1 Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK
2 Department of Molecular Microbiology, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK
3 Centre for Metalloprotein Spectroscopy and Biology, School of Biological Sciences, University of East Anglia, Norwich Research Park, Norwich NR4 7TJ, UK
Correspondence
D. J. Richardson
d.richardson{at}uea.ac.uk
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ABSTRACT |
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INTRODUCTION |
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In continuous culture, periplasmic nitrate reductase activity in P. denitrificans Pd1222 was found to be high during carbon-limited growth on butyrate as compared to the more oxidized substrate malate. Expression was observed at all oxygen concentrations tested between 0 and 100 % air saturation (Sears et al., 1997). Nap activity in batch cultures of P. pantotrophus and P. denitrificans Pd1222 has been found to be comparable. In response to aerobic growth on the reduced carbon substrate butyrate, Nap activities increase by at least an order of magnitude, compared to levels seen with relatively oxidized substrates such as malate or succinate (Richardson & Ferguson, 1992
; Sears et al., 1993
, 1995
). Further studies in P. pantotrophus have shown transcription to occur differentially from two transcriptional initiation start sites upstream of napE in response to carbon substrate and oxygen (Sears et al., 2000
). The upstream site, P1, is utilized at a low level during aerobic growth on succinate, whilst the downstream site, P2, is utilized more during aerobic growth on butyrate. During anaerobic growth both P1 and P2 are inactive. Quantification of nap expression in batch cultures of P. pantotrophus (pnaplacZ) has demonstrated expression to be positively responsive to oxygen and to be tightly regulated in response to the carbon substrate. Correlation between Nap activity and nap expression has demonstrated transcription from the nap promoter to be the major point of regulation for cellular Nap activity (Richardson & Ferguson, 1992
; Ellington et al., 2002
). In these batch culture experiments transcription appeared to be independent of growth phase, since activity reached a maximum during the exponential phase of growth and did not increase during the deceleration or stationary phases of growth (Ellington et al., 2002
). However, there was an inverse relationship between the maximum specific growth rate (µmax) during exponential phase and nap expression. Thus µmax decreased in the order succinate > acetate > butyrate, whilst levels of nap expression increased in the same order, succinate < acetate < butyrate. In light of this, the present study sought to assess the effect of growth rate (µ) on nap expression independently of batch culture growth phase. The effect of the dilution rate (D; which is equivalent to µ under steady-state conditions) on nap expression and Nap activity in carbon-limited chemostat cultures is reported.
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METHODS |
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The culture was allowed at least 4 vessel-volume changes before being judged to be at steady state. To check for carbon limitation, culture supernatants were analysed using a Dionex anion-exchange HPLC chromatography system, whereby known concentration standards were used to quantitate substrate concentration. Elution profiles revealed each carbon substrate to be undetectable. Moreover, at the end of operation of each chemostat an addition of 10 mM carbon substrate was made, and cultures were seen rapidly to increase their biomass to double or more of that seen previously under carbon-limited conditions.
Analytical procedures.
Culture samples of 5, 10, 20 or 50 ml were withdrawn through a small-bore tube attached to a sample bottle. Culture samples of 5 ml were placed on ice for immediate determination of nitrite concentration and -galactosidase activity. Cells from 2x10 ml, 2x20 ml and 50 ml samples were harvested by centrifugation at 10 000 g. The 50 ml sample was resuspended in 500 µl 50 mM phosphate buffer pH 7·0, dried overnight at 80 °C and weighed for biomass determination. Cell pellets and supernatants from the two 20 ml samples were frozen separately at -80 °C. Cell pellets were used to prepare DNA and RNA for relative plasmid copy number and primer extension analysis, respectively. Supernatants from these samples were used for HPLC determination of the residual carbon source and nitrate concentrations in the reservoir medium. The 10 ml samples were utilized for protein determination and methylviologen assays.
NOx assays.
Residual nitrate concentration was measured using a Dionex anion-exchange HPLC system with a UV detector, whereby a standard of known concentration was used to facilitate the quantification of nitrate concentration. Nitrite concentrations were determined spectrophotometrically by a colorimetric change assay (Coleman et al., 1978). From assays of nitrite concentration in the culture vessel at steady state (above) nitrite accumulation rates were calculated and determined as a function of biomass at steady state.
Enzyme activity.
Periplasmic nitrate reductase activity was determined spectrophotometrically using reduced methylviologen (MV+) as the electron donor (Sears et al., 1993). Absorption spectra were collected using a Varian spectrophotometer. The difference between starting and steady-state concentrations of nitrate was used to calculate the steady-state rates for nitrate reduction, which were given as a function of the steady-state biomass of the culture.
Transcription analysis.
-Galactosidase activity from pnaplacZ (Ellington et al., 2002
) was determined spectrophotometrically according to Miller (1972)
using 0·1 ml of culture sample. RNA samples isolated from 20 ml cell culture using an RNeasy RNA isolation kit (Qiagen) were used for primer extension analysis. This was carried out exactly as described previously (Sears et al., 2000
).
