ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTKinetics and Thermodynamics of CO Binding to Cytochrome P450norYoshitsugu Shiro, Minoru Kato, Tetsutaro Iizuka, Kazuhiko Nakahara, and Hirofumi ShounCite this: Biochemistry 1994, 33, 29, 8673–8677Publication Date (Print):July 1, 1994Publication History Published online1 May 2002Published inissue 1 July 1994https://pubs.acs.org/doi/10.1021/bi00195a007https://doi.org/10.1021/bi00195a007research-articleACS PublicationsRequest reuse permissionsArticle Views117Altmetric-Citations22LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose Get e-Alerts
The induction of fungal denitrification byFusarium oxysporum requires a minimal amount of O2, although excess O2 completely represses this process (Zhou, Z., Takaya, N., Sakairi, M. A. C., and Shoun, H. (2001) Arch. Microbiol. 175, 19–25). Here we describe another metabolic mechanism of nitrate in fungal cells, termed ammonia fermentation, that supports growth under conditions more anoxic than those of denitrification. The novel nitrate metabolism of eukaryotes consists of the reduction of nitrate to ammonium coupled with the catabolic oxidation of electron donors to acetate and substrate-level phosphorylation. F. oxysporum thus has two pathways of dissimilatory nitrate reduction that are alternatively expressed in response to environmental O2 tension. F. oxysporum prefers O2 respiration when the O2 supply is sufficient. We discovered that this fungus is the first eukaryotic, facultative anaerobe known to express one of three distinct metabolic energy mechanisms closely depending on environmental O2 tension. We also showed that ammonia fermentation occurs in many other fungi that are common in soil, suggesting that facultative anaerobes are widely distributed among fungi that have been considered aerobic organisms. The induction of fungal denitrification byFusarium oxysporum requires a minimal amount of O2, although excess O2 completely represses this process (Zhou, Z., Takaya, N., Sakairi, M. A. C., and Shoun, H. (2001) Arch. Microbiol. 175, 19–25). Here we describe another metabolic mechanism of nitrate in fungal cells, termed ammonia fermentation, that supports growth under conditions more anoxic than those of denitrification. The novel nitrate metabolism of eukaryotes consists of the reduction of nitrate to ammonium coupled with the catabolic oxidation of electron donors to acetate and substrate-level phosphorylation. F. oxysporum thus has two pathways of dissimilatory nitrate reduction that are alternatively expressed in response to environmental O2 tension. F. oxysporum prefers O2 respiration when the O2 supply is sufficient. We discovered that this fungus is the first eukaryotic, facultative anaerobe known to express one of three distinct metabolic energy mechanisms closely depending on environmental O2 tension. We also showed that ammonia fermentation occurs in many other fungi that are common in soil, suggesting that facultative anaerobes are widely distributed among fungi that have been considered aerobic organisms. Institute for Fermentation Osaka alcohol dehydrogenase acetaldehyde dehydrogenase (acylating) acetate kinase Rapid changes in O2 supply are an ongoing challenge for many organisms living in environments such as soil. Facultative anaerobes are widely distributed among prokaryotes and can adapt immediately to rapid changes in aeration by altering their energy metabolism. On the contrary, much less is known about such adaptive techniques in eukaryotes, although many lower eukaryotes can survive under anoxic conditions (1Embley T.M. Martin W.A. Nature. 1998; 396: 517-519Crossref PubMed Scopus (38) Google Scholar, 2Tielens A.G.M. Van Hellemond J.J. Biochim. Biophys. Acta. 1998; 1365: 71-783Crossref PubMed Scopus (120) Google Scholar, 3Muller M. J. Gen. Microbiol. 