Edible plant oil (EPO)-consumption activity of the isolate Fusarium keratoplasticum EN01 and other relative Fusarium species.

Edible oil is used in humans' daily lives, and the degradation of edible oil is a key process in sewage water treatment and in compost production from food wastes. In this study, a mixed microbial strain EN00, which showed high edible plant oil (EPO)-consumption activity, was obtained from soil via enrichment cultivation. A fungal strain EN01 was isolated from EN00 and relegated to Fusarium keratoplasticum, based on the nucleotide sequences of the TEF1-α gene. Strain EN01 eliminated more than 90% of hydrophobic compounds from the medium containing 1.0% (w/v) EPO within 10 days at 30 °C. The rate of consumption of EPO by EN01 was comparable with that of EN00, suggesting that EN01 was the main microorganism involved in the EPO-consumption ability of EN00. Strain EN01 efficiently utilized EPO as a sole carbon source. The EPO-consumption rate of EN01 was highest among six tested strains of Fusarium solani species complex (FSSC), while two FSSC strains of F. mori and F. cuneirostrum, whose phylogenetic relationships were relatively distant from EN01, had little EPO-eliminating activity. This data implies that the potent EPO-eliminating activity is not general in FSSC strains but is restricted to selected members of this complex. EN01 showed good growth at 25-30 °C, in media with an initial pH of 4-10, and in the presence of 0-3% (w/v) sodium chloride. Although the safety including pathogenicity must be strictly evaluated, some FSSC strains including EN01 have potentials for use in the degradation and elimination of edible oil.

Crystal structure of the GH-46 subclass III chitosanase from Bacillus circulans MH-K1 in complex with chitotetraose.

BACKGROUND: Chitosanases (EC 3.2.1.132) hydrolyze chitosan which is a polymer of glucosamine (GlcN) linked by ß - 1,4 bonds, and show cleavage specificity against partially acetylated chitosan containing N-acetylglucosamine (GlcNAc) residues. Chitosanases' structural underpinnings for cleavage specificity and the conformational switch from open to closed structures are still a mystery. METHODS: The GH-46 subclass III chitosanase from Bacillus circulans MH-K1 (MH-K1 chitosanase), which also catalyzes the hydrolysis of GlcN-GlcNAc bonds in addition to GlcN-GlcN, has had its chitotetraose [(GlcN)4]-complexed crystal structure solved at 1.35 Å resolution. RESULTS: The MH-K1 chitosanase's (GlcN)4-bound structure has numerous structural similarities to other GH-46 chitosanases in terms of substrate binding and catalytic processes. However, subsite -1, which is absolutely specific for GlcN, seems to characterize the structure of a subclass III chitosanase due to its distinctive length and angle of a flexible loop. According to a comparison of the (GlcN)4-bound and apo-form structures, the particular binding of a GlcN residue at subsite -2 through Asp77 causes the backbone helix to kink, which causes the upper- and lower-domains to approach closely when binding a substrate. CONCLUSIONS: Although GH-46 chitosanases vary in the finer details of the subsites defining cleavage specificity, they share similar structural characteristics in substrate-binding, catalytic processes, and potentially in conformational change. GENERAL SIGNIFICANCE: The precise binding of a GlcN residue to the -2 subsite is essential for the conformational shift that occurs in all GH-46 chitosanases, as shown by the crystal structures of the apo- and substrate-bound forms of MH-K1 chitosanase.

Characterization of antifungal activity of the GH-46 subclass III chitosanase from Bacillus circulans MH-K1.

