Polysaccharide hydrolase of the hadal zone amphipods Hirondellea gigas.

Hirondellea species are common inhabitants in the hadal region deeper than 7,000 m. We found that Hirondellea gigas thrived in the Challenger Deep possessed polysaccharide hydrolases as digestive enzymes. To obtain various enzymes of other H. gigas, we captured amphipods from the Japan Trench, and Izu-Ogasawara (Bonin) Trench. A phylogenetic analysis based on the cytochrome oxidase I gene showed close relationships among amphipods, despite the geographic distance between the localities. However, several differences in enzymatic properties were observed in these H. gigas specimens. We also carried out RNA sequencing of H. gigas from the Izu-Ogasawara Trench. The cellulase gene of H. gigas was highly homologous to cellobiohydrolase of Glucosyl Hydrolase family 7 (GH7). On the other hand, enzymatic properties of H. gigas's cellulase were different from those of typical GH7 cellobiohydrolase. Thus, these results indicate that hadal-zone amphipod can be good candidates as the new enzyme resource.

Nutrients Associated with Sleep Bruxism.

BACKGROUND: The purpose of the present research was to identify nutrients related to sleep bruxism and to establish a hypothesis regarding the relationship between sleep bruxism and nutrients. METHODS: We recruited 143 Japanese university students in 2021 and assigned them to sleep bruxism (n = 58) and non-sleep bruxism groups (n = 85), using an identical single-channel wearable electromyography device. To investigate nutrient intakes, participants answered a food frequency questionnaire based on food groups. We assessed differences in nutrient intakes between the sleep bruxism and non-sleep bruxism groups. RESULTS: Logistic regression modeling showed that sleep bruxism tended to be associated with dietary fiber (odds ratio, 0.91; 95% confidence interval, 0.83-1.00; p = 0.059). In addition, a subgroup analysis selecting students in the top and bottom quartiles of dietary fiber intake showed that students with sleep bruxism had a significantly lower dietary fiber intake (10.4 ± 4.6 g) than those without sleep bruxism (13.4 ± 6.1 g; p = 0.022). CONCLUSION: The present research showed that dietary fiber intake may be related to sleep bruxism. Therefore, we hypothesized that dietary fiber would improve sleep bruxism in young adults.

Cultured and uncultured fungal diversity in deep-sea environments.

The importance of fungi found in deep-sea extreme environments is becoming increasingly recognized. In this chapter, current scientific findings on the fungal diversity in several deep-sea environments by conventional culture and culture-independent methods are reviewed and discussed, primarily focused on culture-independent approaches. Fungal species detected by conventional culture methods mostly belonged to Ascomycota and Basidiomycota phyla. Culture-independent approaches have revealed the presence of highly novel fungal phylotypes, including new taxonomic groups placed in deep branches within the phylum Chytridiomycota and unknown ancient fungal groups. Future attempts to culture these unknown fungal groups may provide key insights into the early evolution of fungi and their ecological and physiological significance in deep-sea environments.

Geofilum rubicundum gen. nov., sp. nov., isolated from deep subseafloor sediment.

A novel, facultatively anaerobic bacterium (strain JAM-BA0501(T)) was isolated from a deep subseafloor sediment sample at a depth of 247 m below seafloor off the Shimokita Peninsula of Japan in the north-western Pacific Ocean (Site C9001, 1180 m water depth). Cells of strain JAM-BA0501(T) were gram-negative, filamentous, non-spore-forming and motile on solid medium by gliding. Phylogenetic analysis based on the 16S rRNA gene sequence of strain JAM-BA0501(T) indicated a distant relationship to strains representing genera within the order Bacteroidales, such as Alkaliflexus imshenetskii Z-7010(T) (91.1 % similarity), Marinilabilia salmonicolor ATCC 19041(T) (86.2 %) and Anaerophaga thermohalophila Fru22(T) (89.3 %). The new isolate produced isoprenoid quinones with menaquinone MK-7 as the major component, and the predominant fatty acids were iso-C(15 : 0) and anteiso-C(15 : 0). The DNA G+C content of the isolate was 42.9 mol%. Based on its taxonomic distinctiveness, strain JAM-BA0501(T) is considered to represent a novel species of a new genus within the family Marinilabiliaceae, for which the name Geofilum rubicundum gen. nov., sp. nov. is proposed. The type strain of Geofilum rubicundum is JAM-BA0501(T) ( = JCM 15548(T)  = NCIMB 14482(T)).

Molecular evidence that deep-branching fungi are major fungal components in deep-sea methane cold-seep sediments.

