Diversity and potential host-interactions of viruses inhabiting deep-sea seamount sediments.

Seamounts are globally distributed across the oceans and form one of the major oceanic biomes. Here, we utilized combined analyses of bulk metagenome and virome to study viral communities in seamount sediments in the western Pacific Ocean. Phylogenetic analyses and the protein-sharing network demonstrate extensive diversity and previously unknown viral clades. Inference of virus-host linkages uncovers extensive interactions between viruses and dominant prokaryote lineages, and suggests that viruses play significant roles in carbon, sulfur, and nitrogen cycling by compensating or augmenting host metabolisms. Moreover, temperate viruses are predicted to be prevalent in seamount sediments, which tend to carry auxiliary metabolic genes for host survivability. Intriguingly, the geographical features of seamounts likely compromise the connectivity of viral communities and thus contribute to the high divergence of viral genetic spaces and populations across seamounts. Altogether, these findings provides knowledge essential for understanding the biogeography and ecological roles of viruses in globally widespread seamounts.

Characterization and Comparative Genomic Analysis of a Deep-Sea Bacillus Phage Reveal a Novel Genus.

As the most abundant biological entities, viruses are the major players in marine ecosystems. However, our knowledge on virus diversity and virus-host interactions in the deep sea remains very limited. In this study, vB_BteM-A9Y, a novel bacteriophage infecting Bacillus tequilensis, was isolated from deep-sea sediments in the South China Sea. vB_BteM-A9Y has a hexametric head and a long, complex contractile tail, which are typical features of myophages. vB_BteM-A9Y initiated host lysis at 60 min post infection with a burst size of 75 PFU/cell. The phage genome comprises 38,634 base pairs and encodes 54 predicted open reading frames (ORFs), of which 27 ORFs can be functionally annotated by homology analysis. Interestingly, abundant ORFs involved in DNA damage repair were identified in the phage genome, suggesting that vB_BteM-A9Y encodes multiple pathways for DNA damage repair, which may help to maintain the stability of the host/phage genome. A BLASTn search of the whole genome sequence of vB_BteM-A9Y against the GenBank revealed no existing homolog. Consistently, a phylogenomic tree and proteome-based phylogenetic tree analysis showed that vB_BteM-A9Y formed a unique branch. Further comparative analysis of genomic nucleotide similarity and ORF homology of vB_BteM-A9Y with its mostly related phages showed that the intergenomic similarity between vB_BteM-A9Y and these phages was 0-33.2%. Collectively, based on the comprehensive morphological, phylogenetic, and comparative genomic analysis, we propose that vB_BteM-A9Y belongs to a novel genus under Caudoviricetes. Therefore, our study will increase our knowledge on deep-sea virus diversity and virus-host interactions, as well as expanding our knowledge on phage taxonomy.

Preparation and Embedding Characterization of Hydroxypropyl-ß-cyclodextrin/Menthyl Acetate Microcapsules with Enhanced Stability.

OBJECTIVE: Hydroxypropyl-ß-cyclodextrin (HP-ß-CD)/menthyl acetate (MA) microcapsules were developed to overcome the volatile and unstable defects of MA and improve the ease of use and storage. METHODS: MA microcapsules were prepared via spray drying using HP-ß-CD as the wall material. The embedding rate of MA microcapsules was determined through gas chromatography. The embedding characteristics were studied using phase solubility and nuclear magnetic resonance (NMR). The stability was characterized via differential scanning calorimetry (DSC) and the release and retention rates of MA microcapsules at different temperatures. RESULTS: The embedding rate of HP-ß-CD /MA microcapsules was 96.3%. The Gibbs free energy change, enthalpy change and entropy change of the embedding reaction between HP-ß-CD and MA were all less than zero, indicating that the embedding process was a spontaneous exothermic reaction. NMR spectra showed that MA entered the cavity of HP-ß-CD through the large opening end and interacted with the inner wall of the small opening end. DSC and the release and retention rates of MA microcapsules at different temperatures showed that the stability of MA was significantly enhanced after being embedded in HP-ß-CD. CONCLUSION: The HP-ß-CD/MA microcapsules are able to significantly improve the stability of MA and reduce the volatilization of MA.