We characterized the antifungal activity of the Bacillus circulans subclass III MH-K1 chitosanase (MH-K1 chitosanase), which is one of the most intensively studied glycoside hydrolases (GHs) that belong to GH family 46. MH-K1 chitosanase inhibited the growth of zygomycetes fungi, Rhizopus and Mucor, even at 10 pmol (0.3 µg)/ml culture probably via its fungistatic effect. The amino acid substitution E37Q abolished the antifungal activity of MH-K1 chitosanase, but retained binding to chitotriose. The E37Q mutant was fused with green fluorescent protein (GFP) at its N-terminus and proved to act as a chitosan probe in combination with wheat-germ agglutinin (WGA), which is a chitin-specific binding lectin. The GFP-fused MH-K1 chitosanase mutant E37Q (GFP-E37Q) bound clearly to the hyphae of the Rhizopus and Mucor strains, indicating the presence of chitosan. In contrast, Cy5-labelled WGA (Cy5-WGA), but not GFP-E37Q, stained the hyphae of non-zygomycetes species, i.e. Fusarium oxysporum, Penicillium expansum, and Aspergillus awamori. When the mycelia of Rhizopus oryzae were treated with wild type MH-K1 chitosanase, they could not bind to GFP-E37Q but were stained instead by Cy5-WGA. We conclude that chitin is covered by chitosan in the cell walls of R. oryzae.

Enzymatic and genetic characterization of the DasD protein possessing N-acetyl-ß-d-glucosaminidase activity in Streptomyces coelicolor A3(2).

The dasD gene is located just downstream of the dasABC gene cluster, encoding components of an ABC transporter for uptake of a chitin-degradation product N,N'-diacetylchitobiose [(GlcNAc)(2) ] in Streptomyces coelicolor A3(2). To clarify the roles of the DasD protein in the degradation and assimilation of chitin, we obtained and characterized a recombinant DasD protein and a dasD-null mutant of S. coelicolor A3(2). The recombinant DasD protein produced in Escherichia coli showed N-acetyl-ß-d-glucosaminidase (GlcNAcase) activity and its optimum temperature and pH were 40 °C and 7, respectively. dasD transcription was strongly induced in the presence of chitin, weakly by chitosan, but not by cellulose or xylan in S. coelicolor A3(2). Immuno-blot analysis demonstrated that DasD is a cytoplasmic protein. The dasD-null mutant exhibited cellular GlcNAcase activity which was comparable with that of the parent strain M145. DasD, thus, did not seem to be a major GlcNAcase. Induced extracellular chitinase activity in the dasD-null mutant was, interestingly, higher than M145, in the presence of colloidal chitin or (GlcNAc)(2) . In contrast to M145, (GlcNAc)(2) temporally accumulated in the culture supernatant of the dasD-null mutant in the presence of colloidal chitin.

Classification of chitosanases by hydrolytic specificity toward N¹,N4-diacetylchitohexaose.

The hydrolytic specificities of chitosanases were determined using N¹,N4-diacetylchitohexaose [(GlcN)2-GlcNAc-(GlcN)2-GlcNAc]. The results for the hydrolytic specificities of chitosanases belonging to subclasses I, II, and III toward chitohexaose and N¹,N4-diacetylchitohexaose agreed with previous results obtained by analysis of the hydrolysis products of partially N-acetylated chitosan. N¹,N4-Diacetylchitohexaose is a useful substrate to determine the hydrolytic specificity of chitosanase. On the other hand, chitosanases from Amycolatopsis sp. CsO-2 and Pseudomonas sp. A-01 showed broad cleavage specificity. They cleaved both the GlcNAc-GlcN and the GlcN-GlcNAc bonds in addition to the GlcN-GlcN bond in the substrate. Thus, both enzymes were new chitosanases. The chitosanases were divided into four subclasses according to their specificity for hydrolysis of the ß-glycosidic linkages in partially N-acetylated chitosan.

Aleuria aurantia lectin exhibits antifungal activity against Mucor racemosus.

