Genome sequence of Vibrio sp. strain 10N, an ulvan-degrading bacterium isolated from coastal seawater collected at Ise Bay, Japan.

Genome sequence of an ulvan-degrading bacterium, Vibrio sp. strain 10N, is presented. The genome is 5,358,550 bp with a G + C content of 46.5%. A total of 4,712 coding sequences, including two novel ulvan lyase genes encoding a BNR4 and a glycoside hydrolase (GH88) motif, are known to be involved in the degradation of green algae.

An easy-to-implement, non-invasive head restraint method for monkey fMRI.

Functional magnetic resonance imaging (fMRI) in behaving monkeys has a strong potential to bridge the gap between human neuroimaging and primate neurophysiology. In monkey fMRI, to restrain head movements, researchers usually surgically implant a plastic head-post on the skull. Although time-proven to be effective, this technique could create burdens for animals, including a risk of infection and discomfort. Furthermore, the presence of extraneous objects on the skull, such as bone screws and dental cement, adversely affects signals near the cortical surface. These side effects are undesirable in terms of both the practical aspect of efficient data collection and the spirit of "refinement" from the 3R's. Here, we demonstrate that a completely non-invasive fMRI scan in awake monkeys is possible by using a plastic head mask made to fit the skull of individual animals. In all of the three monkeys tested, longitudinal, quantitative assessment of head movements showed that the plastic mask has effectively suppressed head movements, and we were able to obtain reliable retinotopic BOLD signals in a standard retinotopic mapping task. The present, easy-to-make plastic mask has a strong potential to simplify fMRI experiments in awake monkeys, while giving data that is as good as or even better quality than that obtained with the conventional head-post method.

The BCL-2 family protein BCL-RAMBO interacts and cooperates with GRP75 to promote its apoptosis signaling pathway.

The BCL-2 family protein BCL-RAMBO, also known as BCL2-like 13, anchors at the outer mitochondrial membrane and regulates apoptosis, mitochondrial fragmentation, and mitophagy. However, the mechanisms underlying the proapoptotic role of BCL-RAMBO remain unclear. In the present study, we demonstrated that BCL-RAMBO interacted with glucose-regulated protein 75 (GRP75), also known as heat shock protein family A member 9, and mortalin using co-immunoprecipitation and glutathione S-transferase-based pull-down assays. BCL-RAMBO interacted with GRP75 via its No BCL-2 homology domain. The interaction between BCL-RAMBO and GRP75 was confirmed by genetic interactions in Drosophila because a rough eye phenotype caused by the ectopic expression of BCL-RAMBO was partially suppressed by mutations in Hsc70-5, a mammalian GRP75 ortholog. In human embryonic kidney 293T cells, the co-expression of BCL-RAMBO and GRP75 facilitated an elevation in executioner caspase activity and poly (ADP-ribose) polymerase 1 (PARP-1) cleavage. In contrast, the knockdown of GRP75 suppressed elevated executioner caspase activity and PARP-1 cleavage in BCL-RAMBO-transfected cells. The mitochondrial release of cytochrome c induced by BCL-RAMBO was also attenuated by the knockdown of GRP75. These results indicate that GRP75 interacts with BCL-RAMBO and plays a crucial role in the BCL-RAMBO-dependent apoptosis signaling pathway.

Cell-Penetrating Peptide-Peptide Nucleic Acid Conjugates as a Tool for Protein Functional Elucidation in the Native Bacterium.

Approximately 30% or more of the total proteins annotated from sequenced bacteria genomes are annotated as hypothetical or uncharacterized proteins. However, elucidation on the function of these proteins is hindered by the lack of simple and rapid screening methods, particularly with novel or hard-to-transform bacteria. In this report, we employed cell-penetrating peptide (CPP) -peptide nucleotide acid (PNA) conjugates to elucidate the function of such uncharacterized proteins in vivo within the native bacterium. Paenibacillus, a hard-to-transform bacterial genus, was used as a model. Two hypothetical genes showing amino acid sequence similarity to ι-carrageenases, termed cgiA and cgiB, were identified from the draft genome of Paenibacillus sp. strain YYML68, and CPP-PNA probes targeting the mRNA of the acyl carrier protein gene, acpP, and the two ι-carrageenase candidate genes were synthesized. Upon direct incubation of CPP-PNA targeting the mRNA of the acpP gene, we successfully observed growth inhibition of strain YYML68 in a concentration-dependent manner. Similarly, both the function of the candidate ι-carrageenases were also inhibited using our CPP-PNA probes allowing for the confirmation and characterization of these hypothetical proteins. In summary, we believe that CPP-PNA conjugates can serve as a simple and efficient alternative approach to characterize proteins in the native bacterium.