The genome of a vestimentiferan tubeworm (Ridgeia piscesae) provides insights into its adaptation to a deep-sea environment.

BACKGROUND: Vestimentifera (Polychaeta, Siboglinidae) is a taxon of deep-sea worm-like animals living in deep-sea hydrothermal vents, cold seeps, and organic falls. The morphology and lifespan of Ridgeia piscesae, which is the only vestimentiferan tubeworm species found in the hydrothermal vents on the Juan de Fuca Ridge, vary greatly according to endemic environment. Recent analyses have revealed the genomic basis of adaptation in three vent- and seep-dwelling vestimentiferan tubeworms. However, the evolutionary history and mechanism of adaptation in R. piscesae, a unique species in the family Siboglinidae, remain to be investigated. RESULT: We assembled a draft genome of R. piscesae collected at the Cathedral vent of the Juan de Fuca Ridge. Comparative genomic analysis showed that vent-dwelling tubeworms with a higher growth rate had smaller genome sizes than seep-dwelling tubeworms that grew much slower. A strong positive correlation between repeat content and genome size but not intron size and the number of protein-coding genes was identified in these deep-sea tubeworm species. Evolutionary analysis revealed that Ridgeia pachyptila and R. piscesae, the two tubeworm species that are endemic to hydrothermal vents of the eastern Pacific, started to diverge between 28.5 and 35 million years ago. Four genes involved in cell proliferation were found to be subject to positive selection in the genome of R. piscesae. CONCLUSION: Ridgeia pachyptila and R. piscesae started to diverge after the formation of the Gorda/Juan de Fuca/Explorer ridge systems and the East Pacific Rise. The high growth rates of vent-dwelling tubeworms might be derived from their small genome sizes. Cell proliferation is important for regulating the growth rate in R. piscesae.

Isolation, characterization, and comparative genomic analysis of vB_BviS-A10Y, a novel bacteriophage from mangrove sediments.

Mangrove is among the most carbon-rich biomes on earth, and viruses are believed to play a significant role in modulating local and global carbon cycling. However, few viruses have been isolated from mangrove sediments to date. Here, we report the isolation of a novel Bacillus phage (named phage vB_BviS-A10Y) from mangrove sediments. Phage vB_BviS-A10Y has a hexameric head with a diameter of ~ 79.22 nm and a tail with a length of ~ 548.56 nm, which are typical features of siphophages. vB_BviS-A10Y initiated host lysis at 3.5 h postinfection with a burst size of 25 plaque-forming units (PFU)/cell. The genome of phage vB_BviS-A10Y is 162,435 bp long with 225 predicted genes, and the GC content is 34.03%. A comparison of the whole genome sequence of phage vB_BviS-A10Y with those of other phages from the NCBI viral genome database showed that phage vB_BviS-A10Y has the highest similarity (73.7% identity with 33% coverage) to Bacillus phage PBC2. Interestingly, abundant auxiliary metabolic genes (AMGs) were identified in the vB_BviS-A10Y genome. The presence of a ß-1,3-glucosyltransferase gene in the phage genome supported our previous hypothesis that mangrove viruses may manipulate carbon cycling directly through their encoded carbohydrate-active enzyme (CAZyme) genes. Therefore, our study will contribute to a better understanding of the diversity and potential roles of viruses in mangrove ecosystems.

Agarose-Degrading Characteristics of a Deep-Sea Bacterium Vibrio Natriegens WPAGA4 and Its Cold-Adapted GH50 Agarase Aga3420.