Aleuria aurantia lectin (AAL) is an L-fucose-specific lectin produced in the mycelia and fruit-bodies of the widespread ascomycete fungus Aleuria aurantia. It is extensively used in the detection of fucose, but its physiological role remains unknown. To investigate this, we analyzed the interaction between AAL and, a zygomycete fungus Mucor racemosus, which is assumed to contain fucose in its cell wall. AAL specifically bound to the hyphae of M. racemosus, because binding was inhibited by L-fucose but not by D-fucose. It inhibited the growth of the fungus at 1 µM, and the M. racemosus cells were remarkably disrupted at 7.5 µM. In contrast, two other fucose-specific lectins, Anguilla anguilla agglutinin and Ulex europaeus agglutinin, did not inhibit the growth of M. racemosus. These results suggest that the growth inhibition activity is unique to AAL, and that AAL could act as an antifungal protein in natural ecosystems.

Expression of a bacterial chitosanase in rice plants improves disease resistance to the rice blast fungus Magnaporthe oryzae.

Plant fungal pathogens change their cell wall components during the infection process to avoid degradation by host lytic enzymes, and conversion of the cell wall chitin to chitosan is likely to be one infection strategy of pathogens. Thus, introduction of chitosan-degradation activity into plants is expected to improve fungal disease resistance. Chitosanase has been found in bacteria and fungi, but not in higher plants. Here, we demonstrate that chitosanase, Cho1, from Bacillus circulans MH-K1 has antifungal activity against the rice blast fungus Magnaporthe oryzae. Introduction of the cho1 gene conferred chitosanase activity to rice cells. Transgenic rice plants expressing Cho1 designed to be localized in the apoplast showed increased resistance to M. oryzae accompanied by increased generation of hydrogen peroxide in the infected epidermal cells. These results strongly suggest that chitosan exists in the enzyme-accessible surface of M. oryzae during the infection process and that the enhancement of disease resistance is attributable to the antifungal activity of the secreted Cho1 and to increased elicitation of the host defense response.

Molecular and enzymatic characterization of a subfamily I.4 lipase from an edible oil-degrader Bacillus sp. HH-01.

An edible-oil degrading bacterial strain HH-01 was isolated from oil plant gummy matter and was classified as a member of the genus Bacillus on the basis of the nucleotide sequence of the 16S rRNA gene. A putative lipase gene and its flanking regions were cloned from the strain based on its similarity to lipase genes from other Bacillus spp. The deduced product was composed of 214 amino acids and the putative mature protein, consisting of 182 amino acids, exhibited 82% amino acid sequence identity with the subfamily I.4 lipase LipA of Bacillus subtilis 168. The recombinant product was successfully overproduced as a soluble form in Escherichia coli and showed lipase activity. The gene was, therefore, designated as lipA of HH-01. HH-01 LipA was stable at pH 4-11 and up to 30°C, and its optimum pH and temperature were 8-9 and 30°C, respectively. The enzyme showed preferential hydrolysis of the 1(3)-position ester bond in trilinolein. The activity was, interestingly, enhanced by supplementing with 1 mM CoCl(2), in contrast to other Bacillus lipases. The lipA gene seemed to be constitutively transcribed during the exponential growth phase, regardless of the presence of edible oil.

Streptomyces coacervatus sp. nov., isolated from the intestinal tract of Armadillidium vulgare.

A Gram-staining-positive bacterium, designated AS-0823(T), which formed spiral spore chains on the aerial mycelium, was isolated from the intestinal tract of Armadillidium vulgare, a small terrestrial crustacean commonly found on the ground around houses in Japan. 16S rRNA gene sequence analysis showed that the isolate belonged to the genus Streptomyces and was most closely related to Streptomyces longisporus ISP 5166(T) (98.6 % 16S rRNA gene sequence similarity), Streptomyces curacoi NBRC 12761(T) (98.4 %) and Streptomyces griseoruber NBRC 12873(T) (98.4 %). The affiliation of strain AS-0823(T) to the genus Streptomyces was supported by chemotaxonomic data: iso-C(16 : 0), anteiso-C(15 : 0), C(16 : 0), iso-C(15 : 0) and anteiso-C(17 : 0) as the major cellular fatty acids, ll-diaminopimelic acid as the characteristic diamino acid in the peptidoglycan and the absence of mycolic acids. DNA-DNA hybridization and physiological and biochemical analysis supported the differentiation of strain AS-0823(T) from S. longisporus JCM 4395(T). Therefore, strain AS-0823(T) represents a novel species, for which the name Streptomyces coacervatus sp. nov. is proposed. The type strain is AS-0823(T) ( = IFM 11055(T)  = DSM 41983(T)  = JCM 17138(T)).

