1 Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Rua da Quinta Grande, 6 Apartado 127, 2780-156 Oeiras, Portugal
2 Estação Agronómica Nacional, Instituto Nacional de Investigação Agrária, Quinta do Marquês, 2780-156 Oeiras, Portugal
3 Instituto de Biologia Molecular e Celular, Universidade do Porto, 4150 Porto, Portugal
4 Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602, USA
Correspondence
Helena Santos
santos{at}itqb.unl.pt
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ABSTRACT |
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INTRODUCTION |
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The ability of Desulfovibrio and other sulfate-reducing bacteria to utilize oxygen may be essential for survival in their natural habitats. In fact, the presence of communities of sulfate-reducing bacteria in oxic environments has been widely reported, especially in the oxicanoxic interfaces of microbial mats or sediments, as well as in periodically oxygenated zones of aquatic environments (Battersby et al., 1985; Fukui & Takii, 1990
; Sass et al., 1997
; Manz et al., 1998
; Teske et al., 1998
; Minz et al., 1999
).
Despite the ability of these organisms to cope with oxygen, sustainable growth of pure cultures in the presence of oxygen has never been unequivocally demonstrated (Cypionka, 2000). Several key enzymes in the pathways for substrate oxidation are oxygen sensitive (Stams & Hansen, 1982
; Kremer et al., 1989
; Hensgens et al., 1993
). Moreover, some Desulfovibrio strains undergo morphological alterations in the presence of oxygen (Sass et al., 1998
), which could result from inactivation of enzymes involved in cell division (Cypionka, 2000
). Several highly reactive derivatives of oxygen are toxic and able to damage essential cell components if not scavenged (Fridovich, 1983
; Imlay & Linn, 1988
; Farr & Kogoma, 1991
). To counteract these deleterious effects, many anaerobic organisms have developed defence systems similar to those found in aerobes (Storz et al., 1990
). One well-known type of protection system comprises enzyme activities that diminish or eliminate oxygen derivatives and radicals. Such enzymes have been found in many strictly anaerobic micro-organisms, including sulfate-reducing bacteria (Hewitt & Morris, 1975
; Hatchikian et al., 1977
; Rocha et al., 1996
; Dos Santos et al., 2000
; Pan & Imlay, 2001
), in which they can play a major role in oxygen detoxification.
Following our early results on the aerobic metabolism of D. gigas, it was deemed important to assess oxygen toxicity and cellular responses to oxygen in this organism. Cell survival and growth after exposure to oxygen were examined, as well as the effect of oxygen on substrate utilization. Energy levels were monitored in real time by in vivo 31P-NMR in cells exposed to oxygen. Furthermore, enzyme activities related to the oxidative stress response, and total protein profiles, were examined to evaluate alterations in gene expression induced by oxygen.
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METHODS |
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NMR spectroscopy.
Freshly prepared cell suspensions were transferred to 10 mm NMR tubes under argon. Before the acquisition of the initial spectrum, 5 µl antifoam (silicone) and 5 % (v/v) 2H2O was added to the cell suspensions. Gases were delivered by using an airlift device in the NMR tube (Santos & Turner, 1986). 31P-NMR spectra were obtained with a 10 mm broadband probe head in a Bruker DRX-500 spectrometer operating at 202·45 MHz as previously described (Santos et al., 1994
). Spectra were acquired with proton decoupling under fully relaxing conditions for NTP resonances, using a 30° flip angle and a repetition delay of 3·1 s. Chemical shifts were referenced with respect to external 85 % H3PO4. Calibration of the chemical shift of intracellular Pi as a function of pH was carried out with a cell suspension of D. gigas treated with a concentration of 100 µM of the protonophore 3,3',4',5'-tetrachlorosalicylanilide (TCS). To calibrate the chemical shift of external Pi, a titration curve of MOPS buffer containing 1 mM Pi was performed.
1H-NMR spectra were obtained with presaturation of the water signal, using a 10 mm broadband probe head in a Bruker AMX-300 spectrometer. Free induction decays were acquired with a 45° flip angle and a repetition time of 3·3 s. Chemical shifts were referenced with respect to sodium 3-trimethylsilyl[2,2,3,3-2H]propionate. In all experiments, the probe head temperature was kept at 33 °C. In vivo NMR experiments were performed at least twice.
Growth experiments.
