1 Department of Biochemistry, Faculty of Science, Masaryk University, Kotláská 2, CZ-61137 Brno, Czech Republic
2 Department of Molecular Cell Physiology, Faculty of Biology, BioCentrum Amsterdam, Vrije Universiteit, NL-1081 HV Amsterdam, The Netherlands
Correspondence
Igor Kuera
ikucera{at}chemi.muni.cz
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
As yet we do not know exactly how the biological activity of the FNR-like proteins of P. denitrificans is modulated. The [4Fe4S] cluster of the E. coli FNR protein exhibits great sensitivity to molecular oxygen, which rapidly converts it to the [2Fe2S] form in a direct reaction; this is accompanied by dissociation of the FNR dimer into monomers with a loss of the ability to bind DNA (Khoroshilova et al. 1995, 1997
; Green et al., 1996
; Jordan et al., 1997
). An analogous FeS cluster could be reconstituted into the overexpressed FnrP in vitro, confirming that this protein is a true orthologue of FNR from E. coli with a similar mechanism of oxygen sensing (Hutchings et al., 2002
). By studying the
-galactosidase activity in P. denitrificans strains with the nirS and norC promoters fused to the 'lacZ reporter gene, NNR-dependent transcription was shown to be controlled by NO or a related species (van Spanning et al., 1999
), analogous to earlier findings in Rhodobacter sphaeroides (Tosques et al., 1996
; Kwiatkowski et al., 1997
). Primer extension analysis of the nor promoter region revealed the presence of two transcription start sites. A transcript that initiated downstream of the putative NNR-binding site was identified as the major contributor to NNR-dependent anaerobic nor expression. Besides this, a weak transcription from an upstream start site still occurred both aerobically and anaerobically (Hutchings & Spiro, 2000
).
An understanding of the regulatory network in P. denitrificans was initially developed, based upon the use of an artificial FF-melR class II promoter, in which the consensus FNR-binding (FF) site is centred at -41·5 with respect to the transcription start site (Lodge et al., 1990). This promoter could be activated in P. denitrificans during the switch from oxic to anoxic growth conditions (Spiro, 1992
), provided that the culture contained a sufficient amount of iron (Ku
era & Mat'chová, 1997
). An alternative mode of FF-melR activation upon the addition of respiratory inhibitors to highly aerated cultures forced us to consider the role of changes in the intracellular redox state in transcriptional regulation besides the direct interaction of oxygen with the [4Fe4S] cluster(s) (Ku
era et al., 1994
). Here we decided to broaden the studies using FF-melR to include fnrP and nnr mutant strains and a range of culture conditions, i.e. using various terminal electron acceptors. In this way, we attempted to answer two questions. (a) Which of the FNR-like proteins present in P. denitrificans cells activates FF-melR? (b) Are there factors other than oxygen concentration that modulate the extent of this activation?
The second objective of the present study concerns the true role of nitrogenous electron acceptors in the expression of denitrification enzymes. The newly proposed essential activation of NNR by NO has to be reconciled with previous studies showing that oxygen limitation alone was the dominant regulatory factor (Kuera et al., 1984
; Boublíková et al., 1985
). A possible trivial explanation for the previous results could be the presence of traces of nitrate in the culture media used. To check this possibility, we followed the formation of NOR in cells under carefully controlled growth conditions, minimizing the formation of endogenous NO.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Consideration of the amount of plasmid DNA in cells.
Because a plasmid system was used to study FnrP-dependent transcription, we considered the possibility that the various levels of -galactosidase would emerge as a function of the varying plasmid copy number. Therefore, the amount of plasmid DNA per unit of total cellular protein was estimated for several culture samples and correlated with the corresponding specific activity of
-galactosidase. Cells in 2550 ml cultures were pelleted and subjected to the boiling miniprep procedure (Ausubel et al., 1995
). Electrophoresis of the thus obtained plasmid DNA samples was performed in a horizontal 0·5 % agarose gel (10x8x0·5 cm) at 70 V for 2 h. The photographs of the ethidium-bromide-stained gels were scanned using a Personal Densitometer SI and evaluated by the ImageQuant Version 1.2 program (Molecular Dynamics). The absence of positive correlation indicated that the extent of
-galactosidase overproduction was related to an increased transcription efficiency rather than uncontrolled plasmid copy number variations.
