Arch Microbiol (1996) 166 : 23–31
© Springer-Verlag 1996
O R I G I N A L PA P E R
Matthias Koschorreck · Edward Moore · Ralf Conrad
Oxidation of nitric oxide by a new heterotrophic Pseudomonas sp.
Received: 15 November 1995 / Accepted: 24 February 1996
Abstract A new bacterial strain isolated from soil consumed nitric oxide (NO) under oxic conditions by oxidation to nitrate. Phenotypic and phylogenetic characterization of the new strain PS88 showed that it represents a previously unknown species of the genus Pseudomonas, closely related to Pseudomonas fluorescens and Pseudomonas putida. The heterotrophic, obligately aerobic strain PS88 was not able to denitrify or nitrify; however, strain PS88 oxidized NO to nitrate. NO was not reduced to nitrous oxide (N2O). Nitrogen dioxide (NO2) and nitrite (NO2–) as possible intermediates of NO oxidation to nitrate (NO3–) could not be detected. NO oxidation was inhibited under anoxic conditions and by high osmolarity, but not by nitrite. NO oxidation activity was inhibited by addition of formaldehyde, HgCl2, and antimycin, and by autoclaving or disintegrating the cells, indicating that the process was enzyme-mediated. However, the mechanism remains unclear. A stepwise oxidation at a metalloenzyme and a radical mechanism are discussed. NO oxidation in strain PS88 seems to be a detoxification or a co-oxidation mechanism, rather than an energy-yielding process. Key words Pseudomonas · Nitric oxide · Nitrogen dioxide · Oxidation mechanism · Denitrification · Nitrification
Introduction Nitric oxide (NO) is only a minor constituent of the earth’s atmosphere, but because of its reactivity, it plays
M. Koschorreck · R. Conrad (Y) Max-Planck-Institut für terrestrische Mikrobiologie, Karl-von-Frisch Strasse, D-35043 Marburg, Germany Tel. +49-6421-178801; Fax +49-6421-178809; e-mail
[email protected] E. Moore Gesellschaft für Biotechnologische Forschung, Bereich Mikrobiologie, Mascheroder Weg 1, D-38124 Braunschweig, Germany
an important role in atmospheric chemistry. Soils may contribute about 13% of the global sources of tropospheric NO (Lelieveld and Crutzen 1994). The net flux of NO between soil and atmosphere is the result of simultaneous production and consumption of NO in soil. Whereas the processes of NO production (mainly nitrification, denitrification, and chemical processes) are wellknown, not much is known about NO consumption processes in soils. The reduction of NO to N2O by denitrifying bacteria is the only well-known mechanism of microbial NO consumption. This process normally takes place under anoxic conditions. Even in well-aerated soils, anaerobic microniches that support denitrification may exist. Consumption of NO in several soils could be explained by denitrification (Remde and Conrad 1991; Schäfer and Conrad 1993). However, evidence of an oxidative mechanism of NO consumption exists in certain soils in which NO consumption is inhibited by anoxic conditions (J. Rudolph et al., unpublished results; Baumgärtner et al. 1996). In one of these soils (a podzol), 90 ± 17% of added 15NO was recovered as 15NO3– (J. Rudolph et al., unpublished results). Consumption of NO under oxic conditions has so far been demonstrated in the autotrophic, nitrite-oxidizing Nitrobacter (Freitag and Bock 1990), in the obligately aerobic Rhizobium hedysari (Casella et al. 1994), and in heterotrophically nitrifying bacteria such as methanotrophs (Krämer et al. 1990) and Alcaligenes faecalis (Anderson et al. 1993). However, as shown for R. hedysari, and probably for heterotrophic nitrifiers in general, NO is reduced by an NO reductase activity. Oxidation of NO to nitrite has only been shown in Nitrobacter, where the process is believed to be coupled to the generation of NADH (Freitag and Bock 1990). The consumption of NO by various aerobic bacteria has been demonstrated recently (Baumgärtner et al. 1996). A Pseudomonas sp. (strain PS88) that consumed NO under oxic rather than anoxic conditions was isolated from soil. Sterile soil amended with this strain converted NO to NO2– and NO3–. Comparison of NO consumption rates by strain PS88 with NO uptake rate constants of soils showed
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that the organism may contribute significantly to total NO consumption in soil. In this publication we further characterize strain PS88 and the unknown NO consumption mechanism under oxic conditions. To avoid autoxidation of NO in the presence of oxygen, we restricted our experiments to low concentrations of NO since chemical oxidation of NO depends on the square of the NO concentration. Hence, autoxidation of NO was insignificant when the atmosphere contained NO at mixing ratios < 4 ppmv (ppmv = parts per million by volume = 10–4%).