Cultures were prepared in a medium containing lactate and sulfate and buffered with 30 mM MOPS (sodium salt), using a 2 litre fermentation vessel at 35 °C with continuous gassing with argon. Cell growth was monitored by measuring either the OD450 or the protein concentration. At the middle of the exponential growth phase, the culture was split in two, and fresh medium was added to each fermentation vessel. Both cultures were maintained under argon for approximately 56 h. One of the cultures was then exposed to defined concentrations of oxygen, by continuous bubbling with a suitable argon/air mixture, and the oxygen partial pressure was controlled with an O2 electrode. The other culture was kept anoxic (control). Each growth experiment was performed at least twice.
Preparation of cell-free extracts and determination of enzyme activities.
Cell suspensions were disrupted either by ultrasonic disintegration in an ice-cold bath or by passing twice through a French pressure cell at 3·3 MPa; cell debris was removed by centrifugation (15 min, 16 000 g) in an Eppendorf centrifuge. The resulting supernatant fraction was dialysed overnight against 10 mM Tris/HCl pH 7·0.
Superoxide-scavenging activity was assayed spectrophotometrically by the method described by McCord & Fridovich (1969) for superoxide dismutase (EC 1.15.1.1), with 1 unit (U) of activity causing a 50 % inhibition of the rate of horse heart cytochrome c reduction by the superoxide anion generated by the system xanthine/xanthine oxidase. Catalase (EC 1.11.1.6) was measured according to the procedure of Beers & Sizer (1952)
. Glutathione reductase (EC 1.8.1.7) was assayed as described by Goldberg & Spooner (1983)
. One unit of catalase or glutathione reductase activity was the amount catalysing the formation of 1 µmol product or the consumption of 1 µmol substrate per minute. Two independent determinations were performed for each enzyme activity in all conditions examined.
Protein labelling and sample preparation for electrophoretic analysis.
Anoxic cell suspensions containing 0·51 mg protein ml-1 were prepared in MOPS buffer (30 mM, sodium salt, pH 7·7) from mid-exponential-phase cultures. For protein labelling in vivo, cell suspensions were split into aliquots (2 ml each) and incubated for 35 min in different conditions: under nitrogen atmosphere at 35 °C (control); under oxygen atmosphere at 35 °C (oxygen shock); under nitrogen atmosphere at 45 °C (heat shock). [35S]Methionine (Amersham Life Science) was added to each sample (0·37x106 Bq ml-1) and cells were incubated for further 10 min. Cell suspensions were centrifuged at 3000 g for 10 min at 4 °C, and washed with 30 mM MOPS buffer, pH 7·7. For one-dimensional electrophoresis, the cell sediment was immediately suspended in a lysis solution [7·5 % (v/v) -mercaptoethanol, 1·5 % (w/v) SDS, 0·75 mM PMSF, 2 µM DTT, 50 mM Tris/HCl pH 6·8]. To prevent protein degradation, a cocktail of protease inhibitors was added (Bossier et al., 1993
). After vigorous vortexing, the samples were submitted to three cycles of freezethawing; the resulting lysates were diluted in sample solution [2 % (w/v) SDS, 10 % (v/v) glycerol, 0·025 % (w/v) bromophenol blue, 1 % (v/v)
-mercaptoethanol, 16 mM Tris/HCl pH 6·8], and boiled for 5 min before loading onto the gel.
For two-dimensional electrophoresis, the same procedure was used, except that after washing with buffer solution, cells were suspended in a lysis solution containing 9 M urea, 7·6 % (w/v) CHAPS, 2 % (w/v) DTT, 2 % (v/v) Pharmalytes non-linear pH range 310 (Pharmacia-Biotechnology), 0·14 % (w/v) PMSF; a cocktail of protease inhibitors was also added. After the freezethawing cycles in liquid nitrogen, the lysates were diluted in a sample solution [9 M urea, 1 % (w/v) DTT, 2 % (v/v) Pharmalytes non-linear pH range 310, 2 % (w/v) CHAPS, and a trace of bromophenol blue] and centrifuged at 16 000 g for 10 min at 4 °C; cell debris was discarded.
One- and two-dimensional electrophoresis.
One-dimensional electrophoresis was carried out on SDS-polyacrylamide gels according to the method described by Laemmli (1970) (5 % stacking gel and 10 % separation gel) in a Bio-Rad Mini Protean II apparatus.