The motile cells of chytrids were once believed to be relics from the time before the colonization of land by fungi. However, the majority of chytrids had not been found in marine but freshwater environments. We investigated fungal diversity by a fungal-specific PCR-based analysis of environmental DNA in deep-sea methane cold-seep sediments, identifying a total of 35 phylotypes, 12 of which were early diverging fungi (basal fungi, ex 'lower fungi'). The basal fungi occupied a major portion of fungal clones. These were phylogenetically placed into a deep-branching clade of fungi and the LKM11 clade that was a divergent group comprised of only environmental clones from aquatic environments. As suggested by Lara and colleagues, species of the endoparasitic genus Rozella, being recently considered of the earliest branching taxa of fungi, were nested within the LKM11 clade. In the remaining 23 phylotypes identified as the Dikarya, the majority of which were similar to those which appeared in previously deep-sea studies, but also highly novel lineages associated with Soil Clone Group I (SCGI), Entorrhiza sp. and the agaricomycetous fungi were recorded. The fungi of the Dikarya may play a role in the biodegradation of lignin and lignin-derived materials in deep-sea, because the characterized fungal species related to the frequent phylotypes within the Dikarya have been reported to possess an ability to degrade lignin.

Microbulbifer chitinilyticus sp. nov. and Microbulbifer okinawensis sp. nov., chitin-degrading bacteria isolated from mangrove forests.

Three chitin-degrading strains representing two novel species were isolated from mangrove forests in Okinawa, Japan. The isolates, ABABA23(T), ABABA211 and ABABA212(T), were Gram-negative, non-spore-forming, strictly aerobic chemo-organotrophs. The novel strains produced Q-8 as the major isoprenoid quinone component. The predominant fatty acids were iso-C15:0 and C16:0. On the basis of 16S rRNA gene sequence analysis, the isolates were closely affiliated with members of the genus Microbulbifer. The DNA G+C contents of strains ABABA23(T) and ABABA212(T) were 57.8 and 60.2 mol%, respectively. DNA-DNA relatedness values between these two strains and Microbulbifer reference strains were significantly lower than 70 %, the generally accepted threshold level below which strains are considered to belong to separate species. Based on differences in taxonomic characteristics, the three isolates represent two novel species of the genus Microbulbifer, for which the names Microbulbifer chitinilyticus sp. nov. (type strain, ABABA212(T) = JCM 16148(T) = NCIMB 14577(T)) and Microbulbifer okinawensis sp. nov. (type strain, ABABA23(T) = JCM 16147(T) = NCIMB 14576(T); reference strain, ABABA211) are proposed.

Cipralphelin, a new anti-oxidative N-cinnamoyl tripeptide produced by the deep sea-derived fungal strain Penicillium brevicompactum FKJ-0123.

A new N-cinnamoyl tripeptide, designated cipralphelin (1), was isolated from a cultured broth of Penicillium brevicompactum FKJ-0123 by physicochemical (PC) screening. Compound 1 was purified by silica gel and ODS column chromatography followed by preparative HPLC. The structure of 1 was determined as N-cinnamoyl-prolyl-alanyl-phenylalanine methyl ester by nuclear magnetic resonance and mass spectrometry analyses. The absolute configurations of three amino acids were determined by an advanced Marfey's method applied to the hydrolysate of 1. Compound 1 was evaluated for its cytotoxicity, anti-microbial activity, and ability to scavenge or quench reactive oxygen species (ROS) such as superoxide anion radicals, hydroxy radicals, and singlet oxygen. Compound 1 exhibited potent scavenging activity against hydroxy radicals.

Phylogenetic relationship within the Erythrobasidium clade: molecular phylogenies, secondary structure, and intron positions inferred from partial sequences of ribosomal RNA and elongation factor-1alpha genes.

Phylogenetic relationships within the Erythrobasidium clade as a lineage of the urediniomycetous yeasts were examined using partial regions of 18S rDNA, 5.8S rDNA, 26S rDNA, internal transcribed spacers (ITSs), and elongation factor (EF)-1alpha. Combined data analysis of all segments successfully yielded a reliable phylogeny and confirmed the cohesion of species characterized by Q-10(H2) as a major ubiquinone. Differences in secondary structure predicted for a variable region in 26S rDNA corresponded to major divergences in the phylogenetic tree based on the primary sequence. The common presence of a shortened helix in this region was considered to be evidence of monophyly for species with Q-10(H2), Sakaguchia dacryoides, Rhodotorula lactosa, and Rhodotorula lamellibrachiae, although it was not as strongly supported by the combined data tree. The information on intron positions in the EF-1alpha gene had potential usefulness in the phylogenetic inference between closely related species.