Materials and methods Organism Pseudomonas sp.strain PS88 has been isolated from soil (Baumgärtner et al. 1996) and deposited with the Deutsche Sammlung von Mikroorganismen und Zellkulturen (Braunschweig, Germany; DSM 10315). The organism was maintained in a mineral medium containing 20% glycerol and small glass beads at –70° C. The mineral medium consisted of (g/l): NH4Cl (0.2), K2HPO4 (1), KH2PO4 (0.78), MgSO4 · 7 H2O (0.2), NaCl (0.1), and FeSO4/ EDTA solution (10 ml). The pH was adjusted to 7.0. The FeSO4/ EDTA solution consisted of Na2EDTA (93 mg) and FeSO4 · 7 H2O (63 mg) in 100 ml demineralized water. Cultivation and harvesting A glass bead from the stock culture was added to mineral medium containing (g/l): yeast extract (1) and glucose (1.8). Cells were subcultured at least three times in the same medium in test tubes incubated on a rotator before harvesting. All incubations were done at 25° C. Cells were harvested by centrifugation at the end of the exponential growth phase, washed, and resuspended in mineral medium to the desired optical density at 500 nm, usually to an OD500 of 0.1. Tests had shown that NO consumption activity was maximal in cells from the late exponential growth phase. Taxonomic characterization Standard procedures were used (Smibert and Krieg 1981) for characterization of strain PS88. Fluorescence was tested at 254 nm on cells grown on King B Agar. The G+C content of isolated DNA (Cashion et al. 1977) was determined with a HPLC system (Tamaoka and Komagata 1984). Cell size and flagellation were determined from electron micrographs of negatively stained cells. Substrate utilization was tested in mineral medium supplemented with 4.5 mM of the substrate. Additionally, substrate utilization was tested with the Biolog microtiter test system (Biolog Inc., Hayward, Calif., USA). Use of different nitrogen sources was tested in mineral medium supplemented with glucose and the nitrogen substrate. The pH and salt optima and tolerances of growth were tested in mineral medium supplemented with succinate and different amounts of KH2PO4 or NaCl. Fermentation of glucose was tested with aerobically grown cells (9.3 µg protein ml–1) in mineral medium plus glucose under an atmosphere of N2. Potential fermentation products (fatty acids, alcohols, H2) were monitored for 4 days (Krumböck and Conrad 1991; Cord-Ruwisch et al. 1988). To test for denitrification, aerobically grown cells were transferred to mineral medium containing glucose and nitrate (4 mM) and incubated under N2. Controls were incubated under air. At the beginning and after 6 days of incubation at 25° C, nitrate, nitrite and OD500 were measured.
Genomic DNA isolation, PCR amplification, and sequence determination of 16S rRNA genes Genomic DNA was isolated from 0.1 g (wet weight) cells using the CTAB miniprep protocol for bacterial DNA preparations (Wilson 1987). Nearly complete 16S rRNA genes were amplified by PCR (Mullis and Falloona 1987; Saiki et al. 1988), using a Hans Landgraf model 5.92 thermocycler and reaction conditions reported previously (Karlson et al. 1993). PCR-amplified DNA was purified using Centricon-100 microconcentrators (Amicon, Witten, Germany) and sequenced directly using an Applied Biosystems, 373A Sequencer and the protocol of the manufacturer (Perkin-Elmer/Applied Biosystems, Weiterstadt, Germany) for “Taq cycle sequencing” with fluorescent dye-labeled dideoxynucleotides. The sequencing primers used have been described previously (Lane 1991). Sequence data were aligned with reference rRNA (and rRNA gene) sequences (Neefs et al. 1993; Olsen et al. 1992) using evolutionary-conserved primary sequence and secondary structure as reference (Gutell et al. 1985; Woese et al. 1983). Evolutionary distances were calculated from sequence-pair similarities (Jukes and Cantor 1969). Dendrograms were generated using a weighted, least-squares, distance method (Olsen 1987).
