Bacterial diversity in deep-sea sediments under influence of asphalt seep at the São Paulo Plateau.

Here we investigated the diversity of bacterial communities from deep-sea surface sediments under influence of asphalt seeps at the Sao Paulo Plateau using next-generation sequencing method. Sampling was performed at North São Paulo Plateau using the human occupied vehicle Shinkai 6500 and her support vessel Yokosuka. The microbial diversity was studied at two surficial sediment layers (0-1 and 1-4 cm) of five samples collected in cores in water depths ranging from 2456 to 2728 m. Bacterial communities were studied through sequencing of 16S rRNA gene on the Ion Torrent platform and clustered in operational taxonomic units. We observed high diversity of bacterial sediment communities as previously described by other studies. When we considered community composition, the most abundant classes were Alphaproteobacteria (27.7%), Acidimicrobiia (20%), Gammaproteobacteria (11.3%) and Deltaproteobacteria (6.6%). Most abundant OTUs at family level were from two uncultured bacteria from Actinomarinales (5.95%) and Kiloniellaceae (3.17%). The unexpected high abundance of Alphaproteobacteria and Acidimicrobiia in our deep-sea microbial communities may be related to the presence of asphalt seep at North São Paulo Plateau, since these bacterial classes contain bacteria that possess the capability of metabolizing hydrocarbon compounds.

Hatsusamides A and B: Two New Metabolites Produced by the Deep-Sea-Derived Fungal Strain Penicillium steckii FKJ-0213.

Two new nitrogen-containing metabolites, designated hatsusamide A (1) and B (2), were isolated from a culture broth of Penicilliumsteckii FKJ-0213 together with the known compounds tanzawaic acid B (3) and trichodermamide C (4) by physicochemical (PC) screening. The structures of 1 and 2 were determined as a tanzawaic acid B-trichodermamide C hybrid structure and a new analog of aspergillazines, respectively. The absolute configuration of 1 was determined by comparing the values of tanzawaic acid B and trichodermamide C in the literatures, such as 1H-nuclear magnetic resonance (1H-NMR) data and optical rotation, after hydrolysis of 1. Compounds 1-4 were evaluated for cytotoxicity and anti-malarial activities. Compounds 1 and 3 exhibited weak anti-malarial activity at half-maximal inhibitory concentration (IC50) values of 27.2 and 78.5 µM against the K1 strain, and 27.9 and 79.2 µM against the FCR3 strain of Plasmodium falciparum, respectively. Furthermore, 1 exhibited cytotoxicity against HeLa S3, A549, Panc1, HT29 and H1299 cells, with IC50 values of 15.0, 13.7, 12.9, 6.8, and 18.7 µM, respectively.

New metabolites, sarcopodinols A and B, isolated from deep-sea derived fungal strain Sarcopodium sp. FKJ-0025.

Fungal strain FKJ-0025 was isolated from deep-sea sediment collected at the Wakamiko Caldera in Kagoshima Bay (water depth: 200 m). The fungal strain FKJ-0025 was identified as the genus Sarcopodium based on its morphology and internal transcribed spacer (ITS) sequence. Two new compounds, designated sarcopodinols A (1) and B (2), were isolated together with the known compound SF-227 (3).

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.

Antifungal profile against Candida auris clinical isolates of tyroscherin and its new analog produced by the deep-sea-derived fungal strain Scedosporium apiospermum FKJ-0499.

A new antifungal compound, named N-demethyltyroscherin (1), was discovered from the static fungal cultured material of Scedosporium apiospermum FKJ-0499 isolated from a deep-sea sediment sample together with a known compound, tyroscherin (2). The structure of 1 was elucidated as a new analog of 2 by MS and NMR analyses. The absolute configuration of 1 was determined by chemical derivatization. Both compounds showed potent in vitro antifungal activity against clinically isolated Candida auris strains, with MIC values ranging from 0.0625 to 4 µg ml-1.

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.

Unique substrate specificity of a thermostable glycosyl hydrolase from an uncultured Anaerolinea, derived from bacterial mat on a subsurface geothermal water stream.

To investigate novel extremozymes encoded by sequenced metagenes from a microbial community in an extreme environment, we have characterized a recombinant glycosyl hydrolase (rGH) from an uncultured bacterium within the order Chloroflexi. rGH formed insoluble bodies in an Escherichia coli protein expression system. The protein was partially dissolved by a surfactant and was enzymatically characterized. The MW of the monomeric peptide was ~62 kDa, and it formed a homodimers in buffer. It was optimally active at 65 °C and from pH 4 to 8. rGH showed hydrolytic activity for α-1,1, α-1,2 and α-1,6 linkages, including isomaltose, but not α-1,4 and ß-linkages.

Ecological and Oceanographic Perspectives in Future Marine Fungal Taxonomy.