Up until now, the characterizations of GH50 agarases from Vibrio species have rarely been reported compared to GH16 agarases. In this study, a deep-sea strain, WPAGA4, was isolated and identified as Vibrio natriegens due to the maximum similarity of its 16S rRNA gene sequence, the values of its average nucleotide identity, and through digital DNA-DNA hybridization. Two circular chromosomes in V. natriegens WPAGA4 were assembled. A total of 4561 coding genes, 37 rRNA, 131 tRNA, and 59 other non-coding RNA genes were predicted in the genome of V. natriegens WPAGA4. An agarase gene belonging to the GH50 family was annotated in the genome sequence and expressed in E. coli cells. The optimum temperature and pH of the recombinant Aga3420 (rAga3420) were 40 °C and 7.0, respectively. Neoagarobiose (NA2) was the only product during the degradation process of agarose by rAga3420. rAga3420 had a favorable stability following incubation at 10-30 °C for 50 min. The Km, Vmax, and kcat values of rAga3420 were 2.8 mg/mL, 78.1 U/mg, and 376.9 s-1, respectively. rAga3420 displayed cold-adapted properties as 59.7% and 41.2% of the relative activity remained at 10 3 °C and 0 °C, respectively. This property ensured V. natriegens WPAGA4 could degrade and metabolize the agarose in cold deep-sea environments and enables rAga3420 to be an appropriate industrial enzyme for NA2 production, with industrial potential in medical and cosmetic fields.

Neoagaro-Oligosaccharides Ameliorate Chronic Restraint Stress-Induced Depression by Increasing 5-HT and BDNF in the Brain and Remodeling the Gut Microbiota of Mice.

Neoagaro-oligosaccharides (NAOs) belong to the algae oligosaccharides. NAOs have been found to have diverse biological activities. However, the effects of NAOs on depression and their underlying mechanism have not been thoroughly studied. A chronic restraint stress (CRS)-induced C57BL/6J mouse model was used to assess the antidepressant effects of NAOs. Anxiety and depression behaviors were assessed by open field tests (OFT) and forced swimming tests (FST), while interleukin 18 (IL-18), 5-hydroxytryptamine (5-HT) and brain-derived neurotrophic factor (BDNF) were the molecular biomarkers of depression. Fecal microbiota transplantation (FMT) was performed. The results showed that NAO treatment significantly improved the body weight of depressed mice and reduced the central area time in the OFT and immobility time in the FST. NAO treatment decreased the levels of IL-18 in the serum and increased the levels of 5-HT in the serum and whole brain and of BDNF in the whole brain. NAO treatment mitigated the gut microbiota dysbiosis in the depressed mice and reversed the decreased levels of short-chain fatty acids (SCFAs) in the cecum of the depressed mice. FMT indicated that the gut microbiota is, indeed, linked to depression, which was reflected in the changes in weight gain and behaviors. In a word, NAOs effectively reversed the CRS-induced mice model of depression, which depended on the changes in the gut microbiota and SCFAs, as well as its modulation of 5-HT and BDNF.

The importance of conditionally rare taxa for the assembly and interaction of fungal communities in mangrove sediments.

The fungal communities provide the nutrients and drive the cycles of elements in nature, and the rare fungal taxa are proved to be crucial for these communities in many environments. However, the ecological functions of rare taxa for the fungal communities in mangrove ecosystems are poorly assessed until now. This work aims to reveal the importance of rare taxa for the assembly of fungal communities in mangrove sediments by using the amplicon sequencing analysis of different spatiotemporal samples collected from Sanya mangroves, China. The results showed that Ascomycota and Basidiomycota were the dominant phyla in the conditionally rare taxa (CRT). The fungal communities possessed outstanding stability against the spatiotemporal variation and most collected environmental factors. The CRT possessed narrower niches and were more affected by the environmental variables than the abundant taxa. The current work demonstrated that the CRT had significantly higher relative abundances, degrees (the number of adjacent edges), clustering coefficients, and closeness centralities in the top 8 modules of the co-occurrence network (p < 0.05), indicating the important role of the CRT for the interaction of fungal communities in mangrove sediments. These findings indicate the importance of the CRT for the fungal community structures in mangrove sediments, and would deepen our understanding of dynamic functions of mangrove fungi, thereby facilitating the management, utilization, and protection of mangrove ecosystems. KEY POINTS: • Fungal communities in mangrove sediments are stable against environment variations. • The conditionally rare taxa (CRT) possessed narrower niches than the abundant fungal taxa. • The CRT are central for the co-occurrence network and interaction of fungal communities.