Measurement of NO consumption Consumption of NO was measured in 120-ml serum bottles filled with 5 ml of cell suspension. In some experiments, 21-ml pressure tubes with 1 ml cell suspension were used. Cells in the controls were killed by adding 25 or 50 µl of a 10 mM HgCl2 stock solution. The bottles were capped with black rubber stoppers and the headspace was exchanged with synthetic air (20% O2, 80% N2). The bottles were pressurized by adding 5 ml of synthetic air in excess. To obtain a NO mixing ratio of approximately 1 ppmv, 1 ml of a NO calibration gas (105.4 ppmv NO in N2; all gases from Messer Griesheim, Siegen, Germany) was added to the headspace. After a preincubation of 15 min, gas samples (1 ml) were repeatedly taken by means of a gas-tight syringe (Glenco, Houston, Texas, USA) and analyzed for NO. The obtained values were corrected for pressure, and the first-order uptake rate constant was calculated from the logarithmic decrease of the NO mixing ratio with time. The rate constant of chemical oxidation of NO was determined with killed controls and subtracted from the rate constant obtained with living cells. All tests were carried out in triplicate and rate constants are given as mean ± SD. In a flow-through experiment, 53.5 ml of cell supension was incubated in special incubation vessels (Remde und Conrad 1991) and bubbled with synthetic air [flow rate (f) = 655 cm3 min–1]. Liquid samples (1 ml) were repeatedly withdrawn by means of a sterile syringe, filtered through a cellulose acetate membrane filter (0.2 µm, Sartorius, Göttingen, Germany) and stored at –20° C until analyzed. At each time point, three parallel samples were taken and treated separately. To measure NO consumption, NO calibration gas was added to the air stream, resulting in a NO mixing ratio of 1.5 ppmv. NO was measured in the outlet air (ms). Gas flows were adjusted by means of mass-flow controllers (HiTech, Wagner, Offenbach, Germany). To correct for chemical oxidation of NO, the NO mixing ratio at the outlet of an identical incubation vessel filled with sterile medium was measured (mb). NO consumption was calculated by multiplying the difference of the NO mixing ratio at the outlet and at the inlet of the flask with the gas flow rate: J = f (mo – mi)
(Eq. 1) h–1
dw)–1];
where J = NO flux [ng NO-N (g f = gas flow rate (cm3 min–1); mo = NO mixing ratio at the outlet of the flask = mi ms mb–1 (ppbv); mi = NO mixing ratio at the inlet of the flask = 1500 ppbv (ppbv = parts per billion by volume = 10–7%); c = conversion factor. Cell-free extracts were obtained by passing cell suspension in 0.01 M phosphate buffer (pH 7) at 4° C three times through a
25 French press at 1000 psi (ca. 6.9 MPa). Whole cells and cell debris were removed by centrifugation at 20,000 × g for 20 min. For further fractionation, the extract was centrifuged at 140,000 × g for 90 min. The pellet, which contained the membrane fraction, was resuspended in phosphate buffer. Analytical methods Nitrite and nitrate were analyzed by ion chromatography (Bak et al. 1991). The protein content of cell suspensions was determined after alkaline hydrolysis in 1 M NaOH for 10 min at 90° C using the Lowry method (Hanson and Phillips 1981) with bovine serum albumin as standard. Cell numbers were determined by microscopic counting in a Helber chamber. NO was measured with chemoluminescence detectors. Gas samples in syringes were analyzed in a Thermo-Electron detector (series 14, Hopkinton, Mass., USA) especially equipped for syringe samples (Remde and Conrad 1991). The detection limit was about 5 ppbv NO. Continuous measurements during the flowthrough experiment were carried out with a Tecan CLC 770 Al ppt (Hombrechitkon, Switzerland) as described by Schuster and Conrad (1992). The detection limit was 0.2 ppbv NO. NO2 was analyzed with a chemoluminescence detector LMA3 (Scintrex Ltd., Concord, Ontario, Canada; Rudolph et al. 1996). The detection limit was 10 ppbv NO2 for syringe samples and 0.1 ppbv NO2 for continuous measurements. N2O was analyzed by gas chromatography with an electron capture detector (Conrad and Seiler 1980). The detection limit was 5 ppbv. O2 was analyzed with a gas chromatograph equipped with a thermal-conductivity detector (Koschorreck and Conrad 1993).
Results Morphological and biochemical characteristics The cells of strain PS88 were rod-shaped (0.8 × 2.2 µm) and occurred singly or in pairs (Fig. 1). Coccoidal cells were observed in the stationary growth phase. Cultures contained motile and non-motile cells. Electron micro-
Fig. 1 Phase contrast photomicrograph of strain PS88 in the exponential growth phase. Bar 10 µm
graphs of negatively stained cells revealed a single polar flagellum per cell. Cells of strain PS88 stained gram-negative. Spores were never observed. Cells grown on King B Agar, but not in complex medium, fluoresced at 254 nm. The G+C content was 58.2 mol %. The strain possessed catalase, oxidase and arginine hydrolase activities, no lecithinase or lipase (egg yolk reaction) activities, and did not hydrolyze starch or gelatin.