For 2D electrophoresis, isoelectric focusing was run using 13 cm long ready-made gel strips with immobilized pH gradients (pH 310, non-linear) (Immobiline DryStrips, from Pharmacia-Biotechnology), following previously described protocols (Görg et al., 1995). Isoelectric focusing was run in a horizontal electrophoresis unit (Multiphor II, from Pharmacia-Biotechnology) at 20 °C. For improved sample entry, voltage was set at 300 V for the first 1 h, and at 500 V for the next 2 h. Isoelectric focusing was continued at 3500 V for 6 h. Current and power settings were limited to 0·5 mA and 2 W, respectively, per gel strip. After the run, gel strips were equilibrated according to Görg et al. (1995)
.
The second dimension was performed as described earlier (Görg et al., 1995). SDS-PAGE was carried out by the method of Laemmli (1970)
, as modified by Studier (1973)
, using 7·520 % polyacrylamide SDS slab gels in a vertical electrophoresis unit (SE 600 Series, Hoefer Scientific Instruments). [14C]Methylated proteins (Amersham) were used as molecular mass standards within the range 14·3220 kDa. Electrophoresis was performed at 20 °C for 10 h at 15 mA per gel. After fixation in 7 % (v/v) acetic acid for 30 min, fluorography was carried out either by the methods described by Laskey & Mills (1975)
or by incubation with the fluorographic reagent Amplify (Pharmacia-Biotechnology) for 20 min. Proteins labelled with [35S]methionine were detected by exposing the dried gels to Kodak BioMax films at -70° for 57 days. Experiments were repeated at least five times, since for each of the tested conditions, the protein profiles showed some variability among replicates.
Western immunoblot analysis.
Intracellular proteins were separated by one- or two-dimensional electrophoresis and electroblotted from gels to nitrocellulose membranes. Transfers were done overnight at 80 mV. The membranes were treated first with antibodies (monoclonal mouse anti-heat-shock protein 60, Hsp60, from Sigma; or polyclonal sheep anti-human catalase, from The Binding Site), and then with the appropriate horseradish-peroxidase-conjugated immunoglobulins (anti-mouse IgG, from Amersham; or anti-sheep IgG, from Sigma). Antigenantibody associations were revealed by enhanced chemiluminescence (ECL Detection Reagents, Amersham), followed by exposure to Kodak BioMax films.
Other analytical methods.
Total glutathione (both reduced and oxidized forms) in cell-free extracts of D. gigas was determined according to Tietze (1969). Protein concentration was measured by the method of Bradford (1976)
using BSA as standard. For dry cell mass determination, cells were collected by filtration in nitrocellulose membrane filters (0·2 µm pore size) and dried at 100 °C to constant mass.
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RESULTS |
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Effect of oxygen on growth
Mid-exponential-phase cultures grown with lactate and sulfate as substrates were submitted to several concentrations of oxygen (5, 10, 20, 40 or 120 µM) for 8 h, and compared with control cultures maintained under an argon atmosphere. Upon supply of oxygen to the cultures, growth ceased immediately (Fig. 1); similar results were obtained for all the oxygen partial pressures examined. The values of OD450 and protein concentration measured during exposure to oxygen were constant. However, upon switching to an anoxic atmosphere, growth resumed and the mean growth rate observed in these conditions was 0·14 h-1, corresponding to 70 % of the mean value determined for control cultures not exposed to oxygen (0·2 h-1). The same behaviour was observed when the length of the exposure to oxygen was extended up to 24 h, the longest duration examined.
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Effect of oxygen on the induction of defence systems against oxidative stress
The effect of oxygen on enzyme activities involved in oxidative stress defence and on glutathione levels was assessed. Assays were carried out in cell-free extracts prepared from mid-exponential-phase cultures submitted to the following conditions: (i) different concentrations of oxygen (0, 5, 10, 20, 40 and 120 µM) for 8 h; or (ii) saturating concentrations of oxygen for different periods of time (0, 15, 30, 60 and 120 min).
High levels of superoxide-scavenging activity were found in all samples assayed (Tables 2 and 3), but no significant differences were detected between cultures exposed to different concentrations of oxygen or maintained under argon atmosphere. Moreover, the duration of exposure to oxygen had no significant effect on the activity level.
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Low levels of glutathione reductase activity were detected in D. gigas cell-free extracts [0·0025±0·0005 U (mg protein)-1] and no changes were observed under the wide range of conditions examined.