The first complete genome sequence of Microbulbifer celer KCTC12973T, a type strain with multiple polysaccharide degradation genes.

Genus Microbulbifer plays important roles in element cycling process in marine environments, and the first type strain KCTC 12973T (=ISL-39T = CCUG 54356T) of M. celer was isolated and identified in 2007. However, the genome sequence of M. celer KCTC 12973T is still unclear, which complicates the functional exploration and new species identification of other species belonged to this genus. This study reported the complete genome sequence of M. celer KCTC 12973T with a genome size of 4,346,001 bp. A total of 3601 protein-coding genes were annotated in the genome. The potential genes involved in the polysaccharide degradation, including cellulose, chitin, xylan, and pectate, were found in the protein-coding genes. Besides, the reductase genes of nitrate and nitrite were also annotated in the genome. These findings indicated the potential crucial ecological functions of M. celer KCTC 12973T for carbon and nitrogen cycles in marine ecosystems.

Expression and Characterization of a Novel Cold-Adapted and Stable ß-Agarase Gene agaW1540 from the Deep-Sea Bacterium Shewanella sp. WPAGA9.

The neoagaro-oligosaccharides, degraded from agarose by agarases, are important natural substances with many bioactivities. In this study, a novel agarase gene, agaW1540, from the genome of a deep-sea bacterium Shewanella sp. WPAGA9, was expressed, and the recombinant AgaW1540 (rAgaW1540) displayed the maximum activity under the optimal pH and temperature of 7.0 and 35 °C, respectively. rAgaW1540 retained 85.4% of its maximum activity at 0 °C and retained more than 92% of its maximum activity at the temperature range of 20-40 °C and the pH range of 4.0-9.0, respectively, indicating its extensive working temperature and pH values. The activity of rAgaW1540 was dramatically suppressed by Cu2+ and Zn2+, whereas Fe2+ displayed an intensification of enzymatic activity. The Km and Vmax of rAgaW1540 for agarose degradation were 15.7 mg/mL and 23.4 U/mg, respectively. rAgaW1540 retained 94.7%, 97.9%, and 42.4% of its maximum activity after incubation at 20 °C, 25 °C, and 30 °C for 60 min, respectively. Thin-layer chromatography and ion chromatography analyses verified that rAgaW1540 is an endo-acting ß-agarase that degrades agarose into neoagarotetraose and neoagarohexaose as the main products. The wide variety of working conditions and stable activity at room temperatures make rAgaW1540an appropriate bio-tool for further industrial production of neoagaro-oligosaccharides.

Complete Genome Sequence of Bacillus sp. Strain V3, Isolated from Mangrove Sediments in Wenzhou, China.

We present the complete genome sequence of Bacillus sp. strain V3, which was isolated from mangrove sediments from Yanpu Bay, Wenzhou, China. The 4.44-Mb genome sequence of V3 will be useful for studying the functional genes with potential industrial application of this species from mangrove sediments.

Vibrio ziniensis sp. nov., isolated from mangrove sediments.

A novel Gram-staining-negative, catalase- and oxidase-positive, facultatively anaerobic and rod-shaped motile bacterial strain, designated as ZWAL4003T, was isolated from mangrove sediments of the Zini Mangrove Forest, Zhangzhou City, PR China. Phylogenetic analysis based on its 16S rRNA gene sequence indicated that ZWAL4003T was grouped into a separated branch with Vibrio plantisponsor MSSRF60T (97.38% nucleotide sequence identity) and Vibrio diazotrophicus NBRC 103148T (97.27%). The major cellular fatty acids were C14 : 0 (12.6%), C16 : 0 (17.6%), and summed feature 3 (C16 : 1ω6c /C16 : 1 ω7c, 45.6%). Its genome had a length of 4650556 bp with 42.8% DNA G+C content, and contained genes involved in the biosynthesis of bacteriocin, ß-lactone, resorcinol, N-acyl amino acid, and arylpolyene. The in silico DNA-DNA hybridization and average nucleotide identity values for whole-genome sequence comparisons between ZWAL4003T and V. plantisponsor LMG 24470T were clearly below the thresholds used for the delineation of a novel species. The morphological and chemotaxonomic characteristics and the genotypic data of ZWAL4003T indicated that it represented a novel species of the genus Vibrio. Its proposed name is Vibrio ziniensis sp. nov., and the type strain is ZWAL4003T (=KCTC 72971T=MCCC 1A17474T).