Cultural characteristics Strain PS88 grew only under oxic conditions. Growth occurred at 4° C but not at 41° C and was optimal at 20° C. The doubling time was approximately 1.3 h (µ = 0.55 h–1) when measured at 25° C in mineral medium with glucose. The cells grew between pH 5.9 and 9, with an optimum around pH 7. Growth was inhibited by elevated concentrations of NaCl (Fig. 2).
Nutritional characteristics The metabolism of strain PS88 was respiratory with oxygen as the terminal electron acceptor. Strain PS88 was unable to grow autotrophically on H2+CO2 (80:20, v/v). Under anoxic conditions no fatty acids, alcohols, or H2 were produced from glucose. Growth was tested on substrates with diagnostic value according to the guidelines given by Holt et al. (1994). Strain PS88 was able to grow on glucose, trehalose, succinate, mannitol, myo-inositol, L-valine, DL-alanine, and DL-arginine, but did not grow on ascorbate and geraniol. Strain PS88 was routinely cultured in a complex medium
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excreted during growth on nitrate as the sole N source. Excretion of nitrite was stimulated in the presence of ammonium, but only up to one thousandth of the ammonium concentration. Only traces of nitrate (< 10 µM) were detected during growth on ammonium as sole N source. Nitrite at a concentration > 2 mM inhibited growth. Strain PS88 was apparently unable to carry out nitrification. No growth occurred with nitrate as electron acceptor in the absence of O2. Nitrate was not consumed under anoxic conditions, and nitrite was not produced from nitrate. Addition of 10% acetylene did not result in N2O production. Thus, strain PS88 was unable to carry out denitrification or nitrate respiration. Fig. 2 Effect of salt concentration on the NO consumption rate constant and on growth by strain PS88. The NO consumption rate constant was determined in pressure tubes (21 ml) containing cell suspensions (1 ml; 55 µg protein ml–1 in mineral medium supplemented with different concentrations of NaCl): (J) living cells, (j) inactivated (500 µM HgCl2) cells. The growth rate µ (+) was determined with strain PS88 growing in mineral medium supplemented with 10 mM glucose and different concentrations of NaCl. Bars give standard deviations of n = 3
containing yeast extract, but also grew on other complex media (NB, TS broth) and on mineral medium containing a single carbon source such as glucose. The oxidation of different substrates by strain PS88 was tested additionally with the Biolog microtiter test system. Strain PS88 could not be assigned to one of the species covered by the Biolog system, but showed a close relationship to Pseudomonas fluorescens biovar C (similarity coefficient 0.54, distinction 5.07). Strain PS88 was able to use ammonium and nitrate as N source, but nitrate was used only in the absence of ammonium. No growth occurred with nitrite or N2 as the only N source. Small amounts of nitrite (< 20 µM) were Fig. 3 Phylogenetic tree based on 16S rRNA gene sequences of strain PS88 and of reference species. The scale gives the evolutionary rate per nucleotide position
Phylogenetic characterization Sequencing of the PCR-amplified 16S rDNA of strain PS88 allowed determination of an estimated 97% (1484 nucleotide positions) of the complete gene. Sequence comparisons demonstrated that strain PS88 clustered with bacteria of the gamma subclass of the Proteobacteria and most closely with species of the genus Pseudomonas (Fig. 3). Table 1 presents the sequence similarities determined from comparisons of the 16S rRNA gene sequences of strain PS88 and reference organisms. Based upon the 16S rRNA gene sequence analysis, strain PS88 is recognized as a member of the genus Pseudomonas. The sequence for the 16S rRNA gene of Pseudomonas sp. PS88 (DSM 10315) is deposited with the EMBL under accession number X92416. NO consumption Cells of strain PS88 consumed NO to mixing ratios lower than the detection limit of our analytical system (Fig. 4).