Inhibition of ergosterol synthesis by novel antifungal compounds targeting C-14 reductase.

The limited number of clinically available antifungal drugs for life-threatening fungal infections has produced an increased demand for new agents. In the course of our screening for novel antifungals, we identified aminopiperidine derivatives which exhibit antifungal activities against the major pathogenic yeasts. Thin layer chromatography (TLC) analysis of the extracted non-saponifiable lipids from Candida albicans showed that these compounds inhibited the ergosterol production in the late step of the synthesis pathway. The results of an LC/Q-Tof MS analysis showed that abnormal sterols including predicted ignosterol, which is known to be accumulated in C. albicans ERG24 deleted mutant, were accumulated in C. albicans treated with one of these derivatives (Compound 1b). Furthermore, the partial disruption of the cell membrane of C. albicans treated with compound 1b was observed by electron microscopy analysis, suggesting its inhibition of ergosterol synthesis. Additionally, a genetic approach demonstrated that ERG24 gene would be responsible for the resistance of Saccharomyces cerevisiae against Compound 1b, strongly indicating that the enzyme targeted by Compound 1b is Erg24p. From all these data, we concluded that these aminopiperidine derivatives are novel antifungal compounds inhibiting C-14 reduction in the ergosterol synthesis pathway.

In vitro and in vivo antifungal activities of aminopiperidine derivatives, novel ergosterol synthesis inhibitors.

Aminopiperidine derivatives, Compound 1a and 1b, are novel small molecules that inhibit C-14 reduction catalyzed by Erg24p in ergosterol synthesis of Candida albicans. We evaluated the properties of the in vitro and in vivo activities of these compounds against pathogenic fungi and compared their activities with those of fluconazole. Compound 1a and 1b exhibited potent in vitro activities against clinically important fungi such as Candida species, including both of fluconazole-resistant strains of C. albicans and non-albicans Candida, Aspergillus fumigatus, and Cryptococcus neoformans. Against C. albicans, its mode of action was fungistatic. Furthermore, orally administered Compound 1b clearly prolonged the survival of infected mice in systemic lethal infection caused by C. albicans. These results suggest that aminopiperidine derivative is a promising lead compound for an orally available novel antifungal drug with a broad spectrum.

Enzymatic synthesis of 4-hydroxyphenyl beta-D-oligoxylosides and their notable tyrosinase inhibitory activity.

We have purified and characterized an oligoxylosyl transfer enzyme (OxtA) from Bacillus sp. strain KT12. In the present study, a N-terminally His-tagged recombinant form of the enzyme, OxtA(H)(E), was overproduced in Escherichia coli and applied to the reaction with xylan and hydroquinone to produce 4-hydroxyphenyl beta-D-oligoxylosides, beta-(Xyl)(n)-HQ (n=1-4), by one step reaction. The obtained beta-(Xyl)(n)-HQ inhibited mushroom tyrosinase, which catalyzes the oxidation of L-DOPA to L-DOPA quinine, and the IC(50) values of beta-Xyl-HQ, beta-(Xyl)(2)-HQ, beta-(Xyl)(3)-HQ, and beta-(Xyl)(4)-HQ were 3.0, 0.74, 0.48, and 0.18 mM respectively. beta-(Xyl)(4)-HQ showed 35-fold more potent inhibitory activity than beta-arbutin (4-hydroxyphenyl beta-D-glucopyranoside), of which the IC(50) value was measured to be 6.3 mM. Kinetic analysis revealed that beta-(Xyl)(2)-HQ, beta-(Xyl)(3)-HQ, and beta-(Xyl)(4)-HQ competitively inhibited the enzyme, and the corresponding K(i) values were calculated to be 0.20, 0.29, and 0.057 mM respectively.