Marine fungi are an ecological rather than a taxonomic group that has been widely researched. Significant progress has been made in documenting their phylogeny, biodiversity, ultrastructure, ecology, physiology, and capacity for degradation of lignocellulosic compounds. This review (concept paper) summarizes the current knowledge of marine fungal diversity and provides an integrated and comprehensive view of their ecological roles in the world's oceans. Novel terms for 'semi marine fungi' and 'marine fungi' are proposed based on the existence of fungi in various oceanic environments. The major maritime currents and upwelling that affect species diversity are discussed. This paper also forecasts under-explored regions with a greater diversity of marine taxa based on oceanic currents. The prospects for marine and semi-marine mycology are highlighted, notably, technological developments in culture-independent sequencing approaches for strengthening our present understanding of marine fungi's ecological roles.

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.

Biochemical synthesis of novel, self-assembling glycolipids from ricinoleic acid by a recombinant α-glucosidase from Geobacillus sp.

PURPOSE OF WORK: To explore a novel glycolipid, we performed biochemical reactions using a recombinant α-glucosidase from Geobacillus sp. which shows excellent transglycosylation reaction to hydroxyl groups in a variety of compounds. Two different glycolipids (GL-1 and GL-2) were prepared from ricinoleic acid using a recombinant α-glucosidase from Geobacillus sp. The molecular structure of GL-1 was confirmed as 12-O-α-D-glucopyranosyl-9-hexadecenoic acid by 1D and 2D NMR analyses. According to MALDI-TOF/MS, GL-1 and GL-2 showed single major peaks at m/z 483.82 and 645.97, respectively. The peaks corresponded to the [M + Na](+) ions of the glycolipids. GL-2 was estimated as 12-O-α-D-glucopyranosyl-(4'-O-α-glucopyranosyl)-9-hexadecenoic acid. Light polarization microscopy revealed that GL-2 easily formed self-assembled vesicles in aqueous solution.

Splitting of RNA-dependent RNA polymerase is common in Narnaviridae: Identification of a type II divided RdRp from deep-sea fungal isolates.

Until recently, it was accepted that RNA-dependent RNA polymerase (RdRp) is the only essential gene for non-retro RNA viruses and is encoded by a single open reading frame (ORF) in their genomes. However, divided RdRps that are coded by two ORFs were discovered in fungal RNA viruses in a few independent reports. This discovery showed higher plasticity of viral RdRp than was expected. Among these divided RdRps, the division site was common; specifically, the first part of the RdRp contains motifs F, A, and B, whereas the latter part possesses motifs C and D. These RdRps are designated as type I divided RdRp and have been limited to viruses in a specific clade of Narnaviridae. In this study, to further understand the plasticity of RdRp, we explored viruses from deep sea-derived fungal strains as an untapped resource with a focus on Aspergillus section Versicolores. Seven strains were found to be infected by a total of 13 viruses, and the viral RNA genomes were determined by fragmented and primer-ligated double-stranded RNA sequencing technology. Among them, six strains belong to Narnaviridae. One of the strains, Aspergillus tennesseensis narnavirus 1, which infects an Aspergillus tennesseensis, has a divided RdRp with a new division site (referred to as type II divided RdRp). A couple of sequences for possible type II divided RdRps were also detected in public metagenomic data sets. Our findings reveal that different types of divisions in RdRp are present in the virosphere, and two types of RdRp splitting occurred independently within Narnaviridae.

Structural Comparison of Diplonemid Communities around the Izu Peninsula, Japan.

Diplonemea (diplonemids) is one of the most abundant and species-rich protist groups in marine environments; however, their community structures among local and seasonal samples have not yet been compared. In the present study, we analyzed four diplonemid community structures around the Izu Peninsula, Japan using barcode sequences amplified from environmental DNA. These sequences and the results of statistical analyses indicated that communities at the same site were more similar to each other than those in the same season. Environmental variables were also measured, and their influence on diplonemid community structures was examined. Salinity, electrical conductivity, and temperature, and their correlated variables, appeared to influence the structures of diplonemid communities, which was consistent with previous findings; however, since the results obtained did not reach statistical significance, further studies are required. A comparison of each diplonemid community indicated that some lineages were unique to specific samples, while others were consistently detected in all samples. Members of the latter type are cosmopolitan candidates and may be better adapted to the environments of the studied area. Future studies that focus on the more adaptive members will provide a more detailed understanding of the mechanisms by which diplonemids are widely distributed in marine environments and will facilitate their utilization as indicator organisms to monitor environmental changes.

Optimization of environmental DNA extraction and amplification methods for metabarcoding of deep-sea fish.

