of N22DNAR+ in the presence and absence of nitrate during each of the dark periods shown in Figs 1 and 2. Initial rates of culture washout, as measured by c.f.u. counts, are lower in the presence of nitrate. In all three periods of darkness the presence of nitrate slows the rate of culture washout for 4–6 h. This initial rate of culture washout is lower than the theoretically expected rate of culture washout for a non-growing culture (calculated from equation 4, see Discussion), as well as the initial rate for N22DNAR+ in the absence of nitrate (Fig. 3). At the end of a period of darkness viable cell counts are equivalent to theoretically predicted values in the presence of nitrate. Furthermore the data showed viable cell counts to be higher in the presence of nitrate than in its absence (Fig. 3). This suggests that nitrate reduction has a role in cell survival during dark, chemoheterotrophic, conditions.

The growth characteristics of R. capsulatus N22, a non-nitrate-respiring strain
Having established the characteristics of R. capsulatus N22DNAR+ in response to nitrate during light–dark transitions in continuous culture, a further experiment was carried out with a non-nitrate-respiring strain, R. capsulatus N22, in the presence of nitrate. The N22DNAR+ strain was a spontaneous gain-of-function mutant, which was obtained following prolonged culture of N22 under photoheterotrophic conditions in the presence of nitrate (McEwan et al., 1982). Much shorter culture times were used in the present work so that a similar mutation would not occur; to ensure that N22 cells could be distinguished from N22DNAR+ a rifampicin-resistant strain of the former was isolated. The trends for R. capsulatus N22 in the presence of nitrate (Fig. 4) were similar to those seen for R. capsulatus N22DNAR+ with no nitrate (Fig. 1). No more than 8 µM nitrite accumulated in the culture (Fig. 4c). That no significant nitrate reduction occurred indicated that the N22 nitrate-reductase-negative phenotype was stable for the duration of the experiment. OD660 and c.f.u. ml-1 counts showed similar trends during culture, indicating that both reflect cell growth in the culture and that rifampicin resistance was stable in R. capsulatus N22 for the duration of the experiment. Growth and washout kinetics from c.f.u. ml-1 counts gave a mean growth rate of 16x107 c.f.u. ml-1 h-1, and a mean initial washout rate of -22x107 c.f.u. ml-1 h-1. These values are similar to the averaged rates seen previously in the N22DNAR+ culture with no added nitrate: 14x107 c.f.u. ml-1 h-1 for growth and -19x107 c.f.u. ml-1 h-1 for initial washout (Table 1). In addition at the end of a period of darkness the c.f.u. count values reflected the values gained for N22DNAR+ cultures in the absence of nitrate. These levels were lower than both the theoretically expected values and those observed for N22DNAR+ cultures in the presence of nitrate.



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Fig. 4. Growth profile of R. capsulatus N22 and time dependence of nitrite concentration during light–dark cycles under anaerobic heterotrophic conditions, in the presence of 25 mM nitrate. Open and filled bars above and below plots denote periods of light and dark, respectively. Bacterial growth was measured turbidimetrically at 660 nm (a) and serial dilution c.f.u. ml-1 counts made at 10-6 or 10-7 on PYrif agar plates (b). Nitrite was measured colorimetrically (c).

 
Nitrate reduction confers a selective advantage on R. capsulatus N22DNAR+ over N22, during light–dark cycling of a mixed-strain culture, in the presence of nitrate
Upon consideration of the differing rates of initial washout and viable cell counts in the latter stages of washout, the question was raised as to whether nitrate respiration could confer any selectional advantage during light–dark cycles. In order to investigate this, the two strains, R. capsulatus N22DNAR+ and R. capsulatus N22, were co-cultured under nitrate-respiring photoheterotrophic conditions, with malate as the carbon source. Having previously established the stability of the N22 phenotype and rifampicin resistance over the time period needed for this experiment, PY and PYrif platings were used to determine the c.f.u. counts for the two strains. Light–dark cycles were applied as for previous experiments.