Molecular characterization and antifungal activity of a family 46 chitosanase from Amycolatopsis sp. CsO-2.

An actinomycete strain, Amycolatopsis sp. CsO-2, produces a 27-kDa chitosanase. To reveal the molecular characteristics of the enzyme, its corresponding gene ctoA was cloned by a reverse genetic technique, based on the N-terminal amino acid sequence of the protein. The encoded CtoA protein was deduced to be composed of 286 amino acids, including a putative signal peptide (1-48), and exhibited 83% identity in the amino acid sequence with the family 46 chitosanases from Streptomyces sp. N174 or Nocardioides sp. N106. The active recombinant CtoA protein was successfully overproduced in Escherichia coli. The mutant protein E22Q, in which the glutamic acid residue 22 was replaced with glutamine, abolished the chitosanase activity, showing that the Glu22 residue is required for the enzymatic activity. CtoA exhibited antifungal activity against Rhizopus oryzae, which is known to produce chitosan probably as a cell wall component. In contrast, E22Q did not inhibit the growth of the fungus, suggesting that chitosan-hydrolyzing activity is essential for the antifungal activity. It is noteworthy that the antifungal effect of CtoA against R. oryzae was drastically enhanced by the simultaneous addition of the family 19 chitinase ChiC from Streptomyces griseus.

The msiK gene, encoding the ATP-hydrolysing component of N,N'-diacetylchitobiose ABC transporters, is essential for induction of chitinase production in Streptomyces coelicolor A3(2).

The dasABC genes encode an ATP-binding cassette (ABC) transporter, which is one of the uptake systems for N,N'-diacetylchitobiose [(GlcNAc)(2)] in Streptomyces coelicolor A3(2), although the gene encoding the ABC subunit that provides ATP hydrolysis for DasABC has not been identified. In this study, we disrupted the sequence that is highly homologous to the msiK gene, the product of which is an ABC subunit assisting several ABC permeases in other Streptomyces species. Disruption of msiK severely affected the ability of S. coelicolor A3(2) to utilize maltose, cellobiose, starch, cellulose, chitin and chitosan, but not glucose. The msiK null mutant lacked (GlcNAc)(2)-uptake activity, but GlcNAc transport activity was unaffected. The data indicated that msiK is essential for (GlcNAc)(2) uptake, which in S. coelicolor A3(2) is governed by ABC transporters including the DasABC-MsiK system, in contrast to Escherichia coli and Serratia marcescens, in which (GlcNAc)(2) uptake is mediated by the phosphotransferase system. Interestingly, the induction of chitinase production by (GlcNAc)(2) or chitin was absent in the msiK null mutant, unlike in the parent strain M145. The defect in chitinase gene induction was rescued by expressing the His-tagged MsiK protein under the control of the putative native promoter on a multicopy plasmid. The data suggest that uptake of (GlcNAc)(2) is necessary for induction of chitinase production. The msiK gene was constitutively transcribed, whereas the transcription of dasA [(GlcNAc)(2)-binding protein gene], malE (putative maltose-binding protein gene), cebE1 (putative cellobiose-binding protein gene) and bxlE1 (putative xylobiose-binding protein gene) was induced by their corresponding sugar ligands. This is believed to be the first report to indicate that (GlcNAc)(2) uptake mediated by ABC transporters is essential for chitinase production in streptomycetes, which are known to be the main degraders of chitin in soil.

Molecular characterization of a novel family-46 chitosanase from Pseudomonas sp. A-01.