PGAM5 interacts with Bcl-rambo and regulates apoptosis and mitophagy.

Bcl-rambo, also known as BCL2L13, has been reported to regulate apoptosis, mitochondrial fragmentation, and mitophagy. However, the molecular mechanisms by which Bcl-rambo regulates these processes currently remain unclear. In the present study, we identified phosphoglycerate mutase member 5 (PGAM5) as an emerging partner interacting with Bcl-rambo through phenotypic Drosophila screening. The rough eye phenotype induced by human Bcl-rambo was partly rescued by the knockdown of pgam5-2, a mammalian ortholog of PGAM5. Bcl-rambo bound to PGAM5, and their interaction required the Bcl-rambo transmembrane domain. The co-expression of Bcl-rambo and PGAM5 promoted effector caspase activity in human embryonic kidney 293T cells. The transient overexpression of Bcl-rambo increased LC3B-II levels, which had been decreased by the co-expression of PGAM5. These results suggest that PGAM5 promotes Bcl-rambo-dependent apoptosis, but conversely interferes with Bcl-rambo-dependent mitophagy.

Isolation, Diversity and Characterization of Ulvan-Degrading Bacteria Isolated from Marine Environments.

In this study, we aimed to isolate bacteria capable of degrading the polysaccharide ulvan from the green algae Ulva sp. (Chlorophyta, Ulvales, Ulvaceae) in marine environments. We isolated 13 ulvan-degrading bacteria and observed high diversity at the genus level. Further, the genera Paraglaciecola, Vibrio, Echinicola, and Algibacter, which can degrade ulvan, were successfully isolated for the first time from marine environments. Among the 13 isolates, only one isolate (Echinicola sp.) showed the ability not only to produce externally expressed ulvan lyase, but also to be periplasmic or on the cell surface. From the results of the full-genome analysis, lyase was presumed to be a member of the PL25 (BNR4) family of ulvan lyases, and the bacterium also contained the sequence for glycoside hydrolase (GH43, GH78 and GH88), which is characteristic of other ulvan-degrading bacteria. Notably, this bacterium has a unique ulvan lyase gene not previously reported.

Production of 4-Deoxy-L-erythro-5-Hexoseulose Uronic Acid Using Two Free and Immobilized Alginate Lyases from Falsirhodobacter sp. Alg1.

Falsirhodobacter sp. alg1 expresses two alginate lyases, AlyFRA and AlyFRB, to produce the linear monosaccharide 4-deoxy-L-erythro-5-hexoseulose uronic acid (DEH) from alginate, metabolizing it to pyruvate. In this study, we prepared recombinant AlyFRA and AlyFRB and their immobilized enzymes and investigated DEH production. Purified AlyFRA and AlyFRB reacted with sodium alginate and yielded approximately 96.8% DEH. Immobilized AlyFRA and AlyFRB were prepared using each crude enzyme solution and κ-carrageenan, and immobilized enzyme reuse in batch reactions and DEH yield were examined. Thus, DEH was produced in a relatively high yield of 79.6%, even after the immobilized enzyme was reused seven times. This method can produce DEH efficiently and at a low cost and can be used to mass produce the next generation of biofuels using brown algae.

Investigation of Biofilms Formed on Steelmaking Slags in Marine Environments for Water Depuration.

Steelmaking slags are a promising resource as artificial seaweed beds for the reconstitution of marine environments. To grow seaweed well, the formation of biofilms is an essential process in biofouling. This study focused on the formation of initial biofilms on steelmaking slag samples and analyzed the resulting bacterial communities using the next-generation sequencing technique. Three types of steelmaking slag were submerged in an area of Ise Bay in Mie Prefecture, Japan, for 3 and 7 days in the summer and winter seasons to allow the formation of biofilms. The bacterial communities of these biofilms were richer in sulfur-oxidizing bacteria compared to the biofilms formed on polyurethane sponges. It was found that Helicobacteraceae dominantly grew on the biofilms formed on the slag samples. This shows that steelmaking slags have potential to be used as artificial seaweed beds and marine water purifiers.

Microbial community analysis in the gills of abalones suggested possible dominance of epsilonproteobacterium in Haliotis gigantea.