1993; 139: 2879-2889Crossref PubMed Scopus (338) Google Scholar). Most of these anaerobic eukaryotes have adapted permanently to extreme environments such as swamps and intestines where the O2 supply is always poor. Thus, they are obligate, not facultative, anaerobes. Nitrate is generally metabolized by organisms in assimilatory and dissimilatory reductive pathways. Bacteria, fungi, and plants reduce nitrate to ammonium to assimilate nitrogen into their biomass (Scheme 1). Dissimilatory reduction (nitrate respiration) is performed by many bacteria in which nitrate is used as an alternative electron acceptor for respiration when O2 is not available. One form of nitrate respiration results in denitrification (Scheme 2), a strategy that is extensive in facultative anaerobic bacteria (4Zumft W.G. Balows A. Trüper H.G. Dworkin M. Harder W. Schleifer K.-H. The Prokaryotes. 2nd Ed. Springer, Berlin1992: 554-582Google Scholar, 5Berks B.C. Ferguson S.J. Moir J.W.B. Richardson D.J. Biochim. Biophys. Acta. 1995; 1232: 97-173Crossref PubMed Scopus (498) Google Scholar, 6Zumft W.G. Microbiol. Mol. Biol. Rev. 1997; 61: 533-616Crossref PubMed Scopus (2852) Google Scholar). Another form (see Scheme 1) has been identified in enterobacteria and other proteobacteria (7Page L. Griffiths L. Cole J.A. Arch. Microbiol. 1990; 154: 349-354Crossref PubMed Scopus (101) Google Scholar).NO3−→NO2−→NH4+SCHEME1NO3−→NO2−→NO→N2O→N2SCHEME2Many fungi can perform denitrification (8Shoun H. Tanimoto T. J. Biol. Chem. 1991; 266: 11078-11082Abstract Full Text PDF PubMed Google Scholar, 9Shoun H. Kim D.-H. Uchiyama H. Sugiyama J. FEMS Microbiol. Lett. 1992; 94: 277-282Crossref Google Scholar, 10Tsuruta S. Takaya N. Zhang L. Shoun H. Kimura K. Hamamoto M. Nakase T. FEMS Microbiol. Lett. 1998; 168: 105-110Crossref PubMed Google Scholar). Although the anaerobic process was initially thought to be only a prokaryotic feature (11Ferguson S.J. Cur. Opinion Chem. Biol. 1998; 2: 182-193Crossref PubMed Scopus (125) Google Scholar), the fungal denitrifying system is localized to mitochondria where it acts as a mechanism for anaerobic respiration similar to that of bacteria (12Kobayashi M. Matsuo Y. Takimoto A. Suzuki S. Maruo F. Shoun H. J. Biol. Chem. 1996; 271: 16263-16297Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). The finding of denitrification in fungi suggests that such organisms are eukaryotic facultative anaerobes. Here, we present evidence for ammonia fermentation, a second form of dissimilatory nitrate metabolism in denitrifying fungi, and for the alternative expression of ammonia fermentation and denitrification under anaerobic conditions in response to the O2supply. The results show that many fungi, which are common in soil and which have been considered aerobic organisms, should really be classified as facultative anaerobes. Fusarium oxysporumMT-811 (8Shoun H. Tanimoto T. J. Biol. Chem. 1991; 266: 11078-11082Abstract Full Text PDF PubMed Google Scholar) was mainly studied throughout this investigation. Other fungal strains were obtained from Institute for Fermentation Osaka (IFO)1 and Institute of Applied Microbiology, University of Tokyo. The culture medium consisted of basal mineral medium (8Shoun H. Tanimoto T. J. Biol. Chem. 1991; 266: 11078-11082Abstract Full Text PDF PubMed Google Scholar) supplemented with carbon (ethanol, unless otherwise stated) and nitrogen (indicated) sources. Time-dependent changes in components (see Fig. 1) or effects of aeration (see Fig. 2) were determined using cultures at 30 °C in a 1-liter volume jar fermentor containing 500 ml of medium (13Zhou Z. Takaya N. Sakairi M.A.C. Shoun H. Arch. Microbiol. 2001; 175: 19-25Crossref PubMed Scopus (111) Google Scholar). Aeration effects were examined in fed-batch cultures (13Zhou Z. Takaya N. Sakairi M.A.C. Shoun H. Arch. Microbiol. 2001; 175: 19-25Crossref PubMed Scopus (111) Google Scholar) in which nitrate was supplemented as soon as it was exhausted. After repeating two cycles of fed-batch cultures to determine adaptation, the effects of aeration on nitrate metabolism were determined in one cycle of the culture that continued from renewal of the nitrate supplement until its complete consumption.Figure 2Effects of aeration on nitrate metabolism.F. oxysporum was examined in fed-batch cultures in medium supplemented with 10 mm nitrate (without ammonium) under various aeration conditions (0, 10, 20, or 30 ml O2/h). Ammonium (●) or N2O (○) was determined after one cycle of each culture and expressed as % recovery of nitrogen atoms against consumed (i.e. added) nitrate. Nitrate utilized for assimilation (▪) was estimated by subtracting total amounts of formed ammonium and N2O from amount of initially added nitrate. Dry cell matter was estimated as average before and after a one-cycle culture.