Pseudomonas sp. A-01, isolated as a strain with chitosan-degrading activity, produced a 28 kDa chitosanase. Following purification of the chitosanase (Cto1) and determination of its N-terminal amino acid sequence, the corresponding gene (cto1) was cloned by a reverse-genetic technique. The gene encoded a protein, composed of 266 amino acids, including a putative signal sequence (1-28), that showed an amino acid sequence similar to known family-46 chitosanases. Cto1 was successfully overproduced and was secreted by a Brevibacillus choshinensis transformant carrying the cto1 gene on expression plasmid vector pNCMO2. The purified recombinant Cto1 protein was stable at pH 5-8 and showed the best chitosan-hydrolyzing activity at pH 5. Replacement of two acidic amino acid residues, Glu23 and Asp41, which correspond to previously identified active centers in Streptomyces sp. N174 chitosanase, with Gln and Asn respectively caused a defect in the hydrolyzing activity of the enzyme.

Effect of Gb3 in lipid rafts in resistance to Shiga-like toxin of mutant Vero cells.

Shiga-like toxin 1 (Stx1), produced by enterohemorrhagic Escherichia coli, binds to its receptor, globotriaosylceramide (Gb3), on target cell membranes, as a prerequisite for inducing host cell intoxication. To examine further toxin-receptor interactions, we established an Stx1-resistant clone of Vero cells by chemical mutagenesis. The mutant cells, expressed Gb3, but did not bind Stx1. These mutant cells were larger and had more Gb3 per cell than wild-type cells. Gb3 from both wild-type and mutant Vero cells was recovered in lipid rafts, isolated from cell lysates as detergent resistant membranes (DRMs); the DRMs derived from mutant cells had a lower density of Gb3 than did those from wild-type cells. Stx1 did not bind to the DRMs of mutant cells, both by ELISA and surface plasmon resonance. However, Stx1 bound to Gb3 separated by thin-layer chromatograms from the DRMs of mutant cells. The results indicate that not only presence of Gb3 but also Gb3 density on lipid rafts were important for Stx binding.

Structural simulation and protein engineering to convert an endo-chitosanase to an exo-chitosanase.

To obtain an enzyme for the production of chito-disaccharides (GlcN(2)) by converting endo-chitosanase to exo-chitosanase, we chose an endo-chitosanase from Bacillus circulans MH-K1 (Csn) as the candidate for protein engineering. Using molecular modeling, two peptides with five amino acids (PCLGG) and six amino acids (SRTCKP) were designed and inserted after the positions of D(115) and T(222) of Csn, respectively. The inserted fragments are expected to form loops that might protrude from opposite walls of the substrate-binding cleft, thus forming a 'roof' over the catalytic site that might alter the product specificity. The chimeric chitosanase (Chim-Csn) and wild-type chitosanase (WT-Csn) were both over-expressed in Escherichia coli and purified nearly to homogeneity. The products formed from chitosan were analyzed by ESI-MS (electrospray ionization-mass spectrometry). A mixture of GlcN(2), GlcN(3) and GlcN(4) was obtained with WT-Csn, whereas Chim-Csn formed, with a smaller catalytic rate (3% of WT-Csn activity), GlcN(2) as the dominant product. Measurements of viscosity showed that, with similar amounts of enzyme activity, Chim-Csn catalyzed the hydrolysis of chitosan with a smaller rate of viscosity decrease than WT-Csn. The results indicate that, on inserting two surface loops, the endo-type chitosanase was converted into an exo-type chitosanase, which to our knowledge is the first chitosanase that releases GlcN(2) from chitosan as the dominant product.

Smurf1 directly targets hPEM-2, a GEF for Cdc42, via a novel combination of protein interaction modules in the ubiquitin-proteasome pathway.