27 Table 1 16S rRNA gene sequence similarities between Pseudomonas sp. PS88 and reference species. 16S rRNA gene sequence similarities were derived from total sequence comparisons with data deposited in the European Molecular Biology Laboratory (Neefs et al. 1993) and the Ribosomal Database Project (Olsen et al. 1992) Organism
Pseudomonas aeruginosa Pseudomonas fluorescens Pseudomonas chlororaphis Pseudomonas aureofaciens Pseudomonas putida Pseudomonas syringae Pseudomonas viridiflava Pseudomonas cichorii Pseudomonas stutzeri Pseudomonas mendocina Pseudomonas alcaligenes Pseudomonas pseudoalcaligenes Acinetobacter calcoaceticus Escherichia coli Haemophilus influenzae Legionella pneumophila Stenotrophomonas maltophilia Vibrio parahaemolyticus
Strain
DSM 50071T DSM 50090T DSM 50083T DSM 6698T DSM 291T LMG 1247tlT LMG 2352T LMG2162T CCUG 11256T DSM 50017T LMG 1224T DSM 50188T ATCC 33604 ATCC 33391T ATCC 33152T ATCC 13637T ATCC 178.2T
Sequence similarity with Pseudomonas sp. PS88 94.7a 97.5a 98.0a 97.8a 98.5a 97.6a 96.8a 96.2a 96.0 95.7a 95.9a 96.9a 86.4 84.5 80.5 85.5 84.4 84.9
a
Sequence similarities were derived from comparisons with unpublished sequences obtained by E. Moore
Fig. 4 Consumption of NO by strain PS88 in a batch experiment. Suspensions (5 ml) of cells in mineral medium (60.4 µg protein ml–1) were oxically incubated in serum bottles (120 ml) under an atmosphere of 1000 ppbv NO in synthetic air (ppbv = parts per billion by volume = 10–7%) and assayed for NO (J), N2O (P), and NO2 (always below the detection limit). Controls (j, p) were prepared by adding 500 µM HgCl2. Broken line detection limit for NO and NO2. Bars give standard deviations of n = 3
NO decreased approximately logarithmically with time, indicating a first-order consumption. The rate constant of NO consumption determined in seven independent experiments was 547 ± 224 cm3 h–1 (mg protein)–1 [(1.3 ± 0.5) 10–8 cm3 h–1 cell–1]. At 0.1 ppmv NO, this corresponds to a consumption activity of 2 nmol h–1 (mg protein)–1. Consumption of NO was not saturated up to the highest NO mixing ratio measured (4 ppmv; data not shown). NO consumption was inhibited by autoclaving, formaldehyde, and HgCl2 (Table 2), indicating that the activity
Table 2 Inhibition of NO consumption by strain PS88 by different treatments. The NO consumption rate constants were measured in pressure tubes (21 ml) containing 1 ml cell suspension (OD500 = 0.1) under an atmosphere of 1 ppmv NO in synthetic air. Rates were corrected for chemical NO consumption measured in identical vessels without cells and compared with untreated controls Treatment
Inhibition (%)
Autoclaving Formaldehyde (20 µM) HgCl2 (250 µM) Antimycin A [160 µg (mg protein)–1] Cell breakage with French press Preincubation with C2H2 (10%)
94 ± 1.6 95.1 ± 1 93.1 ± 1.2 76 94.8 ± 1.7 1.7
(n = 3) (n = 3) (n = 3) (n = 3)
Table 3 Specific first-order rate constants of NO consumption in different cell fractions of strain PS88 Cell fraction
Rate constant [cm3 h–1 (mg protein)–1]
Whole cells Cell-free extract Cytosol fraction Membrane fraction
192 ± 97 9.3 ± 1.3 3.5 ± 2.7 24 ± 12
was from living cells. Inhibition of the electron transport chain by antimycin also inhibited NO consumption. To test whether NO consumption was catalyzed by low-molecular-weight substances excreted by the cells, PS88 was incubated in mineral medium for 6 h, after which a sample of the growth medium was filtered through a 0.2-µM membrane filter. The NO uptake rate constant of the cellfree filtrate was measured at 0.7 ± 0.3 cm3 h–1, which represented only 6% of the total NO uptake measured in the cell suspension (12 ± 1.3 cm3 h–1). Thus, NO consumption was due to cell activity of strain PS88 and not to substances in the medium. Most of the activity was lost after disintegration of the cells by French press (Table 2) or ultrasonification (data not shown). Further fractionation of the cell-free extract showed that the residual activity was membrane-bound rather than cytoplasmic (Table 3). Consumption of NO did not result in accumulation of either N2O or NO2 (Fig. 4). To test for N2O consumption, cells were prepared as described for the NO consumption assay, but were incubated with 0.8 ppmv N2O instead of NO. No change in the N2O content of the headspace was observed over an incubation period of 36 h, indicating that strain PS88 was not able to consume N2O. To test for an acetylene-sensitive N2O reductase, cells in mineral medium were preincubated for 1.5 h under an atmosphere of 10% acetylene in air. Because high mixing ratios of acetylene interfered with the NO detector, the headspace was exchanged. NO consumption was not affected by preincubation with acetylene (Table 2), but formation of N2O was not observed. We concluded that strain PS88 was unable to reduce NO to N2O. Cells were prepared as described for the NO consumption assay but incubated with 1 ppmv NO2 instead of NO to test for NO2 consumption. NO2 decreased within a few