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Other cultures were maintained in flasks. Precultures were prepared semi-aerobically incubating the fungus at 30 °C for 3 days in glycerol/peptone/nitrate (5 mm) medium (13Zhou Z. Takaya N. Sakairi M.A.C. Shoun H. Arch. Microbiol. 2001; 175: 19-25Crossref PubMed Scopus (111) Google Scholar). The mycelia in the preculture, collected and washed three times with sterilized saline, were used to inoculate the culture to be examined. A portion of the mycelia (corresponding to the amount of cells obtained from a 20-ml preculture) was transferred to a 500-ml Erlenmeyer flask containing 100 ml of medium supplemented with 600 mm ethanol and 10 mm ammonium and/or nitrate. The flask was sealed with a rubber stopper after purging air from the medium and head space by flashing with argon gas (anoxic conditions) or sealed with a cotton plug without purging the air (aerobic conditions). Dry cell weight was determined after drying washed cells at 94 °C for 24 h. Enzyme activities related to nitrate metabolism were determined with each subcellular fraction prepared from the fungal cells grown under various aeration conditions. F. oxysporum was rotary-shaken in a 5-liter Erlenmeyer flask containing 1 liter of medium containing 10 mm nitrate under each aeration condition. Anoxic (ammonia-fermenting) and aerobic (nitrate-assimilating) conditions were obtained, respectively, as described above. Sealing the flask with a rubber stopper without replacing head space air generated hypoxic (denitrifying) conditions (8Shoun H. Tanimoto T. J. Biol. Chem. 1991; 266: 11078-11082Abstract Full Text PDF PubMed Google Scholar). Non-induced cells were prepared by incubating under anoxic conditions in the absence of nitrate (nitrate was replaced with ammonium). Fungal cells were harvested at the early stage of exponential growth and then disrupted and fractionated into subcellular fractions as reported (12Kobayashi M. Matsuo Y. Takimoto A. Suzuki S. Maruo F. Shoun H. J. Biol. Chem. 1996; 271: 16263-16297Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). All enzyme activities were measured anaerobically at 30 °C in subcellular fractions from fungal cells cultured under various conditions. Alcohol dehydrogenase (Ald) was assayed by following the production of NADH from 50 mmethanol and 5 mm NAD+. Acetaldehyde dehydrogenase (acylating) (AddA) was assayed by determining the amount of acetyl-CoA formed during an incubation with 50 mmacetaldehyde, 5 mm NAD+, and 5 mmcoenzyme A. Acetate kinase (Ack) was assayed by determining the amount of ATP formed from 5 mm ADP and 5 mmacetyl-CoA. Acetyl-CoA hydrolyase was assayed by determining the amount of acetate formed from 5 mm acetyl-CoA. Acetaldehyde dehydrogenase activity was measured by incubating with 50 mm acetaldehyde and 5 mm NAD+, and the amount of formed acetate was measured. Acetyl-CoA synthetase was assayed by measuring the amount of acetyl-CoA formed during an incubation with 50 mm acetaldehyde, 5 mmcoenzyme A, and 5 mm ATP. Nitrate reductase and nitrite reductase were, respectively, assayed by incubating with 10 mm sodium nitrate or sodium nitrite and 2 mmNADH, and the amount of generated nitrite or ammonium was determined. Buffers were as follows: 70 mm Tris-HCl (pH 7.2) for Ald, AddA, acetaldehyde dehydrogenase, and acetyl-CoA synthetase assays and 70 mm potassium phosphate (pH 7.2) for Ack, acetyl-CoA hydrolyase, nitrate reductase, and nitrite reductase assays. Nitrate, nitrite, ammonium, and acetate were measured by ion chromatography using a 761 Compact IC (Metrohm). N2O and CO2 were determined by gas chromatography (13Zhou Z. Takaya N. Sakairi M.A.C. Shoun H. Arch. Microbiol. 