A mean value of 1·8±0·6 nmol (mg protein)-1 was determined for the total glutathione content (reduced plus oxidized forms) in D. gigas. No significant differences were observed upon increasing the oxygen concentration in the medium or the duration of the oxic period.
Effect of oxygen on the energy status of non-growing cells
The energetic status of cell suspensions saturated with oxygen (continuous bubbling) was monitored by in vivo 31P-NMR during 12 h and compared with that of cells kept under anoxic conditions. NTP and NDP levels were determined from the intensity of the respective NMR resonances. Information on the evolution of intracellular and extracellular pH was obtained from the chemical shifts of the signals due to intracellular Pi and external Pi, respectively. High levels of energization were maintained in whole cells during the first 56 h of the experiment (Fig. 2); after 12 h the intensity of the NTP resonances had decreased to approximately 40 % of the initial values regardless of the gas atmosphere used (data not shown). Both external and intracellular pH decreased due to glycogen catabolism, which results in the formation of acetic acid.
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The pattern of proteins induced by heat shock was considerably different from that obtained upon oxygen shock. Fourteen proteins were induced (Table 4); all of them, except two, showed molecular masses and isoelectric points different from those of the proteins induced by exposure to oxygen. The common proteins are designated in Table 4
and Fig. 4
as Ox2/Hs1 and Ox10/Hs6, with molecular masses of 9092 kDa and 58 kDa, and pI values of 4·85·1 and 5·86·0, respectively.
Antibodies against mouse Hsp60 and human catalase were tested on Western blots of D. gigas proteins. Anti-Hsp60 cross-reacted specifically with one protein from D. gigas. This protein was present in the control sample not submitted to stress conditions, but the intensity of the respective spot increased upon either heat shock or oxygen shock; it is designated in Fig. 4 and Table 4
as Ox10/Hs6. The immunoassays for catalase detection were performed with blots from one-dimensional SDS-PAGE gels; we were unable to identify the corresponding spots in two-dimensional gels. Anti-human catalase recognized a protein band with a molecular mass estimated at 6070 kDa. The cross-reacting band in the sample exposed to oxygen showed an increase (about 30 %) in intensity compared with the control. On the other hand, the cross-reacting band in the heat-shocked sample showed approximately the same intensity as the control.
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DISCUSSION |
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The response to oxygen in D. gigas involves an increase in the synthesis of several proteins, which may alleviate toxicity and cell injury caused by stress. Most of the induced proteins could also be detected in control conditions at lower levels, suggesting that they probably have a cellular function both in optimal anoxic conditions and in the presence of oxygen. The major proteins whose levels were enhanced by oxygen were not induced in heat-shocked cells, showing that induction of protein synthesis is specific, depending on the type of stress. Thus far, the nature of most of these proteins is unknown. Among the induced proteins detected by two-dimensional electrophoresis, only a homologue of a 60 kDa eukaryotic heat-shock protein (Hsp60) and catalase were identified by immunoassays. The global characterization of the induced proteins and the elucidation of their functional role would be essential for an understanding of the underlying mechanism of adaptation to oxygen in this organism. However, this goal is beyond the scope of the present study and would require information on the amino acid sequence of the induced proteins. In this context, the availability of the genome sequence of D. gigas would be very helpful. The genome sequence determinations of D. desulfuricans (GenBank accession number NZ_AABN00000000) and D. vulgaris (GenBank accession number NC_002937) are in progress and 200 kb in the D. gigas genome have been sequenced thus far (C. Rodrigues-Pousada, personal communication). The sequence homology between D. gigas and the other two organisms is rather low, emphasizing the need for more specific sequence information and reliable genome annotation.
The ability of D. gigas to survive in oxic environments implies the existence of efficient defence systems to counterbalance the cell damage caused by oxygen-derived toxic radicals. It has already been demonstrated that two important enzyme activities involved in the detoxification of reactive oxygen species, superoxide-scavenging and catalase, are constitutive in D. gigas, with high levels of activity (Hatchikian et al., 1977), comparable to those of canonical aerobes (McCord et al., 1971
). Furthermore, the present study shows that glutathione and glutathione reductase activity are also present in this organism. Catalase activity was enhanced by exposure to oxygen, despite the high levels found in control cells under anaerobiosis. An iron superoxide dismutase and a catalase from D. gigas were recently purified and characterized in detail (Dos Santos et al., 2000
). D. gigas and other strict anaerobes are equipped with alternative protection systems: neelaredoxin, the blue-coloured protein first isolated from D. gigas (Chen et al., 1994
), present in all known genomes of anaerobic prokaryotes, has been identified as a scavenger of the superoxide anion radical (Silva et al., 2001b
; Abreu et al., 2002
; Adams et al., 2002
). On the other hand, oxidative stress protection systems involving desulforedoxin (rubredoxin oxidoreductase) with superoxide reductase activity have been found in D. vulgaris (Lumppio et al., 2001
). Superoxide scavenging could be accomplished by one or several proteins that catalyse either the dismutation or the rubredoxin-coupled reduction of the superoxide radical, catalase playing a crucial role in the removal of hydrogen peroxide, the resulting product of superoxide removal.
