Genomic characterization of closely related species in the Rumoiensis clade infers ecogenomic signatures to non-marine environments.

Members of the family Vibrionaceae are generally found in marine and brackish environments, playing important roles in nutrient cycling. The Rumoiensis clade is an unconventional group in the genus Vibrio, currently comprising six species from different origins including two species isolated from non-marine environments. In this study, we performed comparative genome analysis of all six species in the clade using their complete genome sequences. We found that two non-marine species, Vibrio casei and Vibrio gangliei, lacked the genes responsible for algal polysaccharide degradation, while a number of glycoside hydrolase genes were enriched in these two species. Expansion of insertion sequences was observed in V. casei and Vibrio rumoiensis, which suggests ongoing genomic changes associated with niche adaptations. The genes responsible for the metabolism of glucosylglycerate, a compound known to play a role as compatible solutes under nitrogen limitation, were conserved across the clade. These characteristics, along with genes encoding species-specific functions, may reflect the habit expansion which has led to the current distribution of Rumoiensis clade species. Genome analysis of all species in a single clade give us valuable insights into the genomic background of the Rumoiensis clade species and emphasize the genomic diversity and versatility of Vibrionaceae.

Cloning and characterization of a beta-1,4-mannanase 5C possessing a family 27 carbohydrate-binding module from a marine bacterium, Vibrio sp. strain MA-138.

The beta-1,4-mannanase 5C gene (man5C) of Vibrio sp. strain MA-138 was cloned and expressed in Escherichia coli. The man5C gene consisted of 2,010 bp nucleotides encoding a protein of 669 amino acids with a predicted molecular weight of 76,309. beta-1,4-Mannanase (Man5C) is a modular enzyme composed of a catalytic module belonging to glycoside hydrolase family 5, a linker region, and a putative carbohydrate-binding module (CBM) belonging to family 27. Recombinant Man5C exhibited maximal activity at 50 degrees C at pH 7.0, and it had a K(m) of 0.6 mg ml(-1) and a V(max) of 556.2 micromol min(-1) mumol(-1) for glucomannan. Binding studies revealed that the C-terminal putative CBM27 had the ability to bind soluble beta-mannans and contributed to increasing the rate of depolymerization by binding to the polymeric substrate. Man5C of Vibrio sp. MA-138 is the first non-extremophile enzyme to be identified as a beta-mannanase possessing CBM27.

Development of koji by culturing Aspergillus oryzae on nori (Pyropia yezoensis).

Koji is a traditional fermentation culture medium, based on Aspergillus oryzae, which is commonly used in the manufacture process of Japanese fermented products such as soy sauce, miso, and sake, and promote enzymatic degradation. Koji is usually prepared by culturing a mold on cereals such as wheat flour, soybean, or rice, but that cultured on seaweeds has not been developed yet. This study prepared the koji by culturing A. oryzae on seaweed nori (dried piece of Pyropia yezoensis), and, then, characterized on this nori koji. The nori koji contained 0.85 µg N-acetylglucosamine, estimated as 6.1 µg mold cells, per gram dry matter and showed various kind of enzymatic activities in glycosidase, protease, and phosphatase as well as traditional soy sauce koji and rice koji. The suitability of these characteristics for degradation of nori was tested on nori sauce culture with and without the addition of the nori koji. After 167 days of culture, the fermentation tank with the nori koji showed over 74% recovery of supernatant while that without the nori koji had less than 57% recovery. The supernatant of culture mashes contained more than two times larger quantity of total nitrogen compounds in nori koji test group against control group. The present study prepared koji on seaweed nori for the first time and demonstrated its advantages to shorten the culture period and increase taste value in nori sauce manufacture. Development of seaweed koji enables a method to prepare cereal allergen free fermented sauces from seaweeds.

Cloning of a novel gene encoding beta-1,3-xylosidase from a marine bacterium, Vibrio sp. strain XY-214, and characterization of the gene product.

