Deformability-Based Microfluidic Microdroplet Screening to Obtain Agarolytic Bacterial Cells.

Environmental microorganisms possess enzymes that can digest macromolecules such as agarose into smaller molecules that can be utilized for growth. These enzymes could be valuable for the effective utilization of global resources. However, since most of the microorganisms on Earth remain uncultured, there is significant untapped enzymatic potential in nature. Therefore, it is necessary to develop innovative tools and strategies for exploring these enzymatic resources. To address this, we developed a method for screening microbial cells that secrete hydrogel-degrading enzymes using deformability-based microfluidic microdroplet sorting. In this method, microbial cells are encapsulated as single cells in water-in-oil (W/O) microdroplets with a hydrogel whose shape becomes deformable as the hydrogel is progressively degraded into smaller molecules. Screening is achieved using a microfluidic device that passively sorts the deformed W/O microdroplets. Using this method, we successfully sorted agarose-containing microdroplets, encapsulating single bacterial cells that hydrolyzed agarose. This method can be used to screen various hydrogel-degrading microbial cells.

Effects of Collaborative Training Using Virtual Co-embodiment on Motor Skill Learning.

Virtual reality (VR) is a promising tool for motor skill learning. Previous studies have indicated that observing and following a teacher's movements from a first-person perspective using VR facilitates motor skill learning. Conversely, it has also been pointed out that this learning method makes the learner so strongly aware of the need to follow that it weakens their sense of agency (SoA) for motor skills and prevents them from updating the body schema, thereby preventing long-term retention of motor skills. To address this problem, we propose applying "virtual co-embodiment" to motor skill learning. Virtual co-embodiment is a system in which a virtual avatar is controlled based on the weighted average of the movements of multiple entities. Because users in virtual co-embodiment overestimate their SoA, we hypothesized that learning using virtual co-embodiment with a teacher would improve motor skill retention. In this study, we focused on learning a dual task to evaluate the automation of movement, which is considered an essential element of motor skills. As a result, learning in virtual co-embodiment with the teacher improves motor skill learning efficiency compared with sharing the teacher's first-person perspective or learning alone.

Complete Genomic Sequences of Two Agarolytic Vibrio Species Isolates from the Red Algae Gracilaria.

We report the complete genomic sequences of two agarolytic Vibrio species strains, STUT-A11 and STUT-A16, isolated from the red algae Gracilaria. Genomic annotations revealed that both strains harbor four ß-agarases, α-neoagarooligosaccharide hydrolase, and agarolytic ß-galactosidase, which support efficient agarose catabolism.

Simultaneous Methylene Blue Adsorption and pH Neutralization of Contaminated Water by Rice Husk Ash.

In this study, the potential of rice husk ash (RHA) to act as an adsorbent for treating dye-containing wastewater was demonstrated. The RHA used in this study contained 91.7% silica, which was composed of crystalline (cristobalite and tridymite) and amorphous phases. The mechanochemical treatment of RHA led to an increase in its specific surface area from 6.2 to 14.6 m2/g in 15 min and dramatically improved its methylene blue (MB) adsorption ability. Langmuir adsorption isotherms revealed that the maximum adsorption capacity of the treated RHA was 8.59 mg/g, which is 2.45 times higher than that of raw RHA. pH-dependent adsorption studies on the RHA revealed that MB was adsorbed on the deprotonated Q3 silanol through electrostatic interactions. Moreover, the RHA adsorbent showed pH buffering at a pH value of approximately 7; thus, the pH of the solution could be neutralized simultaneously with the adsorptive removal of MB.

Enzymatic Specific Production and Chemical Functionalization of Phenylpropanone Platform Monomers from Lignin.