Survival of deep-sea shrimp (Alvinocaris sp.) during decompression and larval hatching at atmospheric pressure.

We report successful larval hatching of deep-sea shrimp after decompression to atmospheric pressure. Three specimens of deep-sea shrimp were collected from an ocean depth of 1157 m at cold-seep sites off Hatsushima Island in Sagami Bay, Japan, using a pressure-stat aquarium system. Phylogenetic analysis of Alvinocaris sp. based on cytochrome c oxidase subunit gene sequences confirmed that these species were a member of the genus Alvinocaris. All 3 specimens survived to reach atmospheric pressure conditions after stepwise 63-day decompression. Two of the specimens contained eggs, which hatched after 10 and 16 days, respectively, of full decompression. Although no molting of the shrimp larvae was observed during 74 days of rearing under atmospheric pressure, the larvae developed conventional dark-adapted eyes after 15 days.

The Hadal Amphipod Hirondellea gigas possessing a unique cellulase for digesting wooden debris buried in the deepest seafloor.

The Challenger Deep in the Mariana Trench is the deepest point in the ocean (10,994 m). Certain deep-sea animals can withstand the extreme pressure at this great depth. The amphipod Hirondellea gigas is a resident of the Challenger Deep. Amphipods are common inhabitants at great depths and serve as scavengers. However, there is relatively little information available regarding the physiology of H. gigas or this organism's ecological interactions in the hadopelagic zone. To understand the feeding behavior of this scavenger in the deepest oligotrophic hadal zone, we analyzed the digestive enzymes in whole-body extracts. We describe the detection of amylase, cellulase, mannanase, xylanase, and α-glycosidase activities that are capable of digesting plant-derived polysaccharides. Our identification of glucose, maltose, and cellobiose in the H. gigas extracts indicated that these enzymes function under great pressure in situ. In fact, the glucose content of H. gigas averaged 0.4% (w/dry-w). The purified H. gigas cellulase (HGcel) converted cellulose to glucose and cellobiose at an exceptional molar ratio of 2:1 and efficiently produced glucose from dried wood, a natural cellulosic biomass, at 35 °C. The enzyme activity increased under a high hydrostatic pressure of 100 MPa at 2 °C, conditions equivalent to those found in the Challenger Deep. An analysis of the amino acid sequence of HGcel supported its classification as a family 31 glycosyl hydrolase. However, none of the enzymes of this family had previously been shown to possess cellulase activity. These results strongly suggested that H. gigas adapted to its extreme oligotrophic hadal oceanic environment by evolving digestive enzymes capable of digesting sunken wooden debris.

Yeast extract stimulates production of glycolipid biosurfactants, mannosylerythritol lipids, by Pseudozyma hubeiensis SY62.

We improved the culture conditions for a biosurfactant producing yeast, Pseudozyma hubeiensis SY62. We found that yeast extract greatly stimulates MEL production. Furthermore, we demonstrated a highly efficient production of MELs in the improved medium by fed-batch cultivation. The final concentration of MELs reached 129 ± 8.2g/l for one week.

Biosurfactant-producing yeast isolated from Calyptogena soyoae (deep-sea cold-seep clam) in the deep sea.

We describe a detailed structure determination of biosurfactant produced by Pseudozyma hubeiensis SY62, which was newly isolated from Calyptogena soyoae (deep-sea cold-seep clam, Shirouri-gai) at 1156 m in Sagami bay. P. hubeiensis SY62 was taxonomically slightly different from the P. hubeiensis type strain, which produces biosurfactants. Glycolipid production by the strain was also slightly different from those of previously reported strains. BS productivity was estimated to be around 30 g/l from the weight of the crude extract. At least five different spots of glycolipid biosurfactants (BSs) were detected by TLC. Results of nuclear magnetic resonance spectroscopies indicated the major product, namely MEL-C (4-O-[4'-O-acetyl-2',3'-di-O-alka(e)noil-beta-d-mannopyranosyl]-d-erythritol), as a promising BS. By further structural determination, the major fatty acids of MEL-C were estimated to be saturated C(6), C(10), and C(12) acids, which were shorter than those of previously reported MEL-C. Furthermore, (1)H-NMR spectra implied the presence of C(2) acids as acyl groups. According to surface tension determination, the novel MEL-C showed larger critical micelle concentration (1.1x10(-5) M) than conventional MEL-C which bound C(10) and C(12) acids (9.1x10(-6) M). From these results, shorter fatty acids would confer hydrophilicity onto the novel MEL-C.

Amphritea japonica sp. nov. and Amphritea balenae sp. nov., isolated from the sediment adjacent to sperm whale carcasses off Kagoshima, Japan.