2001; 175: 19-25Crossref PubMed Scopus (111) Google Scholar). ATP was assayed using a luminometer (Lumitester K-21; Kikkoman) (12Kobayashi M. Matsuo Y. Takimoto A. Suzuki S. Maruo F. Shoun H. J. Biol. Chem. 1996; 271: 16263-16297Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). Acetyl-CoA was determined as reported (14Klotzsch H.R. Methods Enzymol. 1969; 13: 381-386Crossref Scopus (27) Google Scholar). Transmission electron microscopy photographs were obtained as described (15Takaya N. Suzuki S. Kuwazaki S. Shoun H. Maruo F. Yamaguchim M. Takeno K. Arch. Biochem. Biophys. 1999; 372: 340-346Crossref PubMed Scopus (58) Google Scholar) using a JEM-1200 EX transmission electron microscope (JEOL Ltd., Tokyo, Japan). We showed that the induction of denitrifying activity in the fungus F. oxysporum MT-811 requires a minimal amount of aeration (hypoxic conditions) and that the recovery of nitrate-nitrogen into N2O (denitrification yield) varies considerably depending on the extent of the O2 supply (13Zhou Z. Takaya N. Sakairi M.A.C. Shoun H. Arch. Microbiol. 2001; 175: 19-25Crossref PubMed Scopus (111) Google Scholar). The coexistence of ammonium in the medium should improve the denitrification yield, because ammonium generally represses the assimilatory use of nitrate. Here we incubated hypoxic cultures (13Zhou Z. Takaya N. Sakairi M.A.C. Shoun H. Arch. Microbiol. 2001; 175: 19-25Crossref PubMed Scopus (111) Google Scholar) in the presence of both 10 mm nitrate and 10 mmammonium to examine effects of ammonium on the denitrification yield (Fig. 1A). In agreement with prior observations (13Zhou Z. Takaya N. Sakairi M.A.C. Shoun H. Arch. Microbiol. 2001; 175: 19-25Crossref PubMed Scopus (111) Google Scholar), denitrification (N2O formation) was induced over a long time course (stage 3). This time, however, we observed an unusual phenomenon during the lag period before denitrification was induced. Ammonium is usually preferred over nitrate for use in assimilation, a characteristic of stage 1 cultures. At stage 2, nitrate levels began to decrease, and nitrite and ammonium concomitantly accumulated in the medium. At stage 3, nitrate decreased rapidly with concomitant evolution of N2O. Gas chromatography-mass spectrometry with the stable isotope species of nitrate, 14NO3− and15NO3−, showed that ammonium ions, as well as N2O (8Shoun H. Tanimoto T. J. Biol. Chem. 1991; 266: 11078-11082Abstract Full Text PDF PubMed Google Scholar), were derived from nitrate during anoxic culture (data not shown) (16Nakane R. J. Mass Spectrom. Soc. Jpn. 1963; 22: 51-58Crossref Google Scholar). By contrast, the decrease or increase of each compound was linear over the entire course of the culture when ammonium was the sole nitrogen source (Fig. 1B). Nitrate did not seem to be converted to ammonium for assimilatory purposes, because a sufficient amount of ammonium remained in the medium when the conversion began. On the other hand, cell growth was significantly increased when nitrate was added (cf. Fig. 1,A and B), indicating that nitrate metabolism is an energy-yielding process. These findings show that nitrate is metabolized in a dissimilatory pathway to form ammonium at stage 2 and N2O at stage 3 (Fig. 1A). The evolution of N2O was accompanied by the accelerated evolution of CO2, indicating that the carbon source (ethanol) was decomposed as a respiration substrate for denitrification. In contrast, the rate of CO2 evolution during stage 2 was much lower, suggesting that the reducing equivalent for the formation of ammonium was supplied by oxidizing ethanol to another C-2 compound. The effect of O2 supply on nitrate metabolism by F. oxysporum MT-811 was examined using fed-batch cultures under various aeration conditions (0, 10, 20, or 30 ml O2/h) in which nitrate was the only nitrogen source (Fig.2). Ammonium or N2O was determined for each culture when nitrate and formed nitrite disappeared. The rate of nitrate conversion to ammonium was highest in the complete absence of an O2 supply. With increasing aeration, the recovery of ammonium declined, and conversely, the evolution of N2O increased. Less than 20% of nitrate was used for assimilation under these O2-limited conditions. With an excess O2 supply, neither ammonium nor N2O was formed, although