D. gigas seems to contain all the necessary enzymic machinery for the efficient detoxification of reactive oxygen species, yet it is unable to grow in the presence of oxygen. Studies with Clostridium acetobutylicum showed that exposure of cultures to aerobiosis prevented the net synthesis of DNA, RNA and protein, suggesting that oxygen could inhibit critical enzymes involved in those processes and arrest growth (O'Brien & Morris, 1971). It has been reported that sulfate-reducing bacteria, including some Desulfovibrio strains, are able to oxidize organic substrates, such as pyruvate, formate, ethanol, and even lactate, under microaerophilic concentrations of oxygen (Dannenberg et al., 1992
). Moreover, endogenous reserves can be metabolized in oxic environments (Santos et al., 1993
; Van Niel et al., 1996
). The present study showed that pyruvate could be oxidized to acetate by non-growing D. gigas cell suspensions under saturating concentrations of oxygen, but was unable to sustain growth when provided to either oxic or anoxic cultures as the sole carbon and energy source. The growth rates of anoxic cultures in a medium containing pyruvate and sulfate were not significantly different from those observed in the presence of lactate and sulfate; therefore, failure to grow on pyruvate was probably due to the lower energetic yield associated with the fermentation of pyruvate, the availability of oxygen as an electron acceptor being unable to reverse this behaviour.
When cells of D. gigas are deprived of exogenous substrates, the internal reserve of glycogen that accumulates in high amounts during growth is mobilized, acting as carbon and energy source for cell maintenance (Fareleira et al., 1997). Glycogen plays an essential role in cell survival under oxic conditions. This is supported by the observation that non-growing cell suspensions maintain a high cellular energy charge during exposure to saturating concentrations of oxygen for long time periods in the absence of external substrates. Most interestingly, an increase in the intracellular levels of NTP could be systematically observed after a shift from anoxic to oxic atmosphere, but similar rates of acetate production derived from glycogen reserves were found in both anoxic and oxic conditions (this work). Thus, the higher steady-state levels of NTP established under oxygen atmosphere do not correlate with a higher rate of glycogen mobilization. These observations could be reconciled if a mechanism other than substrate-level phosphorylation for ATP formation were operating during oxygen consumption by D. gigas. However, this hypothesis is apparently not corroborated by our experimental evidence: (i) proton uncouplers (TCS) did not affect the steady-state levels of NTP in whole cells metabolizing glycogen in the presence of oxygen; (ii) the increase in the NTP levels was also observed upon exposure to oxygen of cell-free extracts. Recently, a membrane-bound oxygen respiratory chain was reported in D. gigas cells grown on a culture medium containing fumarate and sulfate, but this respiratory activity was very poor in membrane fractions derived from cells grown on lactate as carbon source (Lemos et al., 2001
). The amount of data available strongly indicates that lactate-grown D. gigas cells utilize oxygen and synthesize NTP via cytoplasmic systems (Chen et al., 1993a
, b). On the other hand, the observed enhancement of steady-state NTP levels under aerobiosis is most likely due to oxygen inhibition of processes leading to ATP dissipation.
In conclusion, D. gigas appears to be suitably equipped to cope with oxygen stress: it is endowed with well-known anti-oxidative stress systems, it can perform very efficient NTP synthesis from the aerobic metabolism of internal reserves, and it is able to resume growth even after long periods of exposure to oxygen. It is still intriguing that this organism is unable to take advantage of these remarkable metabolic features to achieve growth in the presence of oxygen. Thus, it is conceivable that oxygen could have an irreparable effect on vital cell functions.
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ACKNOWLEDGEMENTS |
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Received 27 November 2002;
revised 24 February 2003;
accepted 25 February 2003.