Two novel species were isolated from the sediment adjacent to sperm whale carcasses off Kagoshima, Japan, at a depth of about 230 m. The isolated strains, JAMM 1866(T), JAMM 1548 and JAMM 1525(T), were Gram-negative, rod-shaped, non-spore-forming and motile by means of a single polar or bipolar flagellum. Phylogenetic analysis based on 16S rRNA gene sequences of strains JAMM 1866(T) and JAMM 1548 indicated a relationship to the symbiotic bacterial clone R21 of Osedax japonicus (100 % sequence similarity) and all three isolates were closely related to Amphritea atlantica (97.7-97.8 % similarity) within the class Gammaproteobacteria. The novel isolates were able to produce isoprenoid quinone Q-8 as the major component. The predominant fatty acids were C(16 : 0), C(16 : 1) and C(18 : 1), with C(12 : 1) 3-OH present in smaller amounts. The DNA G+C contents of the three isolated strains were about 47 mol%. Based on differences in taxonomic characteristics, the three isolated strains represent two novel species of the genus Amphritea for which the names Amphritea japonica sp. nov. (type strain JAMM 1866(T)=JCM 14782(T)=ATCC BAA-1530(T); reference strain JAMM 1548) and Amphritea balenae sp. nov. (type strain JAMM 1525(T)=JCM 14781(T)=ATCC BAA-1529(T)) are proposed.

Dipodascus tetrasporeus sp. nov., an ascosporogenous yeast isolated from deep-sea sediments in the Japan Trench.

Dipodascus tetrasporeus sp. nov. is described as a novel yeast species in the family Dipodascaceae to accommodate an isolate recovered from sediments collected on the deep-sea floor in the north-western Pacific Ocean. In the clade comprising the genera Dipodascus, Galactomyces and Geotrichum, this is the only species that forms asci that bear four ascospores. The ascospore is surrounded by an irregular exosporium wall, similar to what is observed in the genus Galactomyces, but they are released by rupture, which is characteristic of Dipodascus and not Galactomyces. D. tetrasporeus is remarkably divergent (>10 % difference) in its D1/D2 26S rDNA sequence from any other known species. Although maximum-likelihood analysis of combined 18S rDNA and D1/D2 26S rDNA sequences cannot elucidate a reliable position for this species, it was placed among Geotrichum carabidarum, Geotrichum cucujoidarum, Geotrichum fermentans and Geotrichum histeridarum, which also have morphological and physiological affinity with the species. The species is homothallic. The type strain of Dipodascus tetrasporeus sp. nov. is strain SY-277T (=NBRC 103136T =CBS 10071T).

Rhodotorula pacifica sp. nov., a novel yeast species from sediment collected on the deep-sea floor of the north-west Pacific Ocean.

A novel species of the genus Rhodotorula was isolated from sediments collected on the deep-sea floor in the north-west Pacific Ocean. Strains SY-96T, isolated from the Yap Trench, and SY-246, isolated from the Iheya Ridge, had almost identical nucleotide sequences for their internal transcribed spacers and their 5.8S rDNA. Their physiological characteristics were also almost identical. The strains were assumed to be related to Rhodotorula mucilaginosa and Rhodotorula dairenensis based on sequence similarities in the D1/D2 region of the 26S rDNA. The low DNA-DNA relatedness and sequence similarity between strain SY-96T and related species revealed that strains SY-96T and SY-246 represent a hitherto unknown species. As ballistoconidia and sexual reproduction were not observed in strains SY-96T and SY-246, these strains are described as Rhodotorula pacifica sp. nov. The type strain is SY-96T (= JCM 10908T = CBS 10070T).

Characterization of cold- and high-pressure-active polygalacturonases from a deep-sea yeast, Cryptococcus liquefaciens strain N6.

A deep-sea yeast, Cryptococcus liquefaciens strain N6, produces two polygalacturonases, p36 and p40 (N6-PGases). These N6-PGases were highly active at 0-10 degrees C in comparison to a PGase from Aspergillus japonicus. The hydrolytic activity of these N6-PGases remained almost unchanged up to a hydrostatic pressure of 100 MPa at 24 degrees C with a very small activation volume of -1.1 ml/mol. At 10 degrees C, however, the activation volume increased to 3.3 or 5.4 ml/mol (p36 and p40, respectively), suggesting that the enzyme-substrate complexes can expand at their transition states. We speculate that such a volume expansion upon forming the enzyme-substrate complexes contributes to decreasing the activation energy for hydrolysis. This can account for the high activity of N6-PGases at low-temperature.