Enzymatic catalysis is an ecofriendly strategy for the production of high-value low-molecular-weight aromatic compounds from lignin. Although well-definable aromatic monomers have been obtained from synthetic lignin-model dimers, enzymatic-selective synthesis of platform monomers from natural lignin has not been accomplished. In this study, we successfully achieved highly specific synthesis of aromatic monomers with a phenylpropane structure directly from natural lignin using a cascade reaction of ß-O-4-cleaving bacterial enzymes in one pot. Guaiacylhydroxylpropanone (GHP) and the GHP/syringylhydroxylpropanone (SHP) mixture are exclusive monomers from lignin isolated from softwood (Cryptomeria japonica) and hardwood (Eucalyptus globulus). The intermediate products in the enzymatic reactions show the capacity to accommodate highly heterologous substrates at the substrate-binding sites of the enzymes. To demonstrate the applicability of GHP as a platform chemical for bio-based industries, we chemically generate value-added GHP derivatives for bio-based polymers. Together with these chemical conversions for the valorization of lignin-derived phenylpropanone monomers, the specific and enzymatic production of the monomers directly from natural lignin is expected to provide a new stream in "white biotechnology" for sustainable biorefineries.

Culture-independent method for identification of microbial enzyme-encoding genes by activity-based single-cell sequencing using a water-in-oil microdroplet platform.

Environmental microbes are a great source of industrially valuable enzymes with potent and unique catalytic activities. Unfortunately, the majority of microbes remain unculturable and thus are not accessible by culture-based methods. Recently, culture-independent metagenomic approaches have been successfully applied, opening access to untapped genetic resources. Here we present a methodological approach for the identification of genes that encode metabolically active enzymes in environmental microbes in a culture-independent manner. Our method is based on activity-based single-cell sequencing, which focuses on microbial cells showing specific enzymatic activities. First, at the single-cell level, environmental microbes were encapsulated in water-in-oil microdroplets with a fluorogenic substrate for the target enzyme to screen for microdroplets that contain microbially active cells. Second, the microbial cells were recovered and subjected to whole genome amplification. Finally, the amplified genomes were sequenced to identify the genes encoding target enzymes. Employing this method, we successfully identified 14 novel ß-glucosidase genes from uncultured bacterial cells in marine samples. Our method contributes to the screening and identification of genes encoding industrially valuable enzymes.

Thalassobius abyssi sp. nov., a marine bacterium isolated from cold-seep sediment.

A novel marine bacterial strain, designated JAMH 043T, was isolated from cold-seep sediment in Sagami Bay, Japan. Cells were Gram-stain-negative, rod-shaped, non-motile and aerobic chemo-organotrophs. The isolate grew optimally at 25 °C, at pH 7.0-7.5 and with 3 % (w/v) NaCl. The major respiratory quinone was ubiquinone-10 (Q-10). The predominant fatty acid was C18 : 1ω7c. On the basis of 16S rRNA gene sequence analysis, the isolated strain was closely affiliated to members of the genus Thalassobius in the class Alphaproteobacteria, and 16S rRNA gene sequence similarity of the novel isolate with the type strain of its closest related species, Thalassobius aestuarii JC2049T, was 98.4 %. The DNA G+C content of the novel strain was 58.0 mol%. The hybridization values for DNA-DNA relatedness between strain JAMH 043T and reference strains belonging to the genus Thalassobius were less than 14.1 ± 2.2 %. Based on differences in taxonomic characteristics, the isolated strain represents a novel species of the genus Thalassobius, for which the name Thalassobius abyssi sp. nov. is proposed. The type strain is JAMH 043T ( = JCM 30900T = DSM 100673T).

Combination of six enzymes of a marine Novosphingobium converts the stereoisomers of ß-O-4 lignin model dimers into the respective monomers.

Lignin, an aromatic polymer of phenylpropane units joined predominantly by ß-O-4 linkages, is the second most abundant biomass component on Earth. Despite the continuous discharge of terrestrially produced lignin into marine environments, few studies have examined lignin degradation by marine microorganisms. Here, we screened marine isolates for ß-O-4 cleavage activity and determined the genes responsible for this enzymatic activity in one positive isolate. Novosphingobium sp. strain MBES04 converted all four stereoisomers of guaiacylglycerol-ß-guaiacyl ether (GGGE), a structural mimic of lignin, to guaiacylhydroxypropanone as an end metabolite in three steps involving six enzymes, including a newly identified Nu-class glutathione-S-transferase (GST). In silico searches of the strain MBES04 genome revealed that four GGGE-metabolizing GST genes were arranged in a cluster. Transcriptome analysis demonstrated that the lignin model compounds GGGE and (2-methoxyphenoxy)hydroxypropiovanillone (MPHPV) enhanced the expression of genes in involved in energy metabolism, including aromatic-monomer assimilation, and evoked defense responses typically expressed upon exposure to toxic compounds. The findings from this study provide insight into previously unidentified bacterial enzymatic systems and the physiological acclimation of microbes associated with the biological transformation of lignin-containing materials in marine environments.