Analyses of environmental DNA (eDNA) from macroorganisms in aquatic environments have greatly advanced in recent years. In particular, eDNA metabarcoding of fish using universal PCR primers has been reported in various waters. Although pumped deep-sea water was used for eDNA metabarcoding of deep-sea fish, conventional methods only resulted in small amounts of extracted eDNA and subsequent few or no PCR amplicons. To optimize eDNA metabarcoding of deep-sea fish from pumped deep-sea water, we modified conventional procedures of eDNA extraction and PCR amplification. Here, we propose a modified eDNA extraction method, in which a filter used for eDNA sampling was shredded and incubated in microtubes for efficient lysis of eDNA sources. Total eDNA yield extracted using the modified protocol was approximately six-fold higher than that extracted by the conventional protocol. The PCR enzyme Platinum SuperFi II DNA Polymerase successfully amplified a target region of fish universal primers (MiFish) from trace amounts of eDNA extracted from pumped deep-sea water and suppressed nonspecific amplifications more effectively than the enzyme used in conventional methods. Approximately 93% of the sequence reads acquired by next generation sequencing of these amplicons were derived from fish. The improved procedure presented here provided effective eDNA metabarcoding of deep-sea fish.•A modified eDNA extraction protocol, in which a filter was shredded and incubated in microtubes, increased eDNA yields extracted from pumped deep-sea water over the conventional method.•The PCR enzyme Platinum SuperFi II DNA polymerase improved the amplification efficiency of trace amounts of MiFish objectives in eDNA extracted from pumped deep-sea water with suppressing nonspecific amplifications.•The use of Platinum SuperFi II DNA polymerase for eDNA metabarcoding using MiFish primers resulted in the acquisition of abundant sequence reads of deep-sea fish through next generation sequencing.

Plastic additives in deep-sea debris collected from the western North Pacific and estimation for their environmental loads.

Plastic waste has become a growing concern in terms of marine pollution, but little information is available on plastic debris and its possible risks of chemical additives exposure in the deep-sea. This study focused on identification of polymer type and additive concentrations in 21 plastic debris collected from deep-sea of Sagami Bay, Japan and West Pacific Ocean under the Kuroshio Extension and its recirculation gyre (KERG) zone (water depth: 1388-5819 m). Polyethylene (PE) was dominant polymer (57% of the total) in samples, followed by polyvinylchloride (PVC), epoxy resin, polyester (PES), and polypropylene. In plastic additives, bis (2-ethylhexyl) phthalate (DEHP) was detected to be contained in a PVC sheet at concentration of 48%. Butylated hydroxytoluene (BHT) was also detected in PE plastic debris with median concentration of 12,000 ng/g. PES clothes were detected to contain dyeing mixtures, 1,2,4-trichlorobenzene (1,2,4-TCB), up to 42,000 ng/g. Knowing the estimated number of plastic debris under KE current, the minimum burden of chemical additives were estimated that 720 kg of dibutyl phthalate, 570 kg of BHT, 230 kg of DEHP, and 160 kg of 1,2,4-TCB exist on the seabed of KERG zone. This result strongly suggests that enormous amount of hazardous additives lie within plastic debris on abyssal level of the ocean.

Massive occurrence of benthic plastic debris at the abyssal seafloor beneath the Kuroshio Extension, the North West Pacific.

The abyss (3500-6500 m) covers the bulk of the deep ocean floor yet little is known about the extent of plastic debris on the abyssal seafloor. Using video imagery we undertook a quantitative assessment of the debris present on the abyssal seafloor (5700-5800 m depth) beneath the Kuroshio Extension current system in the Northwest Pacific. This body of water is one of the major transit pathways for the massive amounts of debris that are entering the North Pacific Ocean from Asia. Shallower sites (1400-1500 m depth) were also investigated for comparison. The dominant type of debris was single-use plastics - mainly bags and food packaging. The density of the plastic debris (mean 4561 items/km2) in the abyssal zone was the highest recorded for an abyssal plain suggesting that the deep-sea basin in the Northwest Pacific is a significant reservoir of plastic debris.

Aquimarina macrocephali sp. nov., isolated from sediment adjacent to sperm whale carcasses.

A Gram-negative, rod-shaped, non-spore-forming, strictly aerobic strain with gliding motility, designated JAMB N27(T), was isolated from sediment adjacent to sperm whale carcasses off Kagoshima, Japan, at a depth of 219 m. Strain JAMB N27(T) contained MK-6 as the major isoprenoid quinone and iso-C15:0, iso-C15:1, C16:1 and iso-C17:1 as the predominant fatty acids. Casein, chitin, gelatin and starch were degraded. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain JAMB N27(T) represented a separate lineage within the genus Aquimarina. The DNA G+C content of strain JAMB N27(T) was 33.1 mol%. DNA-DNA relatedness values between strain JAMB N27(T) and type strains of species of the genus Aquimarina were significantly lower than the cut-off value accepted for the definition of a novel species. Therefore, strain JAMB N27(T) represents a novel species, for which the name Aquimarina macrocephali sp. nov. is proposed. The type strain is JAMB N27(T) (=JCM 15542(T)=NCIMB 14508(T)).