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Fig. 5 Formation of nitrite and nitrate by NO-consuming strain PS88 in a flow-through experiment. Suspensions of cells (136 µg protein ml–1) in mineral medium supplemented with 20 µM NO2– and 20 µM NO3– were bubbled with air. At the time indicated by the arrows, the gas stream was supplemented with 1.5 ppmv NO (ppmv = parts per million by volume = 10–4%). Controls were prepared by adding 500 µM HgCl2. (P) NO3–; (p) NO3– control; (J) NO2–; (j) NO2– control, Broken line theoretical NO3– formation calculated from the simultaneously measured NO consumption. Bars give standard deviations of n = 3 Table 4 Formation of nitrate from NO by strain PS88 in the flowthrough experiment shown in Fig. 5. NO2– and NO3– accumulation during gassing with 1.5 ppmv NO was compared with the total consumption of NO measured PS88
PS88 + HgCl2
Difference
NO2– at 2.5 h NO2– at 5.5 h Nitrite formation (µM)
16.5 15.7 –0.8
19.2 19.0 –0.2
–0.6
NO3– NO3–
at 2.5 h at 5.5 h Nitrate formation (µM)
23.7 27.8 4.1
22.1 21.3 –0.8
4.9
NO consumption (µM) between 2.5 and 5.5 h
–4.2
1.2
–5.4
minutes with both living and HgCl2-inactivated cells, indicating that NO2 underwent rapid dissolution and chemical dismutation in the aqueous solution (Huie 1994). The first-order rate constant of NO2 disappearance was 1518 cm3 h–1. A first-order rate constant of NO consumption of 29 cm3 h–1 could be calculated from the data shown in Fig. 4. Thus, chemical NO2 consumption is much faster than biological NO consumption, and any NO2 that would possibly be produced would not accumulate in the NO consumption assay. Because of the relatively low NO consumption rate and because of the low NO mixing ratios that could be applied under oxic conditions, it was not possible to detect soluble products of NO consumption in batch experiments. Thus, the products of NO consumption were determined in a flow-through, rather than a batch system. Bubbling a cell suspension with NO-containing air resulted in an increase of the nitrate concentration in the suspension while the nitrite concentration remained constant within the experimental error (Fig. 5). NO was consumed at a
Fig. 6 Influence of O2 on NO consumption by strain PS88 in a batch experiment. Suspensions (1 ml) of cells in mineral medium (10.8 µg protein ml–1) were incubated in pressure tubes (21 ml) under a N2 atmosphere containing O2 at different partial pressures. NO was added to a mixing ratio of 1 ppmv, and the first-order rate constant of NO consumption was determined from the logarithmic decrease of NO with time. Sterile mineral medium was used as control. Bars give standard deviations of n = 3
rate of 1.8 µM h–1. Most of the NO consumed (91%) was trapped as NO3– (Table 4). Although this experiment showed relatively large standard deviations for the concentrations of nitrite and nitrate (Fig. 5), the observation that nitrate increased while nitrite stayed constant could be reproduced in similar experiments (data not shown). Thus, we concluded that strain PS88 oxidized NO predominantly to nitrate. NO consumption by strain PS88 was influenced by the O2 partial pressure (Fig. 6). Maximum activity was observed at 4.3% O2 and slightly lower activities at higher O2 partial pressure. However, under anoxic conditions NO consumption was reduced to only 25% of the maximum activity. NO consumption was not influenced by the nitrite concentration in the mineral medium. Addition of 0.5 and 1 mM KNO2 to cell suspensions did not alter the NO consumption rate (data not shown). Schuster and Conrad (1992) observed in a loess soil high NO consumption activities at very low water contents. To test whether NO consumption by PS88 was related to osmotic stress, NO consumption was determined at different concentrations of NaCl (Fig. 2). NO consumption decreased with increasing osmolarity. In the same manner, growth was inhibited by increasing concentrations of NaCl (Fig. 2). To test the effect of elevated NO mixing ratios on the growth of strain PS88, growth rates were determined in cultures bubbled with air containing different amounts of NO. Concentrations of up to 5 ppmv NO did not affect growth of PS88, while higher concentrations of up to 70 ppmv slightly inhibited growth (< 5% inhibition, data not shown).