nitrate was consumed, indicating that nitrate is utilized only for assimilation, and that accelerated cell-growth (cf. Fig. 3,right) depends on O2 respiration. The nitrogen source-dependent growth of flask cultures in the absence of O2 (Fig. 3, left) indicates that nitrate supports more growth than ammonium. Because only slight denitrification occurs without O2 (Fig. 2), nitrate-mediated anoxic growth must depend on nitrate metabolism to form ammonium. Aerobic cultures in the same medium increased enormously the yield of the cells (Fig. 3, right), supporting the conclusion derived from Fig. 2 that the anaerobic nitrate metabolism (ammonia formation and denitrification) is replaced by O2respiration when the O2 supply is sufficient. Fig.4 shows transmission electron microscopy observations of mitochondria. Most mitochondria in the anoxic cells that formed ammonium (Fig. 4B) were immature and exhibited low electron density, in sharp contrast to the apparently intact mitochondria of cells grown under aerobic conditions (Fig.4A) or in denitrifying cells (15Takaya N. Suzuki S. Kuwazaki S. Shoun H. Maruo F. Yamaguchim M. Takeno K. Arch. Biochem. Biophys. 1999; 372: 340-346Crossref PubMed Scopus (58) Google Scholar). These results demonstrate that anoxic nitrate metabolism forms ammonium in a non-respiratory system that produces ATP. We term this method of eukaryotic nitrate metabolism “ammonia fermentation.” We examined ammonia fermentation by F. oxysporum cultured in flasks under anoxic conditions with ethanol, glycerol, or glucose as the carbon source (TableI). Under these conditions, most nitrate-nitrogen was recovered into ammonium, consistent with the results shown in Fig. 2, and recovery of ammonium was high in the presence of ethanol. Ammonia fermentation was accompanied by acetate accumulation, as predicted above from the results in Fig.1A. The stoichiometry of the formed ammonium and acetate was exactly 1:2 except for the culture with glucose. This indicates that the reducing equivalent derived from 4-electron oxidation to form acetate is utilized in the 8-electron reduction of nitrate to ammonium, consistent with the oxidation of ethanol to acetate. The recovery of ammonium was low in cells cultured with glucose, and instead, much more CO2 evolved, suggesting that alcohol fermentation was predominant.Table IStoichiometry between the products because of ammonia fermentation by F. oxysporumCarbon sourceProduction yieldNH4+1-a% yield against consumed nitrate-N.N2O1-a% yield against consumed nitrate-N.CO2Acetatemmol%mmol%mmolmmolEthanol0.8 ± 0.05800.05 ± 0.02100.45 ± 0.11.6 ± 0.2Glycerol0.7 ± 0.05700.06 ± 0.02120.61 ± 0.11.4 ± 0.2Glucose0.2 ± 0.05400.03 ± 0.01126.8 ± 0.80.6 ± 0.1The cells were incubated by the anoxic flask culture as in Fig. 3(left) in the medium containing only nitrate (10 mm, 1 mmol) for 5 days, and each product was determined. Nitrate was completely consumed by the end of each culture with the exception of that with glucose in which a half of nitrate still remained.1-a % yield against consumed nitrate-N. Open table in a new tab The cells were incubated by the anoxic flask culture as in Fig. 3(left) in the medium containing only nitrate (10 mm, 1 mmol) for 5 days, and each product was determined. Nitrate was completely consumed by the end of each culture with the exception of that with glucose in which a half of nitrate still remained. The possible metabolic pathway described in Fig. 5 is based upon the above finding that ethanol is the best electron donor for ammonia fermentation and that the stoichiometry between the fermentation products, ammonium and acetate, is exactly 1:2. We determined the enzyme activities involved in each step using subcellular fractions prepared from cells cultured under aerobic (assimilatory, with respect to nitrate), hypoxic (denitrifying), or anoxic (ammonia-fermenting) conditions (TableII). AddA and the ATP-forming Ack activities were specifically induced only in anoxic cells that fermented ammonia, whereas other acetogenic activities (acetyl-CoA hydrolyase, acetaldehyde dehydrogenase, and acetyl-CoA synthetase) were particularly low (or absent) in