Comparison of techniques to examine the diversity of fungi in adult patients with cystic fibrosis.

This study compares conventional and molecular techniques for the detection of fungi in 77 adult cystic fibrosis (CF) patients. Three different methods were investigated, i.e., (1) conventional microbiological culture (including yeasts and filamentous fungi), (2) mycological culture with CF-derived fungal specific culture media, and (3) Non-culture and direct DNA extraction from patient sputa. Fungi isolated from environmental air samples of the CF unit were compared to fungi in sputa from CF patients. Fungi (n = 107) were detected in 14/77(18%) of patients by method 1, in 60/77 (78%) of patients by method 2 and with method 3, in 77/77(100%) of the patients. The majority of yeasts isolated were Candida albicans and C. dubliniensis. Exophiala (Wangiella) dermatitidis, Scedosporium apiospermum, Penicillium spp., Aspergillus fumigatus, and Aspergillus versicolor were also identified by sequence analysis of the rDNA short internal transcribed spacer (ITS2) region. Conventional laboratory analysis failed to detect fungi in 63 patients mainly due to overgrowth by Gram-negative organisms. Mycological culture with antibiotics dramatically increased the number of fungi that could be detected. Molecular techniques detected fungi such as Saccharomyces cerevisiae, Malassezia spp., Fuscoporia ferrea, Fusarium culmorum, Acremonium strictum, Thanatephorus cucumeris and Cladosporium spp. which were not found with other methods. This study demonstrates that several potentially important fungi may not be detected if mycological culture methods alone are used. A polyphasic approach employing both enhanced mycological culture with molecular detection will help determine the presence of fungi in the sputa of patients with CF and their healthcare environment.

Cryptic fungal diversity revealed in deep-sea sediments associated with whale-fall chemosynthetic ecosystems.

In this study, sediments from whale-fall chemosynthetic ecosystems (two different sites, one naturally occurring at 4200 m water depth in South Atlantic Ocean and one artificially immersed at 100 m water depth in Kagoshima Bay, Japan) were investigated by Ion Torrent PGM sequencing of the ITS region of ribosomal RNA to reveal fungal communities in these unique marine environments. As a result, a total of 107 (897 including singletons) Operational Taxonomic Units (OTUs) were obtained from the samples explored. Composition of the 107 OTUs at the phylum level among the five samples from two different whale-fall sites was assigned to Ascomycota (46%), Basidiomycota (7%), unidentified fungi (21%), non-fungi (10%), and sequences with no affiliation to any organisms in the public database (No-match) (16%). The high detection of the unidentified fungi and unassigned fungi was revealed in the whale-fall environments in this study. Some of these unidentified fungi are allied to early diverging fungi and they were more abundant in the sediments not directly in contact with whalebone. This study suggests that a cryptic fungal community exists in unique whale-fall ecosystems.

Identification of airborne bacterial and fungal species in the clinical microbiology laboratory of a university teaching hospital employing ribosomal DNA (rDNA) PCR and gene sequencing techniques.

Universal or "broad-range" PCR-based ribosomal DNA (rDNA) was performed on a collection of 58 isolates (n = 30 bacteria + 28 fungi), originating from environmental air from several locations within a busy clinical microbiology laboratory, supporting a university teaching hospital. A total of 10 bacterial genera were identified including both Gram-positive and Gram-negative genera. Gram-positive organisms accounted for 27/30 (90%) of total bacterial species, consisting of seven genera and included (in descending order of frequency) Staphylococcus, Micrococcus, Corynebacterium, Paenibacillus, Arthrobacter, Janibacter and Rothia. Gram-negative organisms were less frequently isolated 3/30 (10%) and comprised three genera, including Moraxella, Psychrobacter and Haloanella. Eight fungal genera were identified among the 28 fungal organisms isolated, including (in descending order of frequency) Cladosporium, Penicillium, Aspergillus, Thanatephorus, Absidia, Eurotium, Paraphaeosphaeria and Tritirachium, with Cladosporium accounting for 10/28 (35.7%) of the total fungal isolates. In conclusion, this study identified the presence of 10 bacterial and eight fungal genera in the air within the laboratory sampled. Although this reflected diversity of the microorganisms present, none of these organisms have been described previously as having an inhalational route of laboratory-acquired infection. Therefore, we believe that the species of organisms identified and the concentration levels of these airborne contaminants determined, do not pose a significant health and safety threat for immunocompotent laboratory personnel and visitors.

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.