Gills are important organs for aquatic invertebrates because they harbor chemosynthetic bacteria, which fix inorganic carbon and/or nitrogen and provide their hosts with organic compounds. Nevertheless, in contrast to the intensive researches related to the gut microbiota, much is still needed to further understand the microbiota within the gills of invertebrates. Using abalones as a model, we investigated the community structure of microbes associated with the gills of these invertebrates using next-generation sequencing. Molecular identification of representative bacterial sequences was performed using cloning, nested PCR and fluorescence in situ hybridization (FISH) analysis with specific primers or probes. We examined three abalone species, namely Haliotis gigantea, H. discus and H. diversicolor using seawater and stones as controls. Microbiome analysis suggested that the gills of all three abalones had the unclassified Spirochaetaceae (one OTU, 15.7 ± 0.04%) and Mycoplasma sp. (one OTU, 9.1 ± 0.03%) as the core microbes. In most libraries from the gills of H. gigantea, however, a previously unknown epsilonproteobacterium species (one OTU) was considered as the dominant bacterium, which accounted for 62.2% of the relative abundance. The epsilonproteobacterium was only detected in the gills of H. diversicolor at 0.2% and not in H. discus suggesting that it may be unique to H. gigantea. Phylogenetic analysis performed using a near full-length 16S rRNA gene placed the uncultured epsilonproteobacterium species at the root of the family Helicobacteraceae. Interestingly, the uncultured epsilonproteobacterium was commonly detected from gill tissue rather than from the gut and foot tissues using a nested PCR assay with uncultured epsilonproteobacterium-specific primers. FISH analysis with the uncultured epsilonproteobacterium-specific probe revealed that probe-reactive cells in H. gigantea had a coccus-like morphology and formed microcolonies on gill tissue. This is the first report to show that epsilonproteobacterium has the potential to be a dominant species in the gills of the coastal gastropod, H. gigantea.

Diversity, enumeration, and isolation of Arcobacter spp. in the giant abalone, Haliotis gigantea.

Arcobacter have been frequently detected in and isolated from bivalves, but there is very little information on the genus Arcobacter in the abalone, an important fishery resource. This study aimed to investigate the genetic diversity and abundance of bacteria from the genus Arcobacter in the Japanese giant abalone, Haliotis gigantea, using molecular methods such as Arcobacter-specific clone libraries and fluorescence in situ hybridization (FISH). Furthermore, we attempted to isolate the Arcobacter species detected. Twelve genotypes of clones were obtained from Arcobacter-specific clone libraries. These sequences are not classified with any other known Arcobacter species including pathogenic Arcobacter spp., A. butzleri, A. skirrowii, and A. cryaerophilus, commonly isolated or detected from bivalves. From the FISH analysis, we observed that ARC94F-positive cells, presumed to be Arcobacter, accounted for 6.96 ± 0.72% of all EUB338-positive cells. In the culture method, three genotypes of Arcobacter were isolated from abalones. One genotype had a similarity of 99.2%-100.0% to the 16S rRNA gene of Arcobacter marinus, while the others showed only 93.3%-94.3% similarity to other Arcobacter species. These data indicate that abalones carry Arcobacter as a common bacterial genus which includes uncultured species.

The human Bcl-2 family member Bcl-rambo and voltage-dependent anion channels manifest a genetic interaction in Drosophila and cooperatively promote the activation of effector caspases in human cultured cells.

We previously reported that the Bcl-2 family member human Bcl-rambo, also known as BCL2L13, induces apoptosis in human embryonic kidney 293T cells. Mouse Bcl-rambo has recently been reported to mediate mitochondrial fragmentation and mitophagy. In the present study, we showed that the transfection of human Bcl-rambo and its microtubule-associated protein light chain 3-interacting region motif mutant (W276A/I279A) caused mitochondrial fragmentation and the perinuclear accumulation of fragmented mitochondria in human lung adenocarcinoma A549 cells. In comprehensive screening using the Drosophila model in which human Bcl-rambo was ectopically expressed in eye imaginal discs, voltage-dependent anion channels (VDAC), also known as mitochondrial porin, were found to manifest a genetic interaction with human Bcl-rambo. In addition to human adenine nucleotide translocase (ANT) 1 and ANT2, the human Bcl-rambo protein bound to human VDAC1, albeit to a lesser extent than ANT2. Moreover, human VDAC1 and human VDAC2 in particular promoted the activation of effector caspases only when they were co-expressed with human Bcl-rambo in 293T cells. Bcl-rambo induced the perinuclear accumulation of fragmented mitochondria by the knockdown of VDAC1, VDAC2, and VDAC3 in A549 cells. Thus, the present study revealed that human Bcl-rambo and VDAC cooperatively promote the activation of effector caspases in human cultured cells.