Systematic study of basidiomycetous yeasts - evaluation of the ITS regions of rDNA to delimit species of the genus Rhodosporidium.

Nucleotide sequences of internal transcribed spacer (ITS) regions were determined to establish the guidelines for species identification in the genus Rhodosporidium. Forty-two strains of nine species of the genus Rhodosporidium were used for ITS (ITS1 and ITS2) analysis. Intraspecific length polymorphisms and sequence variations were observed within R. azoricum, R. diobovatum, R. paludigenum, R. sphaerocarpum and R. toruloides, while no variation was observed within R. babjevae and R. kratochvilovae. Based on comparison of the levels of intraspecific and interspecific sequence similarity, strains with identical sequences were considered to represent a single species and strains with 92% or lower similarity of ITS sequences were considered to be distinct species in the genus Rhodosporidium.

Rhodotorula benthica sp. nov. and Rhodotorula calyptogenae sp. nov., novel yeast species from animals collected from the deep-sea floor, and Rhodotorula lysiniphila sp. nov., which is related phylogenetically.

Three novel species of the genus Rhodotorula are described. Rhodotorula benthica sp. nov. (type strain JCM 10901(T) = SY-91(T)) and Rhodotorula calyptogenae sp. nov. (type strain JCM 10899(T) = SY-86(T)) were respectively isolated from the tubeworm Lamellibrachia sp. and the giant white clam Calyptogena sp., collected from the deep-sea floor of the Pacific Ocean off Japan. Rhodotorula lysiniphila sp. nov. (type strain JCM 5951(T)) is proposed for strains isolated previously in Japan and Pakistan. The three species were placed phylogenetically into a species complex comprising Rhodotorula laryngis, Rhodotorula minuta, Rhodotorula pallida and Rhodotorula slooffiae. R. minuta and R. slooffiae are closely related in both the D1/D2 region of the 26S rDNA and the internal transcribed spacer and 5.8S rDNA regions. R. benthica and R. laryngis were closer to R. pallida based on the D1/D2 region. Other relationships were not clear.

Distinct role of nitric oxide and endothelium-derived hyperpolarizing factor in renal microcirculation. Studies in the isolated perfused hydronephrotic kidney.

BACKGROUND: Both nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF) are established as important factors determining the vascular tone. The relative contribution of these factors to the renal microvascular tone, however, has not been delineated. METHODS: Isolated perfused hydronephrotic rat kidneys were used to characterize the relative role of NO and EDHF in mediating the tone of interlobular arteries (ILA) and afferent arterioles (AFF). RESULTS: During the norepinephrine constriction, acetylcholine (ACH, 1 micromol/l) induced a sustained vasodilation of ILA (90 +/- 9% reversal) and AFF (117 +/- 13% reversal). In the presence of nitro-L-arginine methylester (LNAME), the ACH-induced vasodilation of ILA and AFF was converted to transient dilation, with only 53 +/- 7 and 32 +/- 7% reversal observed 10 min after 1 micromol/l ACH (i.e sustained phase). In contrast, LNAME had no effect on the initial phase of ACH-induced dilation. In the presence of apamin + charybdotoxin, the initial vasodilator response to ACH (1 micromol/l) was diminished (ILA, from 108 +/- 8 to 46 +/- 9%; AFF, from 108 +/- 14 to 58 +/- 8%), whereas no impairment was observed in sustained phases. Furthermore, the magnitude of the vasoconstriction caused by LNAME was greater at smaller vessel segments. Finally, the LNAME-induced inhibition of the sustained phase of ACH-induced vasodilation was greater as the vessel diameter decreased. CONCLUSIONS: That the relative contribution of NO and EDHF differs, with a greater role of NO in the basal tone and ACH-induced vasodilation at smaller vascular segments of ILA and AFF.

Cryptococcus surugaensis sp. nov., a novel yeast species from sediment collected on the deep-sea floor of Suruga Bay.

A novel species of the genus Cryptococcus was isolated from sediment collected on the deep-sea floor of Suruga Bay, Japan. Nucleotide sequences of 18S rDNA, internal transcribed spacers, 5.8S rDNA and the D1/D2 region of 26S rDNA of strain SY-260(T) suggested affinities to a phylogenetic lineage that includes Cryptococcus luteolus. Comparisons of the rDNA sequences of each region clarified that strain SY-260(T) is related distantly to Bullera coprosmaensis and Bullera oryzae, but is distinct at the species level. As ballistoconidia and sexual reproduction were not observed in strain SY-260(T), this strain is described as Cryptococcus surugaensis sp. nov. (type strain, SY-260(T)=JCM 11903(T)=CBS 9426(T)).