The degradation activities for three seaweed polysaccharides of Shewanella sp. WPAGA9 isolated from deep-sea sediments.

Seaweed oligosaccharides possess great bioactivities. However, different microbial strains are required to degrade multiple polysaccharides due to their limited biodegradability, thereby increasing the cost and complexity of production. Shewanella sp. WPAGA9 was isolated from deep-sea sediments in this study. According to the genomic and biochemical analyses, the extracellular fermentation broth of WPAGA9 had versatile degradation abilities for three typical seaweed polysaccharides including agar, carrageenan, and alginate. The maximum enzyme activities of the extracellular fermentation broth of WPAGA9 were 71.63, 76.4, and 735.13 U/ml for the degradation of agar, alginate, and carrageenan, respectively. Moreover, multiple seaweed oligosaccharides can be produced by the extracellular fermentation broth of WPAGA9 under similar optimum conditions. Therefore, WPAGA9 can simultaneously degrade three types of seaweed polysaccharides under similar conditions, thereby greatly reducing the production cost of seaweed oligosaccharides. This finding indicates that Shewanella sp. WPAGA9 is an ideal biochemical tool for producing multiple active seaweed oligosaccharides at low costs and is also an important participant in the carbon cycle process of the deep-sea environment.

Characterization of Bacillus phage Gxv1, a novel lytic Salasvirus phage isolated from deep-sea seamount sediments.

Seamounts are hotspots for marine life, but to date, no bacteriophages have been reported. Here, a novel Bacillus podophage (named as Bacillus phage Gxv1) was isolated from deep-sea seamount sediments of the western Pacific Ocean (~ 5790 m). Phage Gxv1 has a hexameric head ~ 42-53 nm in diameter and a short tail of ~ 30 nm long, which is a typical feature of the Podoviridae family. One-step curve analysis showed that Gxv1 is a lytic phage that can initiate host lysis within 3.5 h post-infection, and has a relatively large burst size. The 21,781-bp genome contains 34 predicted genes, and the G + C content of phage Gxv1 is 39.69%. Whole-genome comparison of phage Gxv1 with known bacteriophages, using BlastN analysis against the IMG/VR database, revealed that phage Gxv1 is closely related to Bacillus phage phi29 that infects Bacillus subtilis, and their genome-wide similarity is 93.62%. Phylogenetic analysis based on DNA polymerase showed that phage Gxv1 belongs to the Salasvirus genus. Multiple genome alignment showed that phage Gxv1 shares a high level of sequence similarity and common gene order with Bacillus phage phi29. However, some sequences are unique to phage Gxv1, and this region contains genes encoding DNA packing protein, DNA replication protein, and unknown protein. These sequences exhibit low sequence similarity to known bacteriophages, highlighting an unknown origin of these sequences. This study will help improve our understanding of the Salasvirus genus and phage diversity in deep-sea seamounts.

Amelioration of Androgenetic Alopecia by Algal Oligosaccharides Prepared by Deep-Sea Bacterium Biodegradation.