Enzyme assays.
NAR activity was measured spectrophotometrically by following the oxidation of reduced methyl viologen coupled to the reduction of 10 mM nitrate to nitrite in cells made permeable by including 0·1 % Triton X-100 in the reaction mixture (Kuera & Kaplan, 1996
). NIR activity was measured by determining the amount of nitrite consumed by a suspension of intact cells in the presence of 20 mM succinate as electron donor (Ku
era et al., 1990
). NOR activity was measured amperometrically using a Clark-type electrode sensitive to NO. The reaction mixture contained 5 mM ascorbate in conjunction with 0·2 mM N,N,N',N'-tetramethylphenylene-1,4-diamine as electron donor and redox mediator, respectively; the initial concentration of NO being 50 µM (Ku
era, 1992
). When the sensitivity to antimycin was examined, the artificial electron donor was replaced by 7 mM succinate.
-Galactosidase activity was measured at 30 °C in toluene-permeabilized cells by monitoring the hydrolysis of ONPG (Miller, 1972
). Activities were expressed in terms of cell density (OD600) by using a modified formula of Miller (1972)
: Miller units=1000x(A420-1·57xA550)/(A600xVxt), in which A
denotes an absorbance at a wavelength
(nm), V is the volume of the added cell suspension (ml) and t is the reaction time (min). The activity data presented are means±SEM (n
3).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
Inhibition of FF-lacZ expression by nitroprusside
In heterologous E. coli systems, the NNR- and FnrP-dependent anoxic transcriptions from the FF-melR promoter were reported to be activated and inhibited, respectively, by sodium nitroprusside (SNP) as a source of NO+ (Hutchings et al., 2000, 2002
). These findings prompted us to investigate which type of response would occur in P. denitrificans. Washed oxically grown cells of P. denitrificans PD1222(pRW2A/FF) were resuspended in succinate growth medium without nitrate at 1·2 mg dry wt ml-1 and incubated with various concentrations of SNP under limited aeration for 1 h. If we regarded the
-galactosidase activity increments in control samples without SNP (153 Miller units) as 100 %, for 0·001, 0·01, 0·1 and 1 mM SNP the respective relative increments (means±SEM, n=3) were 99±4, 83±3, 40±3 and 13±1 %. These results show that SNP significantly inhibits the increase of
-galactosidase production under oxygen-limiting conditions, indicating a FnrP-type response.
NOR synthesis in response to oxygen withdrawal and to the availability of nitrogenous compounds
As mentioned in the Introduction, the synthesis of denitrification enzymes observed under micro-oxic conditions could be due to the reduction of trace amounts of nitrate by bacteria, thereby producing the regulatory NO signal. This possibility was minimized in three ways. First, the growth media were pre-incubated with the washed anoxically grown cells to ensure that nitrate concentration was decreased to submicromolar levels where it could not be metabolized easily. Second, we employed a mutant strain devoid of the NO-forming NIR. Third, haemoglobin was included in some experiments as an NO trap. The results obtained using this combined approach are exemplified by Figs 2 and 3. Comparison of panels (a) and (b) in Fig. 2
indicates that a strain containing an NIR mutation behaved like the wild-type strain in its ability to express NOR in response to oxygen limitation without nitrogenous terminal acceptors. The presence of SNP, an NO-generating compound, stimulated a further NOR increase in both strains, at least after 1·5 h treatment, while nitrite was only effective in the wild-type strain. The absence of FnrP influenced neither low-oxygen nor NO response significantly (Fig. 2c
). On the contrary, we found no NOR activity in the NNR-deficient strain (results not shown). Haemoglobin (10 µM) reversed the effects of exogenous NO, but not the effect of hypoxia (Fig. 3
). We also asked whether the observed NOR activity does indeed reflect the presence of the respiratory-chain-linked NOR. This could be unequivocally proven by the finding that with succinate as electron donor, the respiratory inhibitor antimycin at a concentration of 3 µg (mg dry wt)-1 reduced the rate of NO consumption to a value below 5 %. Taken together, these results support the idea that both NO-independent and NO-dependent signals are required for the full expression of NOR.