Smurf1, a member of HECT-type E3 ubiquitin ligases, regulates cell polarity and protrusive activity by inducing ubiquitination and subsequent proteasomal degradation of the small GTPase RhoA. We report here that hPEM-2, a guanine nucleotide exchange factor for the small GTPase Cdc42, is a novel target of Smurf1. Pulse-chase labeling and a ubiquitination experiment using MG132, a proteasomal inhibitor, indicate that Smurf1 induces proteasomal degradation of hPEM-2 in cells. GST pull-down assays with heterologously expressed firefly luciferase-fusion proteins that include partial sequences of hPEM-2 reveal that part of the PH domain (residues 318-343) of hPEM-2 is sufficient for binding to Smurf1. In contrast, the hPEM-2 binding domain in Smurf1 was mapped to the C2 domain. Although it has been reported that the binding activities of some C2 domains to target proteins are regulated by Ca2+, Smurf1 interacts with hPEM-2 in a Ca2+-independent manner. Our discovery that hPEM-2 is, in addition to RhoA, a target protein of Smurf1 suggests that Smurf1 plays a crucial role in the spatiotemporal regulation of Rho GTPase family members.

The dasABC gene cluster, adjacent to dasR, encodes a novel ABC transporter for the uptake of N,N'-diacetylchitobiose in Streptomyces coelicolor A3(2).

N,N'-Diacetylchitobiose [(GlcNAc)(2)] induces the transcription of chitinase (chi) genes in Streptomyces coelicolor A3(2). Physiological studies showed that (GlcNAc)(2) addition triggered chi expression and increased the rate of (GlcNAc)(2) concentration decline in culture supernatants of mycelia already cultivated with (GlcNAc)(2), suggesting that (GlcNAc)(2) induced the synthesis of its own uptake system. Four open reading frames (SCO0531, SCO0914, SCO2946, and SCO5232) encoding putative sugar-binding proteins of ABC transporters were found in the genome by probing the 12-bp repeat sequence required for regulation of chi transcription. SCO5232, named dasA, showed transcriptional induction by (GlcNAc)(2) and N,N',N'''-triacetylchitotriose [(GlcNAc)(3)]. Surface plasmon resonance analysis showed that recombinant DasA protein exhibited the highest affinity for (GlcNAc)(2) (equilibrium dissociation constant [K(D)] = 3.22 x 10(-8)). In the dasA-null mutant, the rate of decline of the (GlcNAc)(2) concentration in the culture supernatant was about 25% of that in strain M145. The in vitro and in vivo data clearly demonstrated that dasA is involved in (GlcNAc)(2) uptake. Upstream and downstream of dasA, the transcriptional regulator gene (dasR) and two putative integral membrane protein genes (dasBC) are located in the opposite and same orientations, respectively. The expression of dasR and dasB, which seemed independent of dasA transcription, was also induced by (GlcNAc)(2) and (GlcNAc)(3).

Acidianus manzaensis sp. nov., a novel thermoacidophilic archaeon growing autotrophically by the oxidation of H2 with the reduction of Fe3+.

A novel thermoacidophilic iron-reducing Archaeon, strain NA-1, was isolated from a hot fumarole in Manza, Japan. Strain NA-1 could grow autotrophically using H2 or S0 as an electron donor and Fe3+ as an electron acceptor, and also could grow heterotrophically using some organic compounds. Fe3+ and O2 served as electron acceptors for growth. However, S0, NO3-, NO2-, SO4(2-), Mn4+, fumarate, and Fe2O3 did not serve as electron acceptors. The ranges of growth temperature and pH were 60-90 degrees C (optimum: 80 degrees C) and pH 1.0-5.0 (optimum: pH 1.2-1.5), respectively. Cells were nearly regular cocci with an envelope comprised of the cytoplasmic membrane and a single outer S-layer. The crenarchaeal-specific quinone (cardariellaquinone) was detected, and the genomic DNA G + C content was 29.9 mol%. From 16S rDNA analysis, it was determined that strain NA-1 is closely related to Acidianus ambivalens (93.1%) and Acidianus infernus (93.0%). However, differences revealed by phylogenetic and phenotypic analyses clearly show that strain NA-1 represents a new species, Acidianus manzaensis, sp. nov., making it the first identified thermoacidophilic iron-reducing microorganism (strain NA-1T = NBRC 100595 = ATCC BAA 1057).