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.

A novel fungal β-glucosidase gene (bgl4) and its homologue (bgl2) were cloned from the cellulolytic fungi Humicola grisea and Trichoderma reesei, respectively. The deduced amino acid sequences of H. grisea BGL4 and T. reesei BGL2 comprise 476 and 466 amino acids, respectively, and share 73.1% identity. These β-glucosidases show significant homology to plant β-glucosidases belonging to the β-glucosidase A (BGA) family. Both genes were expressed in Aspergillus oryzae, and the recombinant β-glucosidases were purified. Recombinant H. grisea BGL4 is a thermostable enzyme compared with recombinant T. reesei BGL2. In addition to β-glucosidase activity, recombinant H. grisea BGL4 showed a significant level of β-galactosidase activity, while recombinant T. reesei BGL2 showed weak β-galactosidase activity. Cellulose saccharification by Trichoderma cellulases was improved by the addition of recombinant H. grisea BGL4.

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.

Two thermophilic strains, designated 607^T and 606b, were isolated from a compost pile in Japan. The novel strains were Gram-positive, aerobic, spore-forming rods. Phylogenetic analyses based on 16S rRNA gene sequences revealed that strains 607^T and 606b were closely related to Bacillus naganoensis (94.0–94.1 % similarity) and separated from clusters of the related genera Bacillus (<91.9 %) and Sporolactobacillus (91.0–92.5 %). In addition, some chemotaxonomic and physiological characteristics of strains 607^T and 606b differed from those of B. naganoensis and the two related genera. Several differences in physiological characteristics and 16S–23S rRNA gene internal transcribed spacer region nucleotide sequences were observed between strains 607^T and 606b; however, DNA–DNA hybridization indicated that these two strains belonged to the same species. From these results, it is proposed that strains 607^T and 606b represent the type species of a new genus, Tuberibacillus calidus gen. nov., sp. nov., with strain 607^T (=JCM 13397^T=DSM 17572^T) as the type strain. In addition, the results of phylogenetic analyses, as well as chemotaxonomic and physiological characterization, indicated that B. naganoensis and Bacillus laevolacticus did not belong to the genus Bacillus. Based on these results, it is proposed that B. naganoensis and B. laevolacticus should be transferred to Pullulanibacillus naganoensis gen. nov., comb. nov. and Sporolactobacillus laevolacticus comb. nov., respectively.

The urease of thermophilic Bacillus sp. strain TB-90 is composed of three subunits with molecular masses of 61, 12, and 11 kDa. By using synthetic oligonucleotide probes based on N-terminal amino acid sequences of each subunit, we cloned a 3.2-kb EcoRI fragment of TB-90 genomic DNA. Moreover, we cloned two other DNA fragments by gene walking starting from this fragment. Finally, we reconstructed in vitro a 6.2-kb DNA fragment which expressed catalytically active urease in Escherichia coli by combining these three DNA fragments. Nucleotide sequencing analysis revealed that the urease gene complex consists of nine genes, which were designed ureA, ureB, ureC, ureE, ureF, ureG, ureD, ureH, and ureI in order of arrangement. The structural genes ureA, ureB, and ureC encode the 11-, 12-, and 61-kDa subunits, respectively. The deduced amino acid sequences of UreD, UreE, UreF, and UreG, the gene products of four accessory genes, are homologous to those of the corresponding Ure proteins of Klebsiella aerogenes. UreD, UreF, and UreG were essential for expression of urease activity in E. coli and are suggested to play important roles in the maturation step of the urease in a co- and/or posttranslational manner. On the other hand, UreH and UreI exhibited no significant similarity to the known accessory proteins of other bacteria. However, UreH showed 23% amino acid identity to the Alcaligenes eutrophus HoxN protein, a high-affinity nickel transporter.

An l-glucose-utilizing bacterium, Paracoccus sp. 43P, was isolated from soil by enrichment cultivation in a minimal medium containing l-glucose as the sole carbon source. In cell-free extracts from this bacterium, NAD⁺-dependent l-glucose dehydrogenase was detected as having sole activity toward l-glucose. This enzyme, LgdA, was purified, and the lgdA gene was found to be located in a cluster of putative inositol catabolic genes. LgdA showed similar dehydrogenase activity toward scyllo- and myo-inositols. l-Gluconate dehydrogenase activity was also detected in cell-free extracts, which represents the reaction product of LgdA activity toward l-glucose. Enzyme purification and gene cloning revealed that the corresponding gene resides in a nine-gene cluster, the lgn cluster, which may participate in aldonate incorporation and assimilation. Kinetic and reaction product analysis of each gene product in the cluster indicated that they sequentially metabolize l-gluconate to glycolytic intermediates, d-glyceraldehyde-3-phosphate, and pyruvate through reactions of C-5 epimerization by dehydrogenase/reductase, dehydration, phosphorylation, and aldolase reaction, using a pathway similar to l-galactonate catabolism in Escherichia coli. Gene disruption studies indicated that the identified genes are responsible for l-glucose catabolism.