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Although subject to the possible limitations of our plasmid system, the current data argue for the existence of additional signals besides oxygen concentration, capable of modulating FnrP function. One of the factors affecting the activity of FnrP under anoxic conditions may be NO, one of the free intermediates in denitrification. This role of NO is supported by the recently demonstrated sensitivity of the [4Fe4S] cluster to low concentrations of NO (Wu et al., 2000; Cruz-Ramos et al., 2002
), by the observed inactivation of FnrP in vivo by artificial NO donors (Hutchings et al., 2002
; this work), by the retarding effect of nitrate on the transcriptional activation of the FF site under microaerobiosis (Fig. 1
) and especially by the notable difference between reporter enzyme activities in cells grown with nitrate or nitrite and in cells grown with N2O (Table 3
), which possibly arises from the ability of the former electron acceptors to produce NO. Since the nnr mutation causes nitrite accumulation via reduced expression of NIR (van Spanning et al., 1995
) and nitrite in turn inhibits the activity of NOR (Ku
era, 1992
), a negative modulation by NO may also underlie the lower activity of the FF-melR promoter in the nnr mutant compared to the wild-type strain (Table 1
). The mechanism by which nitrogenous terminal acceptors forming NO (nitrate and nitrite) exert their effect can be based on (i) a direct degradation of the [4Fe4S] cluster by NO (Cruz-Ramos et al., 2002
; Hutchings et al., 2002
; Wu et al., 2000
) and/or (ii) activation of other transcriptional regulators, which may compete with FnrP for promoter binding, yet only weakly activate expression. Whatever direct or indirect inactivation of FnrP by NO would adversely affect the first reaction of the denitrification pathway, thus providing a negative feedback mechanism that counters the destabilizing positive feedback effect of NO to enhance the expression of NIR and its own production.
Previous measurements of the appearance of enzyme activities, following adaptation of oxically grown batch cultures to the conditions of restricted aeration, have indicated that oxygen limitation alone is the dominant regulatory factor even if nitrate also has some inducing effect on NAR (Kuera et al., 1984
; Boublíková et al., 1985
). Our new data lend further support to this original view by demonstrating a partial induction of NAR (Fig. 1
) and NOR (Fig. 2
) under micro-oxic conditions. Further induction seen upon the addition of nitrogenous compounds suggests the operation of dual control mechanisms involving separate oxygen and nitrogen oxides sensors. For NAR this is in accord with FnrP and NarR acting in concert to regulate the NAR operon (Wood et al., 2001
), although some uncertainty persists as to the relative roles of both transcriptional factors. For NOR, the involvement of the NNR-mediated NO signal is well documented, while the molecular basis for the NO-independent part of NOR expression remains unclear at present. One possibility is that a metabolic signal other than NO can also activate NNR-dependent transcription. Physiologically, the dual control of denitrification enzymes possibly reflects a strategy of the cell to get ready for denitrification when oxygen tension becomes limiting.
