The induction of fungal denitrification byHere 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 O 2 tension. F. oxysporum prefers O 2 respiration when the O 2 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 O 2 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.Rapid changes in O 2 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 (1-3). Most of these anaerobic eukaryotes have adapted permanently to extreme environments such as swamps and intestines where the O 2 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 O 2 is not available. One form of nitrate respiration results in denitrification (Scheme 2), a strategy that is extensive in facultative anaerobic bacteria (4 -6). Another form (see Scheme 1) has been identified in enterobacteria and other proteobacteria (7).Many fungi can perform denitrification (8 -10). Although the anaerobic process was initially thought to be only a prokaryotic feature (11), the fungal denitrifying system is localized to mitochondria where it acts as a mechanism for anaerobic respiration similar to that of bacteria (12). 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 O 2 supply. The results show that many fungi, which are common in soil and which have been considered aerobic organisms, should real...

Fungal ammonia fermentation is a novel dissimilatory metabolic mechanism that supplies energy under anoxic conditions. The fungus Fusarium oxysporum reduces nitrate to ammonium and simultaneously oxidizes ethanol to acetate to generate ATP (Zhou, Z., Takaya, N., Nakamura, A., Yamaguchi, M., Takeo, K., and Shoun, H. (2002) J. Biol. Chem. 277, 1892-1896). We identified the Aspergillus nidulans genes involved in ammonia fermentation by analyzing fungal mutants. The results showed that assimilatory nitrate and nitrite reductases (the gene products of niaD and niiA) were essential for reducing nitrate and for anaerobic cell growth during ammonia fermentation. We also found that ethanol oxidation is coupled with nitrate reduction and catalyzed by alcohol dehydrogenase, coenzyme A (CoA)-acylating aldehyde dehydrogenase, and acetyl-CoA synthetase (Acs). This is similar to the mechanism suggested in F. oxysporum except A. nidulans uses Acs to produce ATP instead of the ADP-dependent acetate kinase of F. oxysporum. The production of Acs requires a functional facA gene that encodes Acs and that is involved in ethanol assimilation and other metabolic processes. We purified the gene product of facA (FacA) from the fungus to show that the fungus acetylates FacA on its lysine residue(s) specifically under conditions of ammonia fermentation to regulate its substrate affinity. Acetylated FacA had higher affinity for acetyl-CoA than for acetate, whereas non-acetylated FacA had more affinity for acetate. Thus, the acetylated variant of the FacA protein is responsible for ATP synthesis during fungal ammonia fermentation. These results showed that the fungus ferments ammonium via coupled dissimilatory and assimilatory mechanisms.

No abstract

The development of high-performance biobased polymers such as polyimides (PIs) is indispensable to establish a sustainable green society, but it is very difficult due to the incompatibility of their monomeric aromatic diamines with microorganisms. Here, we developed biobased PIs from bioavailable aromatic diamines, which were photodimers of 4-aminocinnamic acid (4ACA) derived from genetically manipulated Escherichia coli. These biobased PI films showed ultrahigh thermal resistance with T 10 values over 425°C and no T g values under 350°C, which is the highest value of all biobased plastics reported thus far. The PI films also showed high tensile strength, high Young's moduli, good cell compatibility, excellent transparency, and high refractive indices.

Two chitin synthase genes, designated chsA and chsB, were isolated from Aspergillus nidulans with the Saccharomyces cerevisiae CHS2 gene as the hybridization probe. Nucleotide sequencing showed that chsA and chsB encoded polypeptides consisting of 1013 and 916 amino acid residues, respectively; the hydropathy profiles of the enzymes were similar to those of other fungal chitin synthases. Northern analysis indicated that both genes were transcribed, suggesting that cellular chitin in A. nidulans is synthesized by at least two chitin synthases. For examination of the roles of the chitin synthase genes in cell growth, gene disruption experiments were done. The chsA disruptant grew as well as the wild-type strain, but the chsB disruptant had severe growth defects that could not be overcome by the addition of 1.2M sorbitol as an osmotic stabilizer. These findings suggested that chsB but not chsA is essential for hyphal growth.