With both strains co-existing at steady state, c.f.u. ml-1 values of approximately 150x107 were observed. The total OD660 values fluctuated in a manner similar to the previous experiments, with similar maximum OD660 values of 0·4±0·050 observed. The rate of culture washout during the first light-to-dark transition, as measured by OD660, was found to lie between the rate for washout for R. capsulatus N22, and that for R. capsulatus N22DNAR+ with nitrate (compare Fig. 5a with Figs 2a and 4a). When c.f.u. ml-1 values (Fig. 5b) were used for calculation of initial washout rates in the first period of darkness, the initial rate for R. capsulatus N22DNAR+ was -4x107 c.f.u. ml-1 h-1. The rate for R. capsulatus N22 washout was higher at -14x107 c.f.u. ml-1 h-1. The initial rate of culture washout for N22DNAR+ was lower than the theoretical expectation for a non-growing culture washing out of the chemostat vessel. For strain N22 the rate was higher than the theoretical rate in the latter stages of culture washout. Before re-illumination the c.f.u. count for N22DNAR+ was 32x107 c.f.u. ml-1, and was double that of N22 at 15x107 c.f.u. ml-1. The amount of viable N22DNAR+ cells was close to the theoretically expected value, whilst the value for N22 was significantly lower. Upon illumination of the culture R. capsulatus N22DNAR+ fully recovered (Fig. 5b). R. capsulatus N22 did not fully recover: c.f.u. ml-1 counts reached approximately half those seen for R. capsulatus N22DNAR+ (Fig. 5b). During the second period of darkness the rate of washout for R. capsulatus N22DNAR+ was -4x107 c.f.u. ml-1 h-1. This value is the same as seen for a single-strain culture of R. capsulatus N22DNAR+ in the presence of nitrate (Compare Figs 2b and 5b). Following the poor recovery made by the non-nitrate-respiring strain R. capsulatus N22 the rate of washout was slow, at -5x107 c.f.u. ml-1 h-1 during the second dark period. This slower rate can be attributed to the lower starting number of viable cells. During this washout period the c.f.u. ml-1 values for the non-nitrate-respiring R. capsulatus N22 strain fell to approximately 1x107 c.f.u. ml-1, whilst values for R. capsulatus N22DNAR+ fell no lower than 30x107 c.f.u. ml-1. During the third cycle R. capsulatus N22DNAR+ continued the same pattern of recovery and washout whilst R. capsulatus N22 remained at ca. 1x107 c.f.u. ml-1 (Fig. 5b).



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Fig. 5. Growth profiles of R. capsulatus N22DNAR+ and R. capsulatus N22 and time dependence of nitrite concentration in a mixed-strain culture during repeated light–dark cycling under anaerobic conditions in the presence of 25 mM nitrate. Open and filled bars above and below plots denote periods of light and dark, respectively. Bacterial growth was measured turbidimetrically at 660 nm (a). Serial dilution c.f.u. ml-1 counts of R. capsulatus N22DNAR+ ({blacksquare}) and R. capsulatus N22 (+) were made at 10-6 or 10-7 on PY and PYrif agar plates (b). Nitrite was measured colorimetrically (c).

 
Nitrite accumulation (Fig. 5c) during mixed-strain culture mirrored the pattern seen with R. capsulatus N22DNAR+ in the presence of nitrate (compare Figs 2c and 5c). As seen previously, after re-illumination of the culture, nitrite accumulation did not occur until culture density reached OD660>0·1, indicating that nitrate respiration was low until that point. In recovered cultures the nitrite concentrations reached 1500±150 µM. The amount of nitrite accumulated per c.f.u. reached a maximum during the initial washout phase, due to nitrite concentrations remaining high whilst culture density dropped. This suggests that increased rates of nitrate reduction were occurring during the early stage of washout.