The beta-1,3-xylosidase gene (xloA) of Vibrio sp. strain XY-214 was cloned and expressed in Escherichia coli. The xloA gene consisted of a 1,608-bp nucleotide sequence encoding a protein of 535 amino acids with a predicted molecular weight of 60,835. The recombinant beta-1,3-xylosidase hydrolyzed beta-1,3-xylooligosaccharides to D-xylose as a final product.

Characterization of fermented seaweed sauce prepared from nori (Pyropia yezoensis).

High-salt content seaweed sauces were prepared for the first time using nori (Pyropia yezoensis) by fermentation and characterized. Components and taste of the two nori sauces (NSs) prepared separately were compared with those of soy and fish sauces. The NSs were rich in total nitrogen compounds (1.5 g N/100 ml on average) and potassium (880 mg/100 g), and had a unique free amino acid composition (e.g., taurine 617 mg/100 g), explaining their unique taste as evaluated by a taste sensing system. As for their food function, inhibitory activity of angiotensin-converting enzyme was observed. As for their food safety, arsenic was detected at a 0.8 mg/100 g level in total, but inorganic arsenic was not detected (<0.05 mg/100 g) and not regarded as a problem. Allergy-causing substances contained in wheat, soy beans, and crustaceans were not detected (<0.1 mg/100 g) with NSs. These results suggest that the nori sauce has a high potential as a novel nutritional source for humans.

Vibrio aphrogenes sp. nov., in the Rumoiensis clade isolated from a seaweed.

A novel strain Vibrio aphrogenes sp. nov. strain CA-1004T isolated from the surface of seaweed collected on the coast of Mie Prefecture in 1994 [1] was characterized using polyphasic taxonomy including multilocus sequence analysis (MLSA) and a genome based comparison. Both phylogenetic analyses on the basis of 16S rRNA gene sequences and MLSA based on eight protein-coding genes (gapA, gyrB, ftsZ, mreB, pyrH, recA, rpoA, and topA) showed the strain could be placed in the Rumoiensis clade in the genus Vibrio. Sequence similarities of the 16S rRNA gene and the multilocus genes against the Rumoiensis clade members, V. rumoiensis, V. algivorus, V. casei, and V. litoralis, were low enough to propose V. aphrogenes sp. nov. strain CA-1004T as a separate species. The experimental DNA-DNA hybridization data also revealed that the strain CA-1004T was separate from four known Rumoiensis clade species. The G+C content of the V. aphrogenes strain was determined as 42.1% based on the genome sequence. Major traits of the strain were non-motile, halophilic, fermentative, alginolytic, and gas production. A total of 27 traits (motility, growth temperature range, amylase, alginase and lipase productions, and assimilation of 19 carbon compounds) distinguished the strain from the other species in the Rumoiensis clade. The name V. aphrogenes sp. nov. is proposed for this species in the Rumoiensis clade, with CA-1004T as the type strain (JCM 31643T = DSM 103759T).

Correction: Vibrio aphrogenes sp. nov., in the Rumoiensis clade isolated from a seaweed.

[This corrects the article DOI: 10.1371/journal.pone.0180053.].

Molecular cloning and characterization of a novel beta-1,3-xylanase possessing two putative carbohydrate-binding modules from a marine bacterium Vibrio sp. strain AX-4.

We cloned a novel beta-1,3-xylanase gene, consisting of a 1728-bp open reading frame encoding 576 amino acid residues, from a marine bacterium, Vibrio sp. strain AX-4. Sequence analysis revealed that the beta-1,3-xylanase is a modular enzyme composed of a putative catalytic module belonging to glycoside hydrolase family 26 and two putative carbohydrate-binding modules belonging to family 31. The recombinant enzyme hydrolysed beta-1,3-xylan to yield xylo-oligosaccharides with different numbers of xylose units, mainly xylobiose, xylotriose and xylotetraose. However, the enzyme did not hydrolyse beta-1,4-xylan, beta-1,4-mannan, beta-1,4-glucan, beta-1,3-xylobiose or p-nitrophenyl-beta-xyloside. When beta-1,3-xylo-oligosaccharides were used as the substrate, the kcat value of the enzyme for xylopentaose was found to be 40 times higher than that for xylotetraose, and xylotriose was extremely resistant to hydrolysis by the enzyme. A PSI-BLAST search revealed two possible catalytic Glu residues (Glu-138 as an acid/base catalyst and Glu-234 as a nucleophile), both of which are generally conserved in glycoside hydrolase superfamily A. Replacement of these two conserved Glu residues with Asp and Gln resulted in a significant decrease and complete loss of enzyme activity respectively, without a change in their CD spectra, suggesting that these Glu residues are the catalytic residues of beta-1,3-xylanase. The present study also clearly shows that the non-catalytic putative carbohydrate-binding modules play an important role in the hydrolysis of insoluble beta-1,3-xylan, but not that of soluble glycol-beta-1,3-xylan. Furthermore, repeating a putative carbohydrate-binding module strongly enhanced the hydrolysis of the insoluble substrate.