Electrical Retrieval of Living Microorganisms from Cryopreserved Marine Sponges Using a Potential-Controlled Electrode.

The purpose of this study was to develop a novel electrical retrieval method (ER method) for living sponge-associated microorganisms from marine sponges frozen at -80 °C. A -0.3-V vs. Ag/AgCl constant potential applied for 2 h at 9 °C induced the attachment of the sponge-associated microorganisms to an indium tin oxide/glass (ITO) or a gallium-doped zinc oxide/glass (GZO) working electrode. The electrically attached microorganisms from homogenized Spirastrella insignis tissues had intact cell membranes and showed intracellular dehydrogenase activity. Dead microorganisms were not attracted to the electrode when the homogenized tissues were autoclaved for 15 min at 121 °C before use. The electrically attached microorganisms included cultivable microorganisms retrieved after detachment from the electrode by application of a 9-MHz sine-wave potential. Using the ER method, we obtained 32 phyla and 72 classes of bacteria and 3 archaea of Crenarchaeota thermoprotei, Marine Group I, and Thaumarchaeota incertae sedis from marine sponges S. insignis and Callyspongia confoederata. Employment of the ER method for extraction and purification of the living microorganisms holds potential of single-cell cultivation for genome, transcriptome, proteome, and metabolome analyses of bioactive compounds producing sponge-associated microorganisms.

Involvement of flocculin in negative potential-applied ITO electrode adhesion of yeast cells.

The purpose of this study was to develop novel methods for attachment and cultivation of specifically positioned single yeast cells on a microelectrode surface with the application of a weak electrical potential. Saccharomyces cerevisiae diploid strains attached to an indium tin oxide/glass (ITO) electrode to which a negative potential between -0.2 and -0.4 V vs. Ag/AgCl was applied, while they did not adhere to a gallium-doped zinc oxide/glass electrode surface. The yeast cells attached to the negative potential-applied ITO electrodes showed normal cell proliferation. We found that the flocculin FLO10 gene-disrupted diploid BY4743 mutant strain (flo10Δ /flo10Δ) almost completely lost the ability to adhere to the negative potential-applied ITO electrode. Our results indicate that the mechanisms of diploid BY4743 S. cerevisiae adhesion involve interaction between the negative potential-applied ITO electrode and the Flo10 protein on the cell wall surface. A combination of micropatterning techniques of living single yeast cell on the ITO electrode and omics technologies holds potential of novel, highly parallelized, microchip-based single-cell analysis that will contribute to new screening concepts and applications.

Draft Genome Sequence of Aneurinibacillus tyrosinisolvens LL-002T, Which Possesses Some Pseudouridine Synthases.

We report the 5.7-Mb draft genome sequence of Aneurinibacillus tyrosinisolvens strain LL-002(T), isolated from organic- and methane-rich sea sediments. The draft genome sequence of strain LL-002(T) consists of 5,693,818 bp in 136 contigs, with a G+C content of 44.5%, 5,946 potential coding sequences (CDS), 2 rRNAs, and 39 tRNAs.

Shimia sagamensis sp. nov., a marine bacterium isolated from cold-seep sediment.