An alternative approach to study the expression of denitrification enzymes is to measure individual mRNA levels. In this way, results at variance with the dual control model were obtained by Baumann et al. (1996), who failed to detect any induction during the oxic-to-anoxic switch when working with a nitrate-free medium. We think that the discrepancy between our results and those of Baumann et al. (1996)
arises essentially from the different concentrations of oxygen in the culture medium used. A number of authors (e.g. Payne et al., 1971
; Ku
era et al., 1984
; Boublíková et al., 1985
; Aida et al., 1986
) have already demonstrated that the anoxic adaptation of strictly respiring (non-fermenting) bacteria requires a residual respiration of O2 to produce the metabolic energy inevitable for biosynthesis of the denitrification pathway. This condition was possibly met for our cultures agitated in open flasks but not for Baumann's chemostat culture sparged with helium. Using an experimental set-up for oxygen limitation similar to ours, Härtig & Zumft (1999)
observed a transient accumulation of denitrification gene transcripts in cultures of Pseudomonas stutzeri subjected to a low oxygen tension in the absence of N oxides. Further work is required to characterize such mRNA changes elicited by an appropriately reduced oxygen supply in P. denitrificans.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Arai, H., Kodama, T. & Igarashi, Y. (1997). Cascade regulation of the two CRP/FNR-related transcriptional regulators (ANR and DNR) and the denitrification enzymes in Pseudomonas aeruginosa. Mol Microbiol 25, 11411148.[Medline]
Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A. & Struhl, K. (1995). Short Protocols in Molecular Biology. New York: Wiley.
Baker, S. C., Ferguson, S. J., Ludwig, B., Page, M. D., Richter, O.-M. H. & van Spanning, R. J. M. (1998). Molecular genetics of the genus Paracoccus: metabolically versatile bacteria with bioenergetic flexibility. Microbiol Mol Biol Rev 62, 10461078.
Baumann, B., Snozzi, M., Zehnder, A. J. B. & van der Meer, J. R. (1996). Dynamics of denitrification activity of Paracoccus denitrificans in continuous culture during aerobic-anaerobic changes. J Bacteriol 178, 43674374.[Abstract]
Boublíková, P., Kuera, I. & Dadák, V. (1985). The effect of oxygen and nitrate on the biosynthesis of denitrification enzymes in Paracoccus denitrificans. Biologia (Bratislava) 40, 357363.
Craske, A. & Ferguson, S. J. (1986). The respiratory nitrate reductase from Paracoccus denitrificans. Molecular characterization and kinetic properties. Eur J Biochem 158, 429436.[Abstract]
Cruz-Ramos, H., Crack, J., Wu, G., Hughes, M. N., Scott, C., Thomson, A. J., Green, J. & Poole, R. K. (2002). NO sensing by FNR: regulation of the Escherichia coli NO-detoxifying flavohaemoglobin, Hmp. EMBO J 21, 32353244.
de Boer, A. P., Reijnders, W. N., Kuenen, J. G., Stouthamer, A. H. & van Spanning, R. J. (1994). Isolation, sequencing and mutational analysis of a gene cluster involved in nitrite reduction in Paracoccus denitrificans. Antonie Van Leeuwenhoek 66, 111127.[Medline]
de Vries, G. E., Harms, N., Hoogendijk, J. & Stouthamer, A. H. (1989). Isolation and characterization of Paracoccus denitrificans mutants with increased conjugation frequencies and pleiotropic loss of a (nGATCn)-DNA-modifying property. Arch Microbiol 152, 5257.
Galimand, M., Gamper, M., Zimmermann, A. & Haas, D. (1991). Positive FNR-like control of anaerobic arginine degradation and nitrate respiration in Pseudomonas aeruginosa. J Bacteriol 173, 15981606.[Medline]
Green, J., Bennett, B., Jordan, P., Ralph, E. T., Thomson, A. J. & Guest, J. R. (1996). Reconstitution of the [4Fe4S] cluster in FNR and demonstration of the aerobicanaerobic transcription switch in vitro. Biochem J 316, 887892.[Medline]
Härtig, E. & Zumft, W. G. (1999). Kinetics of nirS expression (cytochrome cd1 nitrite reductase) in Pseudomonas stutzeri during the transition from aerobic respiration to denitrification: evidence for a denitrification-specific nitrate- and nitrite-responsive regulatory system. J Bacteriol 181, 161166.