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Discussion A new bacterial strain that oxidized NO to nitrate with no detectable free intermediates was described. Strain PS88 represents the first definitive example of an organism that is capable of oxidative NO consumption in soil (Baumgärtner et al. 1996). Comparison of phenotypic data with diagnostic tables in Holt et al. (1994) indicate that strain PS88 is closely related to Pseudomonas fluorescens (which is gelatin-hydrolysis-positive) and Pseudomonas putida (which does not grow on trehalose and m-inositol). The sequence data confirmed the initial tentative identification of strain PS88 by phenotypic characterization as a Pseudomonas species. Sequence comparisons with reference organisms for which sequence data exist, as well as with sequence data from more than 30 species of Pseudomonas species (E. Moore, unpublished data), demonstrated that strain PS88 clusters within the taxon of Pseudomonas sp. However, the sequence similarities derived from comparison with Pseudomonas species (Table 1) indicated that strain PS88 is not a species that has been described previously. The highest sequence similarity is with P. putida (98.5%), thus confirming the phenotypic characterization. However, a 16S rRNA gene sequence difference of 1.5% (22 nucleotides difference) is too large for strains of the same species. Analysis of substrate oxidation (Biolog) by strain PS88 suggested a similarity with P. fluorescens biovar C although the 16S rRNA gene sequence comparison between strain PS88 and P. fluorescens (97.5% similarity) did not suggest a particularly close relationship. However, a Biolog similarity coefficient of 0.54 is not sufficient for strain PS88 to be considered a strain of P. fluorescens. The phenotypic and genetic data clearly demonstrated that strain PS88 is sufficiently different from known species of Pseudomonas to be considered as a new species. However, strain PS88 should not be described as a new species until more strains with identical properties are isolated. Strain PS88 does not belong to one of the bacterial groups for which NO consumption has been established, e.g., denitrifiers or nitrifiers. Consumption of NO has recently been shown in various heterotrophic bacteria such as Bacillus subtilis, Escherichia coli, and different Pseudomonas species (Baumgärtner et al. 1996), but it has not been shown whether NO is oxidized or reduced by these organisms. In strain PS88, however, NO was not reduced to N2O but was instead oxidized, the overall reaction of NO consumption being probably best described by the following equation: 4 NO + 2 H2O + 3 O2→4 NO3– + 4 H+ ∆G°′ = –119.1 kJ (mol NO)–1
(Eq. 2)
Because of the high solubility of NO2 in aqueous solution and its high chemical reactivity, it was not possible to determine whether NO2 is a product of NO oxidation. However, in aqueous solution NO2 reacts to equimolar quantities of NO2– and NO3– (Ignarro et al. 1993). Thus, if NO2
is a product of NO oxidation, production of nitrite should be detectable. However, a significant nitrite production was never detected. Baumgärtner et al. (1996), on the other hand, observed production of nitrite in addition to nitrate when strain PS88 was added to sterile soil. Therefore, we can presently not exclude the possibility that nitrite might be an intermediate of NO oxidation to nitrate. In tissues of higher eukaryotes, NO plays several important roles, e.g., for neurotransmission and immune defense (Bredt and Snyder 1994). NO concentrations in tissues may exist in the micromolar range (Wink et al. 1993). Whereas the production of NO is an enzymatic process carried out by NO synthases, the removal of NO is thought to be due to rapid chemical reactions (Wink et al. 1993; Gaston et al. 1994). The reaction with molecular oxygen is assumed to be important because of its high rate constant (6.6 106 M–2 s–1; Gaston et al. 1994). However, due to the low NO mixing ratios applied in our experiments (i.e., < 4 ppmv NO, equivalent to < 7.5 nM NO in aqueous solution), chemical NO oxidation by O2 was not important, as shown by the slow disappearance of NO in the inactivated controls. Furthermore, NO consumption by strain PS88 was first-order with respect to NO as substrate. This kinetic behavior differs from that of chemical oxidation by molecular oxygen, which is a second-order reaction with respect to NO (Lewis and Deen 1994). A reciprocal plot of the data in Fig. 4 showed linearity for the HgCl2inactivated control. This indicated that the chemical reaction of NO in the gas phase was a second-order process with respect to NO with