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
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ABSTRACT |
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INTRODUCTION |
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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
p (McEwan et al., 1982
, 1983
, 1984
. One reason for this observation could be that nitrate-respiration-dependent
p may be too low to support a detectable growth rate in batch culture, as a threshold dependency of growth rate on
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 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 lightdark 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 lightdark cycling of an anaerobic mixed-strain continuous culture of R. capsulatus.
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METHODS |
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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 45 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 814 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.
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RESULTS |
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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 lightdark 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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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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DISCUSSION |
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Nitrate reduction during lightdark 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.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 1 November 2002;
revised 20 December 2002;
accepted 23 December 2002.