A novel marine bacterial strain designated JAMH 011(T) was isolated from the cold-seep sediment in Sagami Bay, Japan. Cells were Gram-stain-negative, rod-shaped, non-spore-forming, aerobic chemo-organotrophs and motile by means of a single polar flagellum. Growth occurred at temperatures below 31 °C, with the optimum at 25 °C. The major respiratory quinone was Q-10. The predominant fatty acid was C18 : 1ω7c. On the basis of 16S rRNA gene sequence analysis, the isolated strain was closely affiliated with members of the genus Shimia in the class Alphaproteobacteria, and the 16S rRNA gene sequence similarity of the novel isolate with the type strain of the closest related species, Shimia haliotis WM35(T), was 98.1%. The DNA G+C content of the novel strain was 57.3 mol%. The hybridization values for DNA-DNA relatedness between strain JAMH 011(T) and reference strains belonging to the genus Shimia were less than 9.4 ± 0.7%. Based on differences in taxonomic characteristics, the isolated strain represents a novel species of the genus Shimia, for which the name Shimia sagamensis sp. nov. is proposed. The type strain is JAMH 011(T) ( = JCM 30583(T) = DSM 29734(T)).

Aneurinibacillus tyrosinisolvens sp. nov., a tyrosine-dissolving bacterium isolated from organics- and methane-rich seafloor sediment.

A novel Gram-positive-staining, strictly aerobic and heterotrophic bacterium, designated strain LL-002T, was isolated from organics- and methane-rich seafloor sediment at a depth of 100 m in Kagoshima Bay, Kagoshima, Japan. Colonies were lustreless and translucent white in colour. The temperature, pH and salt concentration ranges for growth were 10-30 °C, pH 6.0-6.5 and 0-1 % (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences confirmed that strain LL-002T belongs to the genus Aneurinibacillus of the family Paenibacillaceae. 16S rRNA gene sequence similarities between strain LL-002T and the type strains of species of the genus Aneurinibacillus were 92.8-95.7 %; the highest sequence identity was with the type strain of Aneurinibacillus migulanus. The DNA G+C content of strain LL-002T was 46.2 mol%. MK-7 was the predominant menaquinone. The predominant cellular fatty acids were iso-C15 : 0 and anteiso-C15 : 0, and the cell-wall peptidoglycan contained meso-diaminopimelic acid and glutamic acid, glycine and alanine in addition to muramic acid and glucosamine. The peptidoglycan type was A1γ. In DNA-DNA hybridization assays between strain LL-002T and the type strains of the other species of the genus Aneurinibacillus, the level of hybridization was 6.3-30.1 %. On the basis of its biological features and the 16S rRNA gene sequence comparison presented here, strain LL-002T is considered to represent a novel species of the genus Aneurinibacillus, for which the name Aneurinibacillus tyrosinisolvens sp. nov. is proposed; the type strain is LL-002T ( = NBRC 110097T = CECT 8536T).

Draft Genome Sequence of Novosphingobium sp. Strain MBES04, Isolated from Sunken Wood from Suruga Bay, Japan.

This report describes the draft genome sequence of Novosphingobium sp. strain MBES04, isolated from sunken wood from Suruga Bay, Japan, which is capable of degrading a wide range of lignin-related aromatic monomers. The draft genome sequence contains 5,361,448 bp, with a G+C content of 65.4%.

Halarchaeum grantii sp. nov., a moderately acidophilic haloarchaeon isolated from a commercial salt sample.

Three moderately acidophilic, halophilic archaeal strains, MH1-243-3T, MH1-243-5 and MH1-243-6, were isolated from a commercial salt sample made from seawater in Okinawa, Japan. Cells of the three strains were pleomorphic and stained Gram-negative. Colonies of the strains were orange-red-pigmented. Strain MH1-243-3T was able to grow at 15-27 % (w/v) NaCl (optimum 24 °C), at pH 4.5-6.5 (pH 5.5) and at 35-50 °C (45 °C). Strains MH1-243-5 and MH1-243-6 grew within slightly different ranges (shown in text). The 16S rRNA gene sequences of the three strains were identical, and the closest phylogenetic relative was Halarchaeum salinum MH1-34-1T with 97.0 % similarity. The rpoB' gene sequences of the three strains were also identical, and the closest phylogenetic relative was Halarchaeum acidiphilum JCM 16109T with 92.0 % similarity. The DNA G+C content of MH1-243-3T, MH1-243-5 and MH1-243-6 was 65.2 mol%. The levels of DNA-DNA relatedness amongst the three strains were 84.1-99.8 %, while that between MH1-243-3T and H. salinum MH1-34-1T was 30.6 % and 31.6 % (reciprocally), and those between MH1-243-3T and type strains of other species in the genus Halarchaeum were 42.3-29.4 %. Based on the phenotypic, genotypic and phylogenetic analyses, it is proposed that the isolates should represent a novel species of the genus Halarchaeum, for which the name Halarchaeum grantii sp. nov. is proposed. The type strain is MH1-243-3T ( = JCM 19585T = KCTC 4142T), isolated from commercial sea salt produced in Okinawa, Japan. MH1-243-5 ( = JCM 19586) and MH1-243-6 ( = JCM 18422) are additional strains of the species.