A new class of glutathione S-transferase from the hepatopancreas of the red sea bream Pagrus major.

To elucidate drug deposition and metabolism in cultured marine fishes, in a previous study we isolated and purified the GSTs (glutathione S-transferases) from the hepatopancreas of the red sea bream Pagrus major that contained 25 and 28 kDa GST subunits. The 25 kDa GST subunits encoded by two genes (GSTA1 and GSTA2) have been identified as Alpha-class GSTs. In the present study, we performed the molecular cloning and characterization of the GSTR1 gene encoding the 28 kDa GST subunit from the Pa. major hepatopancreas. The nucleotide sequence of GSTR1 was composed of an ORF (open reading frame) of 675 bp encoding a protein of 225 residues with a predicted molecular mass of 25.925 Da. A search of the BLAST protein database revealed that the deduced amino acid sequence of GSTR1 was structurally similar to that of GSTs derived from other fishes such as largemouth bass (Micropterus salmoides) and plaice (Pleuronectes platessa). The genomic DNA containing the GSTR1 gene was found to consist of six exons and five introns quite distinct from mammalian Theta-class GSTs. We have purified and characterized the recombinant GSTR1 enzyme (pmGSTR1-1) which showed activity only towards 1-chloro-2,4-dinitrobenzene, although it had no detectable activity towards cumene hydroperoxide, 1,2-dichloro-4-nitrobenzene, ethacrynic acid, 4-hydroxynonenal and p-nitrobenzyl chloride. Moreover, pmGSTR1-1 revealed remarkable heat instability (melting temperature Tm=30.3+/-0.11 degrees C). Collectively, our results indicated that the characteristic GST genes including GSTR1 have been conserved and functional in fishes. Therefore we designate them 'Rho-class', a new class of GSTs.

The first crystal structure of a family 31 carbohydrate-binding module with affinity to beta-1,3-xylan.

Here, we present the crystal structure of the family 31 carbohydrate-binding module (CBM) of beta-1,3-xylanase from Alcaligenes sp. strain XY-234 (AlcCBM31) determined at a resolution of 1.25A. The AlcCBM31 shows affinity with only beta-1,3-xylan. The AlcCBM31 molecule makes a beta-sandwich structure composed of eight beta-strands with a typical immunoglobulin fold and contains two intra-molecular disulfide bonds. The folding topology of AlcCBM31 differs from that of the large majority of other CBMs, in which eight beta-strands comprise a beta-sandwich structure with a typical jelly-roll fold. AlcCBM31 shows structural similarity with CBM structures of family 34 and family 9, which also adopt structures based on immunoglobulin folds.

The first thermodynamic characterization of beta-1,3-xylanase from a marine bacterium.

Sequence analysis of beta-1,3-xylanase (TxyA) from a marine bacterium, Alcaligenes sp. strain XY-234 implied that an xylan-binding module belonging to carbohydrate-binding module family 31 (TxyA-CBM) is separated from a catalytic module belonging to glycosyl hydrolase family 26 (TxyA-CM) by a putative glycine-rich linker [Okazaki, F., et al. (2002) J. Bacteriol. 184: 2399-2403]. In order to reveal the role of these structural features of TxyA, two modules, TxyA-CBM and TxyA-CM, were constructed, and those modules and full-length TxyA were characterized by thermodynamic studies. TxyA-CBM and TxyA-CM showed full reversible folding from denaturant-induced unfolded forms, exhibited higher thermodynamic stabilities. The conformational stability of both truncated modules is industrially desirable, as well as aiding the understanding of the enzymatic characterization of the two modules of beta-1,3-xylanase separated by a long linker.