Protein determination.
A cell pellet from 10 ml of cell culture was used for total cell protein determination (see above). The cell pellet was resuspended in 0·5 ml 0·7 M NaOH and heated in a boiling water bath for 10 min. After cooling for 5 min, 50 µl aliquots were added to 1·95 ml 0·1 M phosphate buffer pH 7·0, and mixed with 1 ml 0·25 % CuSO4 in 10 M NaOH. A Varian spectrophotometer was used to determine the A290 after a 10 min incubation at 25 °C and protein determination was made against a BSA standard curve.
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RESULTS |
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Biomass increased proportionally to D, regardless of the carbon substrate. In succinate-limited and acetate-limited cultures dry weight increased by approximately twofold over the D range 0·0360·500 h-1 (Fig. 1a, b). In butyrate-limited cultures the same trend was observed, though with higher absolute values, over the D range 0·0360·100 h-1 (Fig. 1c
). Protein concentrations also increased with the dilution rate, regardless of the growth-limiting carbon substrate (Fig. 1a
c). Conversely, relating the total protein content to the biomass of the culture (percentage of biomass as protein) reveals an inverse relationship to the dilution rate (Fig. 1a
c). In succinate-limited and acetate-limited cultures of P. pantotrophus (pnaplacZ) the protein as a percentage of dry weight decreased by 10 % between D=0·036 h-1 and 0·500 h-1 (Fig. 1a, b
). In butyrate-limited cultures a decrease of 25 % was observed between D=0·036 h-1 and 0·100 h-1 (Fig. 1c
). An inverse relationship between the percentage of biomass as protein and D is a commonly observed feature in carbon-limited bacterial chemostats (Harder & Dijkhuzen, 1982
).
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Expression from the nap promoter responded to the D and the carbon substrate in a similar pattern to that observed for Nap activity in P. pantotrophus (pnaplacZ) (Fig. 3ac). Expression maxima occurred at intermediate D on each carbon substrate such that the maximal
-galactosidase activities for each carbon-limited culture were comparable. The largest D-dependent variation in
-galactosidase activity was a fivefold difference, which occurred in response to D during succinate-limited growth.
Whilst the above data indicate that D and carbon substrate can affect Nap regulation independently, the largest variation in nap expression resulted from the combined effects of D and carbon substrate. For example, a sixfold difference is apparent between succinate-limited cultures at high D and butyrate-limited cultures at intermediate D (Fig. 3). This suggests that nap regulation is affected by a physiological signal which integrates aspects of growth rate and carbon substrate utilization.
The -galactosidase activity data suggested that D affected transcription from the nap promoter independently of the carbon substrate. This effect was very marked in succinate-limited cultures and is in clear contrast to the situation in batch cultures utilizing succinate, where transcription levels are comparatively low throughout all the growth phases (Ellington et al., 2002
). To study this phenomenon in more detail in the succinate-limited chemostat cultures, primer extension analysis was undertaken. Previous work has established the differential use of two transcription initiation sites in batch culture in response to the carbon substrate. The upstream site P1 is used weakly during growth on succinate, when low enzyme and promoter activity occur, whilst the downstream P2 site is used more during growth conditions where high levels of enzyme and promoter activity occur (Sears et al., 2000
; Ellington et al., 2002
). Primer extension analysis of total RNA was used to investigate if transcription start site utilization altered in response to the dilution rate in aerobic succinate-limited continuous cultures. The technique was also used to compare the pattern of transcription initiation in continuous culture with that seen in aerobic batch cultures with succinate or butyrate as the carbon substrate. This comparison indicates that the two transcriptional initiation start sites, P1 and P2, previously identified under batch culture conditions, are also utilized under the continuous culture conditions used in this study. During aerobic succinate-limited continuous culture, transcription initiated relatively weakly from only the P1 site at the lowest and highest dilution rates, 0·036 h-1 and 0·500 h-1, respectively. At the intermediate dilution rate 0·133 h-1, where Nap expression was highest, transcription was also initiated from the P2 site (Fig. 4
). This indicates that the differential utilization of the transcription start sites, described previously in response to carbon substrate (Sears et al., 2000
), can also occur in response to the dilution rate and independently of carbon substrate.
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DISCUSSION |
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The analysis of cellular carbon partitioning in P. pantotrophus revealed that a greater rate of carbon consumption for cellular maintenance occurred during growth on butyrate than during growth on succinate. The highest maintenance rate was observed during growth on acetate. This is unsurprising, as acetate is a two-carbon substrate which must be metabolized via the glyoxylate cycle or bypass, requiring an input of four carbons, or two molecules, to complete a cycle. In contrast to this, butyrate is a four-carbon substrate. However, whilst it must also be metabolized via the glyoxylate cycle, a cycle can be completed with one molecule. If the two-carbon nature of acetate is accounted for and the maintenance requirement halved from 506 µmol mg-1 h-1 to 253 µmol mg-1 h-1 then butyrate has the highest maintenance requirement. The observed high culture maintenance rate and slow growth rate observed in batch (Ellington et al., 2002) and continuous culture indicates the challenge that the metabolism of butyrate for growth poses to the cell.
