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Summary: Nitrosophilus labii HRV44T is a thermophilic chemolithoautotroph possessing clade II type nitrous oxide (N2O) reductase (NosZ) that has an outstanding activity in reducing N2O to dinitrogen gas. Here, we attempt to understand molecular responses of HRV44T to N2O. Time course transcriptome and proteomic mass spectrometry analyses under anaerobic conditions revealed that most of transcripts and peptides related to denitrification were constitutively detected, even in the absence of any nitrogen oxides as electron acceptors. Gene expressions involved in electron transport to NosZ were upregulated within 3 h in response to N2O, rather than upregulation of nos genes. Two genes encoding Crp/Fnr transcriptional regulators observed upstream of nap and nor gene clusters had significant negative correlations with nosZ expression. Statistical path analysis further inferred a significant causal relationship between the gene expression of nosZ and that of one Crp/Fnr regulators. Our findings contribute to understanding the transcriptional regulation in clade II type N2O-reducers.

The overexploitation of the wild populations of Apostichopus japonicus has caused a significant decline in their numbers, prompting the establishment of a sea cucumber aquaculture industry that has become a major player in East Asia. Recent research has focused on identifying the various factors that may affect sea cucumber aquaculture, including water quality, diet, and the microbiome associated with their hosts. Notably, studies on host-associated microbiomes have emerged as a prominent area of research, particularly in the context of investigating the relationship of the microbiome with diseases that have caused significant economic losses. Intestinal atrophy of A. japonicus larvae has been reported at a number of sea cucumber farms in Japan, but there have been no factor screenings, and there is currently no efficient mitigation. In this study, we applied metagenomic analyses for the first time to an intestinal atrophic case of the sea cucumber A. japonicus larvae, which occurred in an aquaculture facility in Hokkaido, Japan, elucidating potential causative microbial factors and comparing the diseased microbiome against the healthy pan-microbiome. Through our metagenome comparisons, we have found significant increases in Oceanicoccus, Ruegeria, Nioella, Labrenzia, Tenacibaculum, and Muricauda in the diseased larvae. Among those, Tenacibaculum was estimated to potentially be the most pathogenic bacterium in the development of intestinal atrophic disease due to previous outbreaks of sea urchin diseases in the same facility. Our metagenomic approach demonstrates the further need of microbial screening of such causative agents and identification of the pathogenesis of such diseases, which could contribute to establishing more sustainable aquaculture in the sea cucumber industry.

The genus Thalassotalea is ubiquitous in marine environments, and up to 20 species have been described so far. A Gram-staining-negative, aerobic bacterium, designated strain PTE2T was isolated from laboratory-reared larvae of the Japanese sea cucumber Apostichopus japonicus. Phylogenetic analysis based on the 16S rRNA gene nucleotide sequences revealed that PTE2T was closely related to Thalassotalea sediminis N211T (= KCTC 42588T = MCCC 1H00116T) with 97.9% sequence similarity. ANI and in silico DDH values against Thalassotalea species were 68.5-77.0% and 19.7-24.6%, respectively, indicating the novelty of PTE2T. Based on genome-based taxonomic approaches, strain PTE2T (= JCM 34608T = KCTC 82592T) is proposed as a new species, Thalassotalea hakodatensis sp. nov.

Light is one of the most critical stimuli in the majority of living organisms. In the last two decades, blue light (BL) has become a major subject of attention because of developments in light-emitting diodes (LED). The effects of BL on eukaryotic organisms and phototrophic prokaryotes have been well studied, but the knowledge of its effects on non-phototrophic prokaryotes remains unclear. Since BL can penetrate seawater, it is expected that most prokaryotes living in the ocean possess molecular mechanisms which protect against BL. The aim of this study is to assess the molecular mechanisms of Vibrio parahaemolyticus cells against BL as a marine bacterial model compared to other wavelength light exposures. Physiological and transcriptomic analyses of BL-exposed cells compared to other light treated cells revealed the highest ROS fold change, the highest number of differentially expressed genes (DEGs), and up-regulation in the gene responsible to not only compatible solute such as glycine betaine and ectoine but also iron-sulfur biosynthesis related to ROS formation. Furthermore, red light (RL) up-regulated the expression of cryptochrome DASH, a protein known to be excited by BL, and orange light (OL) decreased the expression of thermostable direct hemolysin (TDH), suggesting that OL attenuates the virulence of V. parahaemolyticus. In addition, the expression of VtrA (V. parahaemolyticus type III secretion system 2 (T3SS2) regulator A) but not VtrB (V. parahaemolyticus T3SS2 regulator B) increased under both light treatments, indicating that light exposure is unlikely to be involved in T3SS2-mediated pathogenicity. These results expand our knowledge on unique light responses in non-phototrophic marine prokaryotes.