D-xylose isomerase from a marine bacterium, Vibrio sp. strain XY-214, and D-xylulose production from ß-1,3-xylan.

The xylA gene from a marine bacterium, Vibrio sp. strain XY-214, encoding D-xylose isomerase (XylA) was cloned and expressed in Escherichia coli. The xylA gene consisted of 1,320-bp nucleotides encoding a protein of 439 amino acids with a predicted molecular weight of 49,264. XylA was classified into group II xylose isomerases. The native XylA was estimated to be a homotetramer with a molecular mass of 190 kDa. The purified recombinant XylA exhibited maximal activity at 60°C and pH 7.5. Its apparent K (m) values for D-xylose and D-glucose were 7.93 and 187 mM, respectively. Furthermore, we carried out D-xylulose production from ß-1,3-xylan, a major cell wall polysaccharide component of the killer alga Caulerpa taxifolia. The synergistic action of ß-1,3-xylanase (TxyA) and ß-1,3-xylosidase (XloA) from Vibrio sp. strain XY-214 enabled efficient saccharification of ß-1,3-xylan to D-xylose. D-xylose was then converted to D-xylulose by using XylA from the strain XY-214. The conversion rate of D-xylose to D-xylulose by XylA was found to be approximately 40% in the presence of 4 mM sodium tetraborate after 2 h of incubation. These results demonstrated that TxyA, XloA, and XylA from Vibrio sp. strain XY-214 are useful tools for D-xylulose production from ß-1,3-xylan. Because D-xylulose can be used as a source for ethanol fermentation by yeast Saccharomyces cerevisiae, the present study will provide a basis for ethanol production from ß-1,3-xylan.

Cell wall regeneration in Bangia atropurpurea (Rhodophyta) protoplasts observed using a mannan-specific carbohydrate-binding module.

The cell wall of the red alga Bangia atropurpurea is composed of three unique polysaccharides (beta-1,4-mannan, beta-1,3-xylan, and porphyran), similar to that in Porphyra. In this study, we visualized beta-mannan in the regenerating cell walls of B. atropurpurea protoplasts by using a fusion protein of a carbohydrate-binding module (CBM) and green fluorescent protein (GFP). A mannan-binding family 27 CBM (CBM27) of beta-1,4-mannanase (Man5C) from Vibrio sp. strain MA-138 was fused to GFP, and the resultant fusion protein (GFP-CBM27) was expressed in Escherichia coli. Native affinity gel electrophoresis revealed that GFP-CBM27 maintained its binding ability to soluble beta-mannans, while normal GFP could not bind to beta-mannans. Protoplasts were isolated from the fronds of B. atropurpurea by using three kinds of bacterial enzymes. The GFP-CBM27 was mixed with protoplasts from different growth stages, and the process of cell wall regeneration was observed by fluorescence microscopy. Some protoplasts began to excrete beta-mannan at certain areas of their cell surface after 12 h of culture. As the protoplast culture progressed, beta-mannans were spread on their entire cell surfaces. The percentages of protoplasts bound to GFP-CBM27 were 3%, 12%, 17%, 29%, and 25% after 12, 24, 36, 48, and 60 h of culture, respectively. Although GFP-CBM27 bound to cells at the initial growth stages, its binding to the mature fronds was not confirmed definitely. This is the first report on the visualization of beta-mannan in regenerating algal cell walls by using a fluorescence-labeled CBM.