Androgenetic alopecia (AGA) is a dihydrotestosterone (DHT)-mediated hair loss disorder characterized by shortened anagen hair cycle. Oligosaccharides derived from seaweeds possess diverse biological functions. However, little is known about their effects on AGA. In this study, algal oligosaccharide (AOS) was characterized for its mitigation effects on key features involved in AGA pathogenesis, such as DHT- mediated cellular signaling and shortened anagen hair cycle. AOS with varying degrees of polymerization (DP), namely, AOS (DP2), AOS (DP4-6), and AOS (DP8-12), were prepared by agar biodegradation with Flammeovirga pacifica WPAGA1, an agarolytic bacterium isolated from deep-sea sediments. In vitro results showed that AOS with varying DPs significantly ameliorated the DHT-induced alterations of regulatory factors in human hair follicle dermal papilla cells in a dose- and DP-dependent manner, as revealed by the normalization of several hair-growth-stimulating or inhibitory factors. In vivo studies showed that AOS (DP2) extended the anagen phase and thereby delayed catagen progression in mice. Furthermore, AOS (DP2) stimulated dorsal hair growth in mice by increasing hair length, density, and thickness. Therefore, our findings indicated that AOS antagonized key factors involved in AGA pathogenesis, suggesting the potential application of AOS in the prevention and the treatment of AGA.

Production of Neoagaro-Oligosaccharides With Various Degrees of Polymerization by Using a Truncated Marine Agarase.

Bioactivities, such as freshness maintenance, whitening, and prebiotics, of marine neoagaro-oligosaccharides (NAOS) with 4-12 degrees of polymerization (DPs) have been proven. However, NAOS produced by most marine ß-agarases always possess low DPs (≤6) and limited categories; thus, a strategy that can efficiently produce NAOS especially with various DPs ≥8 must be developed. In this study, 60 amino acid residues with no functional annotation result were removed from the C-terminal of agarase AgaM1, and truncated recombinant AgaM1 (trAgaM1) was found to have the ability to produce NAOS with various DPs (4-12) under certain conditions. The catalytic efficiency and stability of trAgaM1 were obviously lower than the wild type (rAgaM1), which probably endowed trAgaM1 with the ability to produce NAOS with various DPs. The optimum conditions for various NAOS production included mixing 1% agarose (w/v) with 10.26 U/ml trAgaM1 and incubating the mixture at 50°C in deionized water for 100 min. This strategy produced neoagarotetraose (NA4), neoagarohexaose (NA6), neoagarooctaose (NA8), neoagarodecaose (NA10), and neoagarododecaose (NA12) at final concentrations of 0.15, 1.53, 1.53, 3.02, and 3.02 g/L, respectively. The NAOS served as end-products of the reaction. The conditions for trAgaM1 expression in a shake flask and 5 L fermentation tank were optimized, and the yields of trAgaM1 increased by 56- and 842-fold compared with those before optimization, respectively. This study provides numerous substrate sources for production and activity tests of NAOS with high DPs and offers a foundation for large-scale production of NAOS with various DPs at a low cost.

Identification and Characterization of a Novel Thermostable and Salt-Tolerant ß-1,3 Xylanase from Flammeovirga pacifica Strain WPAGA1.

ß-1,3 xylanase is an important enzyme in the biorefinery process for some algae. The discovery and characterization of new ß-1,3 xylanase is a hot research topic. In this paper, a novel ß-1,3 xylanase (Xyl88) is revealed from the annotated genome of Flammeovirga pacifica strain WPAGA1. Bioinformatic analysis shows that Xyl88 belongs to the glycoside hydrolase 26 (GH26) with a suspected CBM (carbohydrate-binding module) sequence. The activity of rXyl88 is 75% of the highest enzyme activity (1.5 mol/L NaCl) in 3 mol/L NaCl buffer, which suggests good salt tolerance of rXy188. The optimum reaction temperature in the buffer without NaCl and with 1.5 mol/L NaCl is 45 °C and 55 °C, respectively. Notably, the catalytic efficiency of rXyl88 (kcat/Km) is approximately 20 higher than that of the thermophilic ß-1,3 xylanase that has the highest catalytic efficiency. Xyl88 in this study becomes the most efficient enzyme ever found, and it is also the first reported moderately thermophilic and salt-tolerant ß-1,3 xylanase. Results of molecular dynamics simulation further prove the excellent thermal stability of Xyl88. Moreover, according to the predicted 3D structure of the Xyl88, the surface of the enzyme is distributed with more negative charges, which is related to its salt tolerance, and significantly more hydrogen bonds and Van der Waals force between the intramolecular residues, which is related to its thermal stability.