   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
This paper reports a study of nitrate respiration by R. capsulatus N22DNAR+ in continuous cultures exposed to alternate cycles of light and dark conditions. The results demonstrate that nitrate reduction to nitrite, catalysed by the periplasmic nitrate reductase, takes place if nitrate is present in the medium. Comparison of steady-state biomass under photoheterotrophic conditions revealed that the presence or absence of nitrate did not significantly affect culture yield. Consideration of the reduction of nitrate linked to the oxidation of malate reveals that a small fraction of the malate flux into the vessel is oxidized. Given that the steady-state reduction rate of nitrate is approximately 150 µmol l-1 h-1 (calculated from Fig. 2) and that 1 mol of malate could in principle provide reduction for 6 mol of nitrate:

the rate of nitrate reduction accounts for only 25 µmol malate l-1 h-1. This is a small fraction of the total 1 mmol l-1 h-1 flux of malate into the culture vessel. Whilst this low rate of nitrate reduction did not appear to affect the steady-state biomass during photoheterotrophic growth it was sufficient to influence the transition to anaerobic dark culture conditions. Under such conditions, if growth ceases entirely the change in biomass (x) as a function of time would be given by:

where µ and D are the specific growth rate and dilution rate, respectively. If µ=0 and D=0·1 then

Thus in the case of N22DNAR+ if the steady state c.f.u. ml-1 count during illumination was approximately 220x107 the initial biomass decrease if µ=0 would be 22 c.f.u. ml-1 h-1. This is close to that observed in the absence of nitrate, but higher than that observed when nitrate was present (Table 1). In the case of N22, which cannot reduce nitrate, the initial rate of washout was close to the theoretical rate for µ=0 in both the presence and absence of nitrate. The data also suggest that a slow rate of growth and cell division is maintained for a few hours after the onset of dark conditions when nitrate respiration is possible: using the steady-state c.f.u. ml-1 count of 220x107 c.f.u. and the initial rate of decrease, -6x107 c.f.u. ml-1 from Table 1, in equation 3 (above) gives µ=0·082 h-1. Thus, for a transient period the periplasmic nitrate reductase can provide the cell with a means to sustain a low rate of growth. Consideration of the theoretical washout of the culture as a function of time, determined from the equation

where x is the biomass at time t and x0 is the initial biomass at time t=0 reveals that R. capsulatus N22DNAR+ reducing nitrate can transiently sustain itself at levels above those expected during culture washout and thus maintain cell viability for longer (Fig. 3). This is evidenced in this study from viable cell counts in the latter stages of a dark period. Previously it has been proposed that the physiological advantage of nitrate reduction in R. capsulatus may be gained during photoheterotrophic growth at low light intensities (Jones et al., 1990). Additionally, under anaerobic dark conditions the extra oxidant, nitrate, may supply an exogenous terminal electron acceptor for disposal of reductant under energy-limiting conditions. This would dispose of reducing equivalents via an electron-transport pathway that can conserve energy at the NADH–ubiquinone oxidoreductase step rather than energy consuming CO2 fixation (Jones et al., 1990; Berks et al., 1995a). Thus electron flux to Nap may provide the cell with a method both of maintaining redox poise and of generating a transmembrane electrochemical gradient when the UQ/UQH2 pool is reduced. This would maximize the conversion of the available reducing power to ATP, or other endergonic processes, during dark conditions via the anaerobic respiration of nitrate. It follows that N22DNAR+ cells would be better placed to survive longer or even recover faster to grow under more energy-sufficient conditions, provided that nitrate is present.

Nitrate reduction during light–dark transitions could be important in many environments which Rhodobacter species occupy. This additional role for Nap in R. capsulatus can be viewed in the context that the role of Nap varies amongst different bacteria. Recent evidence in E. coli has suggested that the Nap system can utilize its various components for interacting with different physiological substrates (Brondijk et al., 2002). Thus, it seems likely that due to the versatility of the Nap system the role of the enzyme could not only vary between organisms, but could vary in the same organism under different metabolic conditions.


   ACKNOWLEDGEMENTS
 
We thank the Biotechnology and Biological Sciences Research Council for supporting this work through a PMS committee studentship to M. J. K. E.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Alef, K., Jackson, J. B., McEwan, A. G. & Ferguson, S. J. (1984). The activities of two pathways of nitrate respiration in Rhodopseudomonas capsulata. Arch Microbiol 142, 403–408.

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Received 1 November 2002; revised 20 December 2002; accepted 23 December 2002.