a rate constant of 52.2 l mol–1 s–1, which is in the range of the reported rate constant (61 l mol–1 s–1) for the reaction of NO with O2 (Huie 1994). The conversion of NO to NO3– might also be explained by a reaction with oxygen radicals, which is thought to be an important mechanism of NO removal in tissues (Oury et al. 1992; Bondy and Naderi 1994; Murphy and Sies 1991). The reaction of NO with O2– (Huie and Padmaja 1993), H2O2 (Stamler et al. 1992), or organic peroxyl radicals (Padmaja and Huie 1993) results in the formation of peroxynitrite, which is rearranged to nitrate as the main product under physiological conditions (Radi et al. 1993; Crow et al. 1994; Gatti et al. 1994). Our results show that the mechanism of NO consumption in strain PS88 is at least partially enzymatic. Thus, if a radical mechanism applies, at least one of the reaction steps, most likely the formation of the peroxyl, should be enzyme-mediated. Another possible mechanism of NO consumption is a stepwise oxidation by a metalloenzyme. NO is readily coordinated at heme (Stamler et al. 1992), non-heme iron, or copper centers (Kroneck and Zumft 1990). In metal-nitrosyl complexes, the metal can accept an electron from the NO•, which formally binds as a nitrosonium ion (Stamler et al. 1992). The following oxidation of the NO+ corresponds to an inverse nitrite reductase; the oxidation of nitrite to nitrate corresponds to the nitrite oxidoreductase of nitrifiers. A final decision on the reaction mechanism of NO oxidation by strain PS88 will require experiments in cell-free systems.
30
According to Eq. 2, the bacteria should be able to get energy from the oxidation of NO. But a simple estimation shows that the energy yield would be too low to significantly increase the growth yield. With the measured firstorder rate constant of NO consumption [547 cm3 h–1 (mg protein)–1] and the maximum energy yield of 119.1 kJ mol–1, the cells would receive 2.66 J h–1 (g protein)–1 from the oxidation of NO at a NO mixing ratio of 1 ppmv. Assuming a minimal substrate demand of 0.31 mmol glucose h–1 (g cells)–1 (as in Escherichia coli; Pirt 1965), a yield of 24 mol ATP per mol glucose, an energy yield of –30 kJ (mol ATP)–1 (Lehninger 1987), and a protein content of 50% of the cell dry weight (Schlegel 1985), a bacterium such as E.coli needs 0.45 kJ h–1 (g protein)–1. Thus, the energy yield from NO consumption at 1 ppmv NO would be only 0.6% of the maintenance energy of the bacterium. If one takes into account that naturally occurring NO mixing ratios are in the parts per trillion to parts per billion range (Conrad 1990), this small energy yield is further reduced. We conclude that oxidation of NO is not an energy-yielding process for strain PS88. Elevated NO mixing ratios are toxic to living organisms. Excretion of NO is thought to be a mechanism by which macrophages kill bacteria, viruses, and cancer cells (Bredt and Snyder 1994). NO can change protein conformation (Girard and Potier 1993), cause mutations (Arroyo et al. 1992), or inhibit bacterial growth (Mancinelli and McKay 1983). However, strain PS88 was fairly resistant against elevated NO mixing ratios up to 70 ppmv. NO consumption by strain PS88 might be a detoxification mechanism to keep NO concentrations low. However, it is also possible that the oxidation of NO is not of any use for the bacteria, but is the result of a co-oxidation. Nitric oxide was consumed by an apparent first-order reaction up to NO mixing ratios of > 4 ppmv, equivalent to aqueous concentrations of > 7.5 nM NO (at 20° C). Therefore, the Km of the NO-oxidizing system must have been at least 7.5 nM NO or higher. The Km for NO of the NO-oxidizing strain PS88 thus appeared to be higher than those (0.2–7.5 nM) reported for NO-reducing denitrifying bacteria (Remde and Conrad 1991; Schäfer and Conrad 1993; McKenney et al. 1994). If this difference is real, then the Km value may be a diagnostic tool for the distinction of oxidative and reductive pathways of NO consumption in soil. In fact, we recently observed that a heathland soil exhibited much lower Km values for NO consumption under oxic (Km > 7.5 nM) than under anoxic (Km < 1.6 nM) incubation conditions (J. Rudolph et al. 1996). Acknowledgements We thank K. Zinkan and I. Arth for occasional technical assistance and P. M. H. Kroneck for helpful discussion. This study was financially supported by the Fonds der Chemischen Industrie. E. Moore was supported in part by the German Ministry of Research and Technology (BMFT Vorhaben 0319–433A) and by the European Union (BIO2-CT94-3098).
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