Hasegawa, N., Arai, H. & Igarashi, Y. (1998). Activation of a consensus FNR-dependent promoter by DNR of Pseudomonas aeruginosa in response to nitrite. FEMS Microbiol Lett 166, 213217.[CrossRef][Medline]
Hutchings, M. I. & Spiro, S. (2000). The nitric oxide regulated nor promoter of Paracoccus denitrificans. Microbiology 146, 26352641.
Hutchings, M. I., Shearer, N., Wastell, S., van Spanning, R. J. M. & Spiro, S. (2000). Heterologous NNR-mediated nitric oxide signalling in Escherichia coli. J Bacteriol 182, 64346439.
Hutchings, M. I., Crack, J. C., Shearer, N., Thompson, B. J., Thompson, A. J. & Spiro, S. (2002). Transcription factor FnrP from Paracoccus denitrificans contains an ironsulfur cluster and is activated by anoxia: identification of essential cysteine residues. J Bacteriol 184, 503508.
Jordan, P. A., Thomson, A. J., Ralph, E. T., Guest, J. R. & Green, J. (1997). FNR is a direct oxygen sensor having a biphasic response curve. FEBS Lett 416, 349352.[CrossRef][Medline]
Khoroshilova, N., Beinert, H. & Kiley, P. (1995). Association of a polynuclear ironsulfur center with a mutant FNR protein enhances DNA binding. Proc Natl Acad Sci U S A 92, 24992503.[Abstract]
Khoroshilova, N., Popescu, C., Münck, E., Beinert, H. & Kiley, P. (1997). Ironsulfur cluster disassembly in the FNR protein of Escherichia coli by O2: [4Fe4S] to [2Fe2S] conversion with loss of biological activity. Proc Natl Acad Sci U S A 94, 60876092.
Kuera, I. (1992). Oscillations of nitric oxide concentration in the perturbed denitrification pathway of Paracoccus denitrificans. Biochem J 286, 111116.[Medline]
Kuera, I. & Kaplan, P. (1996). A study on the transport and dissimilatory reduction of nitrate in Paracoccus denitrificans using viologen dyes as electron donors. Biochim Biophys Acta 1276, 203209.
Kuera, I. & Mat'chová, I. (1997). Iron as a possible mediator of the oxic-to-anoxic transition in Paracoccus denitrificans. Biochem Mol Biol Int 43, 305310.[Medline]
Kuera, I., Boublíková, P. & Dadák, V. (1984). Function of terminal acceptors in the biosynthesis of denitrification pathway components in Paracoccus denitrificans. Folia Microbiol 29, 108114.
Kuera, I., Mat'chová, I. & Dadák, V. (1990). Respiratory rate as a regulatory factor in the biosynthesis of the denitrification pathway of the bacterium Paracoccus denitrificans. Biocatalysis 4, 2937.
Kuera, I., Mat'chová, I. & Spiro, S. (1994). Respiratory inhibitors activate an FNR-like regulatory protein in Paracoccus denitrificans: implications for the regulation of the denitrification pathway. Biochem Mol Biol Int 32, 245250.[Medline]
Kwiatkowski, A. V., Laratta, W. P., Toffanin, A. & Shapleigh, J. P. (1997). Analysis of the role of the nnR gene product in the response of Rhodobacter sphaeroides 2.4.1 to exogenous nitric oxide. J Bacteriol 179, 56185620.[Abstract]
Lodge, J., Williams, R., Bell, A., Chan, B. & Busby, S. (1990). Comparison of promoter activities in Escherichia coli and Pseudomonas aeruginosa: use of a new broad-host-range promoter-probe plasmid. FEMS Microbiol Lett 67, 221226.[CrossRef]
Miller, J. H. (1972). Experiments in Molecular Genetics. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Nedoma, J. (1983). Determination of nitrates in waters using the salicylate method and chromotropic acid. Chem Listy 77, 638641 (in Czech).