Xylanase B from Clostridium cellulovorans 743B: overexpression, purification, crystallization and X-ray diffraction analysis.

Clostridium cellulovorans produces multi-enzyme complexes called cellulosomes capable of efficiently degrading cellulosic biomass. There are three xylanase genes containing a sequence corresponding to a dockerin domain that are necessary for constructing cellulosomes in the genome. Among the xylanases encoded by these genes, xylanase B (XynB) contains a catalytic domain belonging to glycoside hydrolase family 10 and a carbohydrate-binding module (CBM) at the N-terminus, making it a member of CBM family 22. In this study, XynB was cloned, overexpressed, purified and crystallized. XynB was crystallized using the hanging-drop vapour-diffusion method in the presence of 0.2 M sodium acetate trihydrate, 0.1 M Tris-HCl pH 8.5, 32%(w/v) PEG 4000 at 293 K. X-ray diffraction analysis revealed that the crystal diffracted to 1.95 Šresolution and belonged to space group P212121, with unit-cell parameters a = 74.28, b = 77.55, c = 88.20 Å, α = ß = γ = 90°. The data-evaluation statistics revealed high quality of the collected data, thereby establishing a solid basis for determination of the structure of cellulosomal xylanase from C. cellulovorans.

Arcobacter haliotis sp. nov., isolated from abalone species Haliotis gigantea.

A Gram-negative, aerobic, polar-flagellated and rod-shaped, sometimes slightly curved bacterium, designated MA5T, was isolated from the gut of an abalone of the species Haliotis gigantea collected in Japan. Phylogenetic analyses based on 16S rRNA, gyrB, hsp60 and rpoB gene sequences placed strain MA5T in the genus Arcobacter in an independent phylogenetic line. Comparison of the 16S rRNA gene sequence of this strain with those of the type strains of the established Arcobacter species revealed A. nitrofigilis (95.1 %) as nearest neighbour. Strain MA5T grew optimally at 25 °C, pH 6.0 to 9.0 and in the presence of 2 to 5 % (w/v) NaCl under both aerobic and microaerobic conditions. The predominant fatty acids found were summed feature 3 (iso-C15 : 0 2-OH and/or C16 : 1 ω7c), C12 : 0 3-OH and C18 : 1 ω7c. Menaquinone-6 (MK-6) and menaquinone-7 (MK-7) were found as the major respiratory quinones. The major polar lipids detected were phosphatidylethanolamine and phosphatidylglycerol. Strain MA5T could be differentiated phenotypically from the phylogenetic closest Arcobacter species by its ability to grow on 0.05 % safranin and 0.01 % 2,3,5-triphenyl tetrazolium chloride (TTC), but not on 0.5 % NaCl. The obtained DNA G+C content of strain MA5T was 27.9 mol%. Based on the phylogenetic, chemotaxonomic and phenotypic distinctiveness of MA5T, this strain is considered to represent a novel species of the genus Arcobacter, for which the name Arcobacter haliotis sp. nov. is proposed. The type strain is MA5T (=LMG 28652T=JCM 31147T).

Construction of bioengineered yeast platform for direct bioethanol production from alginate and mannitol.

Brown macroalgae are a sustainable and promising source for bioethanol production because they are abundant in ocean ecosystems and contain negligible quantities of lignin. Brown macroalgae contain cellulose, hemicellulose, mannitol, laminarin, and alginate as major carbohydrates. Among these carbohydrates, brown macroalgae are characterized by high levels of alginate and mannitol. The direct bioconversion of alginate and mannitol into ethanol requires extensive bioengineering of assimilation processes in the standard industrial microbe Saccharomyces cerevisiae. Here, we constructed an alginate-assimilating S. cerevisiae recombinant strain by genome integration and overexpression of the genes encoding endo- and exo-type alginate lyases, DEH (4-deoxy-L-erythro-5-hexoseulose uronic acid) transporter, and components of the DEH metabolic pathway. Furthermore, the mannitol-metabolizing capacity of S. cerevisiae was enhanced by prolonged culture in a medium containing mannitol as the sole carbon source. When the constructed strain AM1 was anaerobically cultivated in a fermentation medium containing 6% (w/v) total sugars (approximately 1:2 ratio of alginate/mannitol), it directly produced ethanol from alginate and mannitol, giving 8.8 g/L ethanol and yields of up to 32% of the maximum theoretical yield from consumed sugars. These results indicate that all major carbohydrates of brown macroalgae can be directly converted into bioethanol by S. cerevisiae. This strain and system could provide a platform for the complete utilization of brown macroalgae.