Crystal structure of the catalytic domain of a GH16 ß-agarase from a deep-sea bacterium, Microbulbifer thermotolerans JAMB-A94.

A deep-sea bacterium, Microbulbifer thermotolerans JAMB-A94, has a ß-agarase (MtAgaA) belonging to the glycoside hydrolase family (GH) 16. The optimal temperature of this bacterium for growth is 43-49 °C, and MtAgaA is stable at 60 °C, which is one of the most thermostable enzymes among GH16 ß-agarases. Here, we determined the catalytic domain structure of MtAgaA. MtAgaA consists of a ß-jelly roll fold, as observed in other GH16 enzymes. The structure of MtAgaA was most similar to two ß-agarases from Zobellia galactanivorans, ZgAgaA, and ZgAgaB. Although the catalytic cleft structure of MtAgaA was similar to ZgAgaA and ZgAgaB, residues at subsite -4 of MtAgaA were not conserved between them. Also, an α-helix, designated as α4', was uniquely located near the catalytic cleft of MtAgaA. A comparison of the structures of the three enzymes suggested that multiple factors, including increased numbers of arginine and proline residues, could contribute to the thermostability of MtAgaA.

Brevundimonas denitrificans sp. nov., a denitrifying bacterium isolated from deep subseafloor sediment.

A novel Gram-stain-negative, aerobic, heterotrophic, stalked and capsulated bacterium with potential denitrification ability, designated strain TAR-002(T), was isolated from deep seafloor sediment in Japan. Colonies lacked lustre, and were viscous and translucent white. The ranges of temperature, pH and salt concentration for growth were 8-30 °C, pH 6.0-10.0 and 1-3% (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences confirmed that strain TAR-002(T) belongs to the genus Brevundimonas of the class Alphaproteobacteria. Levels of similarity between the 16S rRNA gene sequence of strain TAR-002(T) and those of the type strains of species of the genus Brevundimonas were 93.5-98.9%; the most closely related species was Brevundimonas basaltis. In DNA-DNA hybridization assays between strain TAR-002(T) and its phylogenetic neighbours, Brevundimonas lenta DS-18(T), B. basaltis J22(T), Brevundimonas subvibrioides ATCC 15264(T) and Brevundimonas alba DSM 4736(T), mean hybridization levels were 6.4-27.7%. The G+C content of strain TAR-002(T) was 70.3 mol%. Q-10 was the major respiratory isoprenoid quinone. The major fatty acids were C(18:1)ω7c and C(16:0), and the presence of 1,2-di-O-acyl-3-O-[D-glucopyranosyl-(1 → 4)-α-D-glucopyranuronosyl]glycerol (DGL) indicates the affiliation of strain TAR-002(T) with the genus Brevundimonas. On the basis of biological characteristics and 16S rRNA gene sequence comparisons, strain TAR-002(T) is considered to represent a novel species of the genus Brevundimonas, for which the name Brevundimonas denitrificans sp. nov. is proposed; the type strain is TAR-002(T) ( =NBRC 110107(T) =CECT 8537(T)).

Enhancing thermostability and the structural characterization of Microbacterium saccharophilum K-1 ß-fructofuranosidase.