Parsonage, D., Greenfield, A. J. & Ferguson, S. J. (1985). The high affinity of Paracoccus denitrificans cells for nitrate as an electron acceptor. Analysis of possible mechanisms of nitrate and nitrite movement across the plasma membrane and the basis for inhibition by added nitrite of oxidase activity in permeabilised cells. Biochim Biophys Acta 807, 8195.
Payne, W. J., Riley, P. S. & Cox, C. D., Jr (1971). Separate nitrite, nitric oxide, and nitrous oxide reducing fractions from Pseudomonas perfectomarinus. J Bacteriol 106, 356361.[Medline]
Saunders, N. F. W., Houben, E. N. G., Koefoed, S., de Weert, S., Reijnders, W. N. M., Westerhoff, H. V., de Boer, A. P. N. & van Spanning, R. J. M. (1999). Transcription regulation of the nir gene cluster encoding nitrite reductase of Paracoccus denitrificans involves NNR and NirI, a novel type of membrane protein. Mol Microbiol 34, 2436.[CrossRef][Medline]
Sears, H. J., Ferguson, S. J., Richardson, S. J. & Spiro, S. (1993). The identification of a periplasmic nitrate reductase in Paracoccus denitrificans. FEMS Microbiol Lett 113, 107112.[CrossRef]
Simon, R., Priefer, U. & Pühler, A. (1983). A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in Gram negative bacteria. Bio/Technology 1, 16.
Spiro, S. (1992). An FNR-dependent promoter from Escherichia coli is active and anaerobically inducible in Paracoccus denitrificans. FEMS Microbiol Lett 98, 145148.[CrossRef]
Tosques, I. E., Shi, J. & Shapleigh, J. P. (1996). Cloning and characterization of nnr, whose product is required for the expression of proteins involved in nitric oxide metabolism in Rhodobacter sphaeroides 2.4.3. J Bacteriol 178, 49584964.[Abstract]
van Spanning, R. J. M., de Boer, A. P. N., Reijnders, W. N. M., Spiro, S., Westerhoff, H. V., Stouthamer, A. H. & van der Oost, J. (1995). Nitrite and nitric oxide reduction in Paracoccus denitrificans is under the control of NNR, a regulatory protein that belongs to the FNR family of transcriptional activators. FEBS Lett 360, 151154.[CrossRef][Medline]
van Spanning, R. J. M., de Boer, A. P. N., Reijnders, W. N. M., Westerhoff, H. V., Stouthamer, A. H. & van der Oost, J. (1997). FnrP and NNR of Paracoccus denitrificans are both members of the FNR family of transcriptional activators but have distinct roles in respiratory adaptation in response to oxygen limitation. Mol Microbiol 23, 893907.[CrossRef][Medline]
van Spanning, R. J. M., Houben, E., Reijnders, W. N. M., Spiro, S., Westerhoff, H. V. & Saunders, N. (1999). Nitric oxide is a signal for NNR-mediated transcription activation in Paracoccus denitrificans. J Bacteriol 181, 41294132.
Wood, N. J., Alizadeh, T., Bennett, S., Pearce, J., Ferguson, S. J., Richardson, D. J. & Moir, J. W. B. (2001). Maximal expression of membrane-bound nitrate reductase in Paracoccus is induced by nitrate via a third FNR-like regulator named NarR. J Bacteriol 183, 36063613.
Wu, G., Cruz-Ramos, H., Hill, S., Green, J., Sawers, G. & Poole, R. K. (2000). Regulation of cytochrome bd expression in the obligate aerobe Azotobacter vinelandii by CydR (Fnr). Sensitivity to oxygen, reactive oxygen species, and nitric oxide. J Biol Chem 275, 46794686.
Received 6 June 2003;
revised 20 August 2003;
accepted 15 September 2003.