the cells. These results demonstrated that ethanol is oxidized successively by Ald, AddA, and Ack to form acetate, coupled with ATP production and the reduction of nitrate to ammonia (Fig. 5), and that ammonia fermentation is an adaptive metabolism of the fungus F. oxysporum. These activities (Ald, AddA, and Ack) were recovered in the soluble and microsome fractions but not in the mitochondrial fraction, which would be consistent with the increasing population of damaged mitochondria in the anoxic cells (Fig. 4). NADH-dependent properties of nitrate reductase and nitrite reductase activities (Scheme 1) are quite distinct from those of the mitochondrial dissimilatory nitrate and nitrite reductases of the denitrifying cells (12Kobayashi M. Matsuo Y. Takimoto A. Suzuki S. Maruo F. Shoun H. J. Biol. Chem. 1996; 271: 16263-16297Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar) but are similar to the assimilatory reductases generally found among fungi.Table IISpecific activity and subcellular distribution of enzymes involved in ammonia fermentationFractionActivity (nmol each product indicated·min−1·mg protein−1)Ald (CH3CHO)AddA (CH3CO·CoA)Ack (ATP)Ath (CH3COOH)Add (CH3COOH)Acs (CH3CO·CoA)Nar (NO2−)Nir (NH4+)Aerobic (assimilation)Soluble175 ± 2000400 ± 201250 ± 1003125 ± 12060 ± 920 ± 6Microsome562 ± 4000310 ± 10300 ± 20760 ± 2610 ± 20Mitochondrion00000020 ± 50Hypoxic (denitrification)Soluble24 ± 403 ± 2260 ± 151000 ± 301040 ± 8824 ± 53 ± 3Microsome76 ± 1005 ± 3240 ± 20260 ± 12241 ± 148 ± 20Mitochondrion00000030 ± 40Anoxic (Ammonia fermentation)Soluble4 ± 1300 ± 1030 ± 12130 ± 1020 ± 5040 ± 1012 ± 6Microsome11 ± 31500 ± 20025 ± 4205 ± 140012 ± 60Mitochondrion00000018 ± 30Anoxic (non-induced cells)Crude extract2 ± 210 ± 2034 ± 821 ± 510 ± 69 ± 42 ± 1 Open table in a new tab Ammonia-fermenting activity was screened in 17 fungal strains closely or distantly related to F. oxysporum MT-811 cultured under anoxic flask conditions as shown in Fig. 3(left). Only two among the 17 strains tested did not exhibit this activity, and the following 15 strains distinctly converted nitrate to ammonium under anoxic conditions: Talaromyces rotundus (IFO; 9142), Trichophyton rubrum(IFO; 5807), Hyalodendron sp. (IFO; 31243),Penicillium abeanum (IFO; 6239), Petriella guttulata (IFO; 8613), Calonectria kyotensis (IFO; 8962), Hypocrea nigricans (IFO; 31290), Hypomyces trichothecoides (IFO; 6892), Neurospora crassa (IFO; 6067), F. oxysporum (IFO; 30710), F. oxysporum (IFO; 9968), Cylindrocarpon tonkinense (IFO; 30561), Gibberella fujikuroi (IFO; 6349), F. oxysporum (Institute of Applied Microbiology, University of Tokyo; 5009), Fusarium lini (Institute of Applied Microbiology, University of Tokyo; 5008), and the following two strains did not:Podospora carbonaria (IFO; 31850), Orbimyces spectabilis (IFO; 32157). Most of these strains are found in subdivisions of ascomycotina and ascomycetous imperfect fungi with the exceptions of Hyalodendron sp. (Mastigomycotina) and P. carbonaria (Zygomycotina). The present study presents evidence that the denitrifying fungusF. oxysporum contains another anaerobic type of nitrate metabolism, which we refer to as ammonia fermentation. We also showed that ammonia fermentation and denitrification are alternatively expressed depending on the extent of the O2 supply. Ammonia fermentation is expressed under the most anoxic conditions even in the complete absence of O2 supply. Small but distinct cell growth during ammonia fermentation (see Fig. 1 and Fig. 3) should depend on substrate-level phosphorylation by Ack (acetate kinase), which is specifically induced in cells that ferment ammonia (Table II). Anoxic nitrate metabolism (ammonia fermentation) is replaced by denitrification (Fig. 2) with concomitant formation of intact mitochondria when the O2 supply is limited, and under a sufficient supply of O2, denitrification is further replaced by aerobic (O2) respiration. Thus the fungusF. oxysporum that expresses diversified pathways of nitrate metabolism closely regulates energy metabolism in response to environmental O2 tension, ammonia fermentation under anoxic conditions, denitrification when hypoxic, and oxygen respiration under aerobic conditions. This study is the first to show that an organism (or eukaryote) can use a multimodal type of respiration (or ATP-producing) system to rapidly adapt to changes in the oxygen supply. Ammonia fermentation was limited when glucose was the carbon source (Table I). The ammonium recovery was low, but far more CO2evolved, suggesting that alcohol fermentation predominates over ammonia fermentation when glucose is available. F. oxysporumferments alcohol, which has been understood for some time (17Singh A. Kumar P.K. Crit. Rev. Biotechnol. 