Characterization and application of carbohydrate-binding modules of beta-1,3-xylanase XYL4.

beta-1,3-Xylanase from Vibrio sp. strain AX-4 (XYL4) is a modular enzyme composed of an N-terminal catalytic module belonging to glycoside hydrolase family 26 and two putative carbohydrate-binding modules (CBMs) belonging to family 31 in the C-terminal region. To investigate the functions of these three modules, five deletion mutants lacking individual modules were constructed. The binding assay of these mutants showed that a repeating unit of the CBM was a non-catalytic beta-1,3-xylan-binding module, while the catalytic module per se was not likely to contribute to the binding activity when insoluble beta-1,3-xylan was used for the assay. The repeating CBMs were found to specifically bind to insoluble beta-1,3-xylan, but not to beta-1,4-xylan, Avicel, beta-1,4-mannan, curdlan, chitin or soluble glycol-beta-1,3-xylan. Both the enzyme and the binding activities for insoluble beta-1,3-xylan but not soluble glycol-beta-1,3-xylan were enhanced by NaCl in a concentration-dependent manner, indicating that the CBMs of XYL4 bound to beta-1,3-xylan through hydrophobic interaction. This property of the CBMs was successfully applied to the purification of a recombinant XYL4 from the cell extracts of Escherichia coli transformed with the xyl4 gene and the detection of beta-1,3-xylan-binding proteins including beta-1,3-xylanase from the extract of a turban shell, Turbo cornutus.

A unique beta-agarase, AgaA, from a marine bacterium, Vibrio sp. strain PO-303.

The agaA gene encoding beta-agarase-a (AgaA) was cloned from the chromosomal DNA of a marine bacterium, Vibrio sp. strain PO-303. The nucleotide sequence of the agaA gene consists of 2,958 bp and encodes a protein of 985 amino acids with a molecular mass of 106,062 Da. The deduced enzyme protein contains a typical N-terminal signal peptide of 29 amino acid residues, followed by a 266 amino acid sequence that is homologous to catalytic module of family 16 glycoside hydrolases, a bacterial immunoglobulin group 2 (Big-2)-like domain of 52 amino acid residues, two carbohydrate-binding modules of family 6 separated from Big-2-like domain by nine times repeated GDDTDP amino acid sequence. AgaA is the first agarase that was identified to possess a Big-2-like domain. The recombinant AgaA (rAgaA) expressed in Escherichia coli exhibited maximal activity around 40 degrees C and pH 7.5, with a specific activity of 16.4 units mg(-1), a K (m) of 1.10 mg ml(-1), and a V (max) of 22.5 micromol min(-1) mg(-1) for agarose. The rAgaA hydrolyzed neoagarohexaose, but did not act on neoagarotetraose and neoagarobiose.

Molecular cloning, expression, and characterization of a beta-agarase gene, agaD, from a marine bacterium, Vibrio sp. strain PO-303.

The beta-agarase-d gene (agaD) from a marine bacterium, Vibrio sp. strain PO-303, was cloned and expressed in Escherichia coli. The gene consists of 1,362 bp and encodes a protein of 453 amino acids with a predicted molecular weight of 50,824. The full length of agarase-d consists of a signal peptide, a glycoside hydrolase family 16 catalytic module (CM), and a carbohydrate binding module (CBM). The full length of agarase-d without the signal peptide (rAgaDDeltafull), the catalytic module (rAgaDCM), or the CBM (rAgaDCBM) was expressed in E. coli as recombinant proteins. rAgaDCM exhibited higher enzyme activity (63.6 units/mg) than rAgaDDeltafull (1.20 units/mg) against agarose. rAgaDCM hydrolyzed agar and porphyran to several oligosaccharides and acted on neoagarohexaose to produce neoagarotetraose and neoagarobiose, but did not act on neoagarotetraose. rAgaDCBM bound to agarose.

Cloning of the novel gene encoding beta-agarase C from a marine bacterium, Vibrio sp. strain PO-303, and characterization of the gene product.

The beta-agarase C gene (agaC) of a marine bacterium, Vibrio sp. strain PO-303, consisted of 1,437 bp encoding 478 amino acid residues. beta-Agarase C was identified as the first beta-agarase that cannot hydrolyze neoagarooctaose and smaller neoagarooligosaccharides and was assigned to a novel glycoside hydrolase family.

Molecular cloning and characterization of alpha-class glutathione S-transferase genes from the hepatopancreas of red sea bream, Pagrus major.