Anaerobic growth of Pseudomonas aeruginosa PAO1 was affected by quorum sensing. Deletion of genes that produce N-acyl-L-homoserine lactone signals resulted in an increase in denitrification activity, which was repressed by exogenous signal molecules. The effect of the las quorum-sensing system was dependent on the rhl quorum-sensing system in regulating denitrification.Bacteria regulate their metabolism to adapt to various conditions by sensing environmental signals. Under anoxic conditions, many bacteria are able to use N-oxides as terminal electron acceptors. Pseudomonas aeruginosa is a denitrifying bacterium capable of anaerobic growth by utilizing N-oxides such as nitrate (NO 3 Ϫ ) and nitrite (NO 2 Ϫ ). Denitrification is induced under low-oxygen conditions when N-oxides are also present (1, 9, 11).Recent studies on bacteria have revealed new types of environmental signals known as cell-to-cell communication signals (25). P. aeruginosa is reported to control gene expression globally in response to cell density by utilizing N-acyl-L-homoserine lactone (AHL) signals. This cell-density-dependent regulation is termed quorum sensing (5). P. aeruginosa possesses at least two quorum-sensing systems: the LasR-LasI (las) and RhlR-RhlI (rhl) systems (20). LasI directs the synthesis of the AHL signal N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C 12 -HSL) (17, 18), and RhlI directs the synthesis of another AHL signal, N-butyryl-L-homoserine lactone (C 4 -HSL) (19). The transcription regulatory proteins, LasR (6) and RhlR (16), are specifically activated by 3-oxo-C 12 -HSL and C 4 -HSL, respectively. Previous studies have indicated that production of virulence factors, such as protease, exotoxin A, rhamnolipids, and siderophores, is regulated by quorum sensing in P. aeruginosa (7,12,26), suggesting that quorum sensing is important in the pathogenesis of infection with this bacterium.Recent transcriptomic and proteomic studies indicate that quorum sensing is a global regulation system in P. aeruginosa (3,23,28). From these findings, it can be presumed that quorum-sensing systems have ecologically important roles in addition to the control of pathogenesis for the bacterium. For example, recent work suggests that quorum sensing regulates the activities of denitrification enzymes. In a recent study by Yoon et al. (31), the authors reported that levels of denitrifying enzyme activities of anaerobically grown P. aeruginosa cells are higher for an rhlR mutant than for its parent strain in an in vitro system. We were interested in further characterizing the potential anaerobic regulation of denitrification by the quorum-sensing system. Here we present a comprehensive analysis of the impact of quorum sensing on the denitrification pathway under anaerobic conditions by using in vivo and in vitro analyses.Effect of quorum sensing on denitrification activity. P. aeruginosa PAO1 was cultured anaerobically in 17-ml Hungate tubes containing 5 ml Luria-Bertani (LB) medium supplemented with 100 mM KNO 3 with shaking at 200 rpm at 37...

Pseudomonas aeruginosa and other Gram-negative bacteria release membrane vesicles (MVs) from their surfaces, and MVs have an ability to interact with bacterial cells. Although it has been known that many bacteria have mechanisms that control their phenotypes with the transition from exponential phase to stationary phase, changes of properties in released MVs have been poorly understood. Here, we demonstrate that MVs released by P. aeruginosa during the exponential and stationary phases possess different physiochemical properties. MVs purified from the stationary phase had higher buoyant densities than did those purified from the exponential phase. Surface charge, characterized by zeta potential, of MVs tended to be more negative as the growth shifted to the stationary phase, although the charges of PAO1 cells were not altered. Pseudomonas quinolone signal (PQS), one of the regulators related to MV production in P. aeruginosa, was lower in MVs purified from the exponential phase than in those from the stationary phase. MVs from the stationary phase more strongly associated with P. aeruginosa cells than did those from the exponential phase. Our findings suggest that properties of MVs are altered to readily interact with bacterial cells along with the growth transition in P. aeruginosa.

Most denitrifiers produce nitrous oxide (N 2 O) instead of dinitrogen (N 2 ) under aerobic conditions. We isolated and characterized novel aerobic denitrifiers that produce low levels of N 2 O under aerobic conditions. We monitored the denitrification activities of two of the isolates, strains TR2 and K50, in batch and continuous cultures. Both strains reduced nitrate (NO 3 ؊ ) to N 2 at rates of 0.9 and 0.03 mol min ؊1 unit of optical density at 540 nm ؊1 at dissolved oxygen (O 2 ) (DO) concentrations of 39 and 38 mol liter ؊1 , respectively. At the same DO level, the typical denitrifier Pseudomonas stutzeri and the previously described aerobic denitrifier Paracoccus denitrificans did not produce N 2 but evolved more than 10-fold more N 2 O than strains TR2 and K50 evolved. The isolates denitrified NO 3؊ with concomitant consumption of O 2 . These results indicated that strains TR2 and K50 are aerobic denitrifiers. These two isolates were taxonomically placed in the ␤ subclass of the class Proteobacteria and were identified as P. stutzeri TR2 and Pseudomonas sp. strain K50. These strains should be useful for future investigations of the mechanisms of denitrifying bacteria that regulate N 2 O emission, the single-stage process for nitrogen removal, and microbial N 2 O emission into the ecosystem.Nitrous oxide (N 2 O) is a gaseous nitrogen oxide that is present at a concentration of about 350 ppb in the atmosphere. The concentration of this compound was maintained below 300 ppb in the global nitrogen cycle before the 20th century. However, recent reports suggest that the atmospheric concentration of N 2 O is now increasing at a rate as high as 0.3% per year (1). N 2 O has a 200-to 300-fold-stronger greenhouse effect than carbon dioxide (CO 2 ) and has the potential to destroy the ozone layer (17). Therefore, the N 2 O balance is critical to the natural environment. The proposed sources of N 2 O are chemical industries, burning fossil fuels, and biomass, as well as soil denitrification of nitrogenous compounds resulting from excess agricultural fertilizer (3,6,25). Another critical source of N 2 O is wastewater treatment plants, in which considerable amounts of nitrogen pollutants removed from treated water are released into the atmosphere as N 2 O, as well as dinitrogen (N 2 ).Currently, nitrogen removal in wastewater treatment plants is essentially based on the activity of nitrifying and denitrifying microorganisms, both of which are inhabitants of activated sludge. Nitrifying bacteria aerobically oxidize ammonium contaminants to nitrite (NO 2 Ϫ ) and nitrate (NO 3 Ϫ ), which are then reduced by denitrifying bacteria to gaseous nitrogen forms such as N 2 O and N 2 . Efficient wastewater treatment relies on successively exposing water to aerobic and anaerobic conditions, since nitrification and denitrification are aerobic and anaerobic processes, respectively (4, 18). These properties represent a shortcoming of current systems since both denitrification and nitrification produce N 2 O as a by-product in the absen...