1991; 11: 129-147Crossref PubMed Scopus (48) Google Scholar). The recovery of ammonium was highest when ethanol was the electron donor (Table I), indicating that ammonia fermentation acts physiologically as a secondary fermentation method that replaces primary alcohol fermentation when nitrate is available. This fungal process is similar to acetogenic fermentation in prokaryotes, a reaction that is coupled to nitrate reduction and substrate-level phosphorylation reactions (18Ishimoto M. Umeyama M. Chiba S. Z. Allg. Mikrobiol. 1974; 14: 115-121PubMed Google Scholar, 19Seifritz C. Daniel S.L. Gossner A. Drake H.L. J. Bacteriol. 1993; 175: 8008-8013Crossref PubMed Google Scholar). However, bacterial nitrate metabolism is highly restricted, arising only in a single genus of obligate anaerobes, Clostridium, and it is regarded as a primitive form of anaerobic respiration in these bacteria (20Hasan S.M. Hall J.B. J. Gen. Microbiol. 1975; 87: 120-128Crossref PubMed Scopus (74) Google Scholar). It is therefore surprising to find anoxic nitrate metabolism (ammonia fermentation) in eukaryotes (fungi) that have been considered to date as aerobic organisms. Recent advances in genome projects, along with our screening results (21Zhang L. Takaya N. Kitazume T. Kondo T. Shoun H. Eur. J. Biochem. 2001; 268: 3198-3204Crossref PubMed Scopus (48) Google Scholar), have revealed that cytochrome P450nor, a characteristic enzyme in fungal denitrifying systems that functions as nitric-oxide reductase, is expressed widely among fungi. This indicates that denitrification is a key mechanism of nitrate metabolism in fungi. We further demonstrate here that ammonia is also fermented in the denitrifying fungus F. oxysporum MT-811 and in many other fungi that are common to soil. These results demonstrated that many such fungi should really be classified as facultative, rather than as obligate aerobes, although most fungi are in fact so far recognized as obligate aerobes. Advances in soil microbiology have revealed that the microbial biomass of temperate soils is usually dominated by fungi (22Ruzicka S. Edgerton D. Norman M. Hill T. Soil Biol. Biochem. 2000; 32: 989-1005Crossref Scopus (132) Google Scholar). Our present conclusion that many soil fungi are facultative anaerobes is consistent with this fact. The fungal prosperity in soil should be supported by a multirespiratory system, because the natural environment in soil with respect to oxygen supply is highly variable, and the ability to immediately adapt to rapid changes should be a potent tool for survival. The present results should thus evoke interest in how eukaryotes have evolved such a variety of metabolic mechanisms to produce energy.
This article is a report on a symposium sponsored by the American Society for Pharmacology and Experimental Therapeutics and held at the Experimental Biology 01 meeting in Orlando, FL. The presentations addressed the mechanisms of inhibition and regulation of cytochrome P450 and flavin monooxygenase enzymes by nitric oxide. They also highlighted the consequences of these effects on metabolism of drugs and volatile amines as well as on important physiological parameters, such as control of blood pressure, renal ion transport, and steroidogenesis. This is achieved via regulation of P450-dependent prostacyclin, hydroxyeicosatetraenoic acid, and epoxyeicosatrienoic acid formation. Conversely, the mechanisms and relative importance of nitric oxide synthases and P450 enzymes in NO production from endogenous and synthetic substrates were also addressed.