A ß-fructofuranosidase from Microbacterium saccharophilum K-1 (formerly known as Arthrobacter sp. K-1) is useful for producing the sweetener lactosucrose (4(G)-ß-D-galactosylsucrose). Thermostability of the ß-fructofuranosidase was enhanced by random mutagenesis and saturation mutagenesis. Clones with enhanced thermostability included mutations at residues Thr47, Ser200, Phe447, Phe470, and Pro500. In the highest stability mutant, T47S/S200T/F447P/F470Y/P500S, the half-life at 60 °C was 182 min, 16.5-fold longer than the wild-type enzyme. A comparison of the crystal structures of the full-length wild-type enzyme and three mutants showed that various mechanisms appear to be involved in thermostability enhancement. In particular, the replacement of Phe447 with Val or Pro induced a conformational change in an adjacent residue His477, which results in the formation of a new hydrogen bond in the enzyme. Although the thermostabilization mechanisms of the five residue mutations were explicable on the basis of the crystal structures, it appears to be difficult to predict which amino acid residues should be modified to obtain thermostabilized enzymes.

Deep-sea Rhodococcus sp. BS-15, lacking the phytopathogenic fas genes, produces a novel glucotriose lipid biosurfactant.

Glycolipid biosurfactant-producing bacteria were isolated from deep-sea sediment collected from the Okinawa Trough. Isolate BS15 produced the largest amount of the glycolipid, generating up to 6.31 ± 1.15 g l(-1) after 4 days at 20 °C. Glucose was identified in the hydrolysate of the purified major component of the biosurfactant glycolipid. According to gas chromatography/mass spectrometry analysis, the hydrophobic moieties in the major component were hexadecanoate, octadecanoate, 3-hydroxyhexadecanoate, 2-hydroxyoctanoate, and succinate. The molecular weight of the purified major glycolipid was calculated to be 1,211, while (1)H and (13)C nuclear magnetic resonance spectra confirmed that the major component consisted of 2 mol of α-glucoside and 1 mol of ß-glucoside. The molecular structure was assigned as novel trisaccharide-type glycolipid biosurfactant, glucotriose lipids. The critical micelle concentration of the purified major glycolipid was 2.3 × 10(-6) M, with a surface tension of 29.5 mN m(-1). Phylogenetic analysis showed isolate BS15 was closely related to a Rhodococcus strains isolated from Antarctica, and to Rhodococcus fascians, a phytopathogen. PCR analysis showed that the fasA, fasB, fasC, fasD, fasE, and fasF genes, which are involved in phytohormone-like cytokinin production, were not present in the genome of BS15; however, analysis of a draft genome sequence of BS15 (5.5 Mb) identified regions with 31 %, 53 %, 46 %, 30 %, and 31 % DNA sequence identity to the fasA, fasB, fasC, and fasD genes, respectively.

Characteristics of established KSG cells derived from the scorpionfish Sebastiscus marmoratus: what happens under the hydrostatic pressure like the deep sea?

Advances in cell biology depend, partly, on the development of new cell lines and culture methods. Our research focused on a fibroblast-like cell line, "KSG," which is derived from scorpionfish fin tissue (Sebastiscus marmoratus). Cells were grown in Leibovitz's L-15 medium with 10% fetal bovine serum following standard procedures. The optimum growth temperatures for these lines ranged from 15°C to 25°C. All cells survived storage for at least 3 yr at -80°C. Subsequently, they were continuously cultured until the 78th generation without evident changes in their morphology. Moreover, we were able to culture KSG cells in the absence of fetal bovine serum in a culture medium containing the fish serum "SeaGrow." Optimum SeaGrow concentrations for these cells ranged from 5% to 20%. The growth rate of KSG cells decreased when the concentration of SeaGrow was reduced to 1%. However, this decrease could be partially reversed by adding 0.5% "Hy-Fish." In addition, the inclusion of Hy-Fish improved cell adhesion. KSG cells that were cultured in serum-free culture media containing 0.5% and 1% Hy-Fish had been added and were able to survive at low densities. Furthermore, we successfully transfected this cell line with a commercial plasmid vector coding a fluorescent protein using the cationic lipid. Finally, the analyses of cell behavior under hydrostatic pressure showed that some pressures (10 MPa) helped the cells to proliferate more.