Characterization of the cellulosomal scaffolding protein CbpC from Clostridium cellulovorans 743B.

Clostridium cellulovorans 743B, an anaerobic and mesophilic bacterium, produces an extracellular enzyme complex called the cellulosome on the cell surface. Recently, we have reported the whole genome sequence of C. cellulovorans, which revealed that a total of 4 cellulosomal scaffolding proteins: CbpA, HbpA, CbpB, and CbpC were encoded in the C. cellulovorans genome. In particular, cbpC encoded a 429-residue polypeptide that includes a carbohydrate-binding module (CBM), an S-layer homology module, and a cohesin. CbpC was also detected in the culture supernatant of C. cellulovorans. Genomic DNA coding for CbpC was subcloned into a pET-22b+ vector in order to express and produce the recombinant protein in Escherichia coli BL21(DE3). Measurement of CbpC adsorption to crystalline cellulose indicated a dissociation constant of 0.60 µM, which is a similar to that of CBM from CbpA. We also subcloned the region encoding xylanase B (XynB) with the dockerin from C. cellulovorans and analyzed the interaction between XynB and CbpC by GST pull-down assay. It was observed that GST-CbpC assembles with XynB to form a minimal cellulosome. The activity of XynB against rice straw tended to be increased in the presence of CbpC. These results showed a synergistic effect on rice straw as a representative cellulosic biomass through the formation of a minimal cellulosome containing XynB bound to CbpC. Thus, our findings provide a foundation for the development of cellulosic biomass saccharification using a minimal cellulosome.

Kinetics of the Inactivation of Vibrio parahaemolyticus in Weakly Acidic Sodium Chlorite Solution.

　The kinetics of the inactivation of Vibrio parahaemolyticus in sodium chlorite (NaClO2) solution was studied in the weakly acidic pH range of 4.0 to 6.5 and at various temperatures. The logarithmic reduction of the survival ratio depended on the concentration-time product, and all the inactivation curves showed a linear reduction phase. The first-order inactivation rate constant (k) increased by approximately twice for every 0.44 unit fall in pH. During the inactivation experiments, no formation of chlorine dioxide occurred. These data indicated that undissociated HClO2 was the active species governing the inactivation of V. parahaemolyticus. It was also shown that the use of weakly acidic NaClO2 solutions containing high concentrations of ionized ClO2- gave slower kinetics of the inactivation, whereas it could achieve the significant reduction of viable cells of more than 4-log. The k value showed an Arrhenius-type temperature dependence in the temperature range of 5 to 40℃. The apparent activation energy for the inactivation of V. parahaemolyticus was estimated to be 43.5 kJ/mol. The k value increased by approximately 1.8 times for every 10℃ rise in temperature.

Genome Sequence of Arcobacter sp. Strain LA11, Isolated from the Abalone Haliotis discus.

Arcobacter sp. strain LA11 was isolated from the gut of the abalone Haliotis discus Here, we present the annotation and analysis of the draft genome of this strain, which is involved in nitrogen metabolism.

Inactivation of Vibrio parahaemolyticus Unattached and Attached to a Solid Surface in pH-Controlled Sodium Hypochlorite Solutions.

　The inactivation of Vibrio parahaemolyticus cells that were unattached or attached to a polyethylene terephthalate (PET) disc in pH-controlled sodium hypochlorite (NaOCl) solutions was studied under turbulent conditions. No significant desorption of attached cells occurred at the free available chlorine (FAC) concentrations from 0.1 to 1.0 mg/l. The number of viable cells was estimated by microbial calorimetry. The logarithmic relative reduction of viable cells was proportional to the product of the FAC concentration and time. In the pH range of 5.6 to 9.3, the first-order inactivation rate constants for unattached and attached cells increased with decreasing solution pH. It was found that the rate constants for unattached cells were approximately 6 to 7 times higher than those for attached cells at all pH values examined. It was confirmed that attached cells were more resistant to NaOCl solutions than unattached cells even when accessibility of attached cells to HOCl/OCl- was enhanced under turbulent conditions.

Genome Sequence of Formosa haliotis Strain MA1, a Brown Alga-Degrading Bacterium Isolated from the Gut of Abalone Haliotis gigantea.

Formosa haliotis is a brown alga-degrading bacterium isolated from the gut of abalone Haliotis gigantea Here, we report the draft genome sequence of this bacterium and pointed out possible important features related to alginate degradation.