The results show that aerobic nitrate reduction occurs under all the carbon-limited continuous culture conditions tested. Furthermore, nitrite accumulation did not match nitrate consumption, indicating that, as observed previously (Sears et al., 1997), nitrite reduction had also occurred. The proportion of succinate oxidation linked to the reduction of nitrate under aerobic conditions at the intermediate dilution rate D=0·133 h-1 is small. The steady-state reduction rate of nitrate is 21 µmol l-1 h-1 (calculated from Figs 1 and 2
). From equation 1 it is apparent that complete oxidation of 1 mol succinate could provide reduction for 7 mol nitrate:
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Therefore, a nitrate reduction rate of 21 µmol l-1 h-1 accounts for only 3 µmol succinate l-1 h-1. This is a small fraction of the carbon substrate influx given that the rate of succinate flux into the culture vessel is 665 µmol l-1 h-1. At the intermediate D=0·056 h-1 during butyrate-limited growth, the proportion of butyrate oxidation linked to nitrate reduction can reach higher levels. Equation 2 shows that complete oxidation of 1 mol of butyrate can provide reduction for 10 mol nitrate:
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During butyrate-limited growth the highest rate of nitrate reduction was 204 µmol butyrate l-1 h-1 (calculated from Figs 1 and 2). From equation 2 this can account for the oxidation of 20·4 µmol l-1 h-1. This figure is around 7 % of the 280 µmol l-1 h-1 influx of butyrate at D=0·056 h-1. The large difference in the proportions of carbon substrate being oxidized by nitrate reduction indicates the importance of nitrate as an auxiliary oxidant during growth on butyrate.
Detailed molecular studies of the regulation of Nap systems have been undertaken in Rhodobacter sp. and Escherichia coli (Darwin et al., 1998; Dobao et al., 1994
; Gavira et al., 2002
; Liu et al., 1999
; Reyes et al., 1998
; Stewart et al., 2002
; Wang et al., 1999
). A notable feature of Nap systems is the variation, both within and between organisms, in the physiological functions, as well as the regulation and expression, of nap. For example, Nap plays a role in anaerobic denitrification in Pseudomonas G-179 and Rhodobacter sphaeroides f. sp. denitrificans, in anaerobic nitrate respiration under nitrate-limited conditions in E. coli, and redox-poising of the photosynthetic electron-transport chain or survival during lightdark transitions in Rhodobacter capsulatus (Jones et al., 1990
; Siddiqui et al., 1993
; Castillo et al., 1996
; Zumft, 1997
; Liu et al., 1999
; Moreno-Vivian et al., 1999
; Philippot & Hojberg, 1999
; Potter et al., 1999
, 2001
; Richardson et al., 2001
; Brondijk et al., 2002
; Ellington et al., 2003
). Previously, the importance of transcriptional regulation for the regulation of Nap activity in P. pantotrophus has been established in batch culture (Ellington et al., 2002
). The evidence presented in this study supports the assertion that transcriptional regulation is the major point of differential regulation for the P. pantotrophus Nap system. Previously, growth on succinate has shown low levels of transcription from the nap promoter under all conditions tested. Furthermore, differential transcriptional start site usage and nap promoter activity has been found only in response to differing carbon substrates (Sears et al., 2000
; Ellington et al., 2002
). A new feature of nap transcriptional regulation highlighted in this study is differential transcriptional start site usage and nap promoter activity in response to the growth rate.
Under the carbon-limited conditions in this study, levels of expression did not fall to those seen in batch culture on any carbon substrate. This suggests that during carbon-limited continuous culture the signal affecting nap transcription is more abundant than during batch culture with the same substrates. Together with the effect of growth rate on Nap activity and transcription in carbon-limited cultures this observation reinforces the suggestion that nap regulation is affected by a signal originating from the metabolism of a substrate. It also suggests that the abundance of this signal can be altered by the growth rate of P. pantotrophus in carbon-limited, continuously growing cultures.
The increased level of transcription and enzyme activity at intermediate growth rates described in this study is a common response of protein synthesis and enzyme activity to growth rate (Harder & Dijkhuizen, 1982). In the case of Nap this phenomenon may be a result of differential reductant accumulation in response to the growth rate of the culture. During carbon-limited growth at low growth rates only a relatively small amount of carbon is available for growth. This could translate into relatively small amounts of reductant accumulating. Likewise, at high dilution rates reductant may not accumulate as readily due to a shift in the equilibrium between oxidized and reduced reducing equivalents as the biosynthesis of new cellular material acts as a sink for excess reductant. However, at intermediate dilution rates sufficient carbon may be available to generate a quantity of excess reducing equivalents. At intermediate growth rates this pool of excess reducing equivalents would not be utilized in biosynthetic reactions.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 31 January 2003;
revised 17 March 2003;
accepted 18 March 2003.
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