Two distinct cDNAs corresponding to GSTA1 and GSTA2 genes encoding glutathione S-transferases (GSTs) from the hepatopancreas of red sea bream, Pagrus major were cloned and sequenced. A comparison of the nucleotide sequences of GSTA1 and GSTA2 revealed 98% identity and their derived amino acid sequences had 96% similarity. Both genes could be classified as alpha-class GSTs on the basis of their amino acid sequence identity with other species. Genomic DNA cloning showed that both GSTA1 and GSTA2 genes consisted of six exons and five introns. In a comparison of genomic DNAs, the structures of GSTA1 and GSTA2 differed. In addition, Southern-blot analysis indicated that at least two kinds of alpha-class GSTs existed in the P. major genome. In order to biochemically characterize the recombinant enzymes (pmGSTA1-1 and pmGSTA2-2), both clones were highly expressed in Escherichia coli. The purified pmGSTA1-1 and pmGSTA2-2 exhibited glutathione conjugating activity toward 1-chloro-2,4-dinitrobenzene and glutathione peroxidase activity toward cumene hydroperoxide, while neither pmGSTs show detectable activity toward 1,2-dichloro-4-nitrobenzene, ethacrynic acid, 4-hydroxynonenal, or p-nitrobenzyl chloride. Despite their high level of amino acid sequence identity, the pmGSTs had quite different enzyme-kinetic parameters.

Purification and characterization of beta-1,3-xylanase from a marine bacterium, Alcaligenes sp. XY-234.

A beta-1,3-xylanase-producing bacterium, Alcaligenes sp. XY-234, was isolated from the marine environment. The organism produced endo-1,3-beta-xylanase at a high level in the culture fluid. The enzyme was purified 292-fold by ammonium sulfate precipitation and several column chromatographies. The final enzyme preparation appeared to be homogeneous on disc gel electrophoresis and SDS-PAGE with a molecular mass of 59 kDa, and the pI was 4.0. The enzyme hydrolyzed beta-1,3-xylan and larger xylooligosaccharides than xylobiose to give several xylooligosaccharides, but it could not hydrolyze xylobiose, p-nitrophenyl-beta-D-xyloside, and beta-1,4-xylan. The Km of the enzyme was 4.0 mg/ml. Optimal pH and temperature were 7.5 and 40 degrees C, respectively. It was stable from pH 6.0 to 10 and at a temperature of less than 40 degrees C. The enzyme was strongly inhibited by 1 mM HgCl(2)., AlCl(3), CuCl(2), FeCl(3), HgCl(2), Pb(CH(3)COO) (2), and N-bromosuccinimide.

Novel carbohydrate-binding module of beta-1,3-xylanase from a marine bacterium, Alcaligenes sp. strain XY-234.

A beta-1,3-xylanase gene (txyA) from a marine bacterium, Alcaligenes sp. strain XY-234, has been cloned and sequenced. txyA consists of a 1,410-bp open reading frame that encodes 469 amino acid residues with a calculated molecular mass of 52,256 Da. The domain structure of the beta-1,3-xylanase (TxyA) consists of a signal peptide of 22 amino acid residues, followed by a catalytic domain which belongs to family 26 of the glycosyl hydrolases, a linker region with one array of DGG and six repeats of DNGG, and a novel carbohydrate-binding module (CBM) at the C terminus. The recombinant TxyA hydrolyzed beta-1,3-xylan but not other polysaccharides such as beta-1,4-xylan, carboxymethylcellulose, curdlan, glucomannan, or beta-1,4-mannan. TxyA was capable of binding specifically to beta-1,3-xylan. The analysis using truncated TxyA lacking either the N- or C-terminal region indicated that the region encoding the CBM was located between residues 376 and 469. Binding studies on the CBM revealed that the K(d) and the maximum amount of protein bound to beta-1,3-xylan were 4.2 microM and 18.2 micromol/g of beta-1,3-xylan, respectively. Furthermore, comparison of the enzymatic properties between proteins with and without the CBM strongly indicated that the CBM of TxyA plays an important role in the hydrolysis of beta-1,3-xylan.