Vibrionaceae is one of the most diverse bacterial families and is currently classified into over 50 clades, some members of which play an important role in the symbiotic relationships with humans and animals. Halioticoli clade, which currently consists of 10 species: 8 species associated with the gut of abalone (symbiotic), 1 species (V. breoganii) from bivalves, and 1 species (V. ishigakensis) from subtropical seawater (planktonic). To accelerate studies in the evolution, ecogenomics, and biotechnology of Halioticoli clade species, the genomic backbones and pangenome analyses based on complete genome sequences are needed. Genome sizes of Halioticoli clade species ranged from 3.5 Mb to 4.8 Mb, with V. ishigakensis the biggest. The evolutionary relationships using multilocus sequence analysis based on eight housekeeping genes and 125 single-copy core genes revealed a division of five sub-clades in this clade; 1) V. breoganii, V. comitans, V. inusitatus and V. superstes, 2) V. ezurae, V. neonatus, and V. halioticoli, 3) V. rarus, 4) V. gallicus, and 5) V. ishigakensis. The pan-genomic analysis combined with function and metabolism estimations showed that the planktonic group (sub-clade 5) contained the greatest number of specific genes, and more genes responsible for carbohydrate metabolisms, especially the genes encoding D-galactonate degradation. These results demonstrated that the genome expanded by acquiring more abilities for utilizing various carbohydrates during the evolution from symbiotic to a planktonic lifestyle. Moreover, according to Carbohydrate-Active enZYmes (CAZy) profiling, genes encoding alginate degrading enzymes (aly), classified into PL6, PL7, PL15, and PL17 were common in the ten genomes, but sub-clade 1 had the most. Meanwhile, sub-clade 1and 5 also possessed abundant genes related to macroalgae substrates degradation (GHs), which are also responsible for the genome expansion of sub-clade 1 and 5.

There is a lot of evidence indicating pioneer microbes in early life having various effects on later host biology. Because of the influential phylogenetic position of sea cucumber, which is a deep branching clade in Deuterostomia, the attention on the microbiome in sea cucumber has been increasing. Although microbes in sea cucumber have been reported in several studies, there is a lack of knowledge regarding the pioneer microbiota in the early life stages of sea cucumber. In this study, microbiota changes during the larval development of sea cucumber were assessed using a laboratory rearing system. Microbial community structure was likely to be related to the developmental stage and significant alterations were detected in the late auricularia stage. The relative abundances of Oceanospirillales, Alteromonadales, and Rhodobacterales significantly varied after gut formation. A total of 257 strains were isolated from larval developmental stages of sea cucumber and affiliated to 124 ASVs in the metagenomic analysis. This data demonstrates for the first-time dynamic changes of sea cucumber microbiota in the developmental stages in early life.

A Gram-staining-negative, aerobic bacterium, designated strain PT3T was isolated from laboratory-reared larvae of the Japanese sea cucumber Apostichopus japonicus. Phylogenetic analysis based on the 16S rRNA gene nucleotide sequences revealed that PT3T was closely related to Amphritea ceti RA1T (= KCTC 42154T = NBRC 110551T) and Amphritea spongicola MEBiC05461T (= KCCM 42943T = JCM 16668T) both with 98.3% sequence similarity, however, average nucleotide identity (ANI) and in silico DNA-DNA hybridization (in silico DDH) values among these three strains were below 95% and 70%, respectively, confirming the novelty of PT3T. Furthermore, the average amino acid identity (AAI) values of PT3T against other Amphritea species were on the reported genus delineation boundary (64-67%). Multilocus sequence analysis using four protein-coding genes (recA, mreB, rpoA, and topA) further demonstrated that PT3T, Amphritea ceti and Amphritea spongicola formed a monophyletic clade clearly separate from other members of the genus Amphritea. Three strains (PT3T, A. ceti KCTC 42154T and A. spongicola JCM 16668T) also showed higher similarities in their core genomes compared to those of the other Amphritea spp. Based on the genome-based taxonomic approach, Aliamphritea gen. nov. was proposed together with the reclassification of the genus Amphritea and Aliamphritea ceti comb. nov. (type strain RA1T = KCTC 42154T = NBRC 110551T), Aliamphritea spongicola comb. nov. (type strain MEBiC05461T = KCCM 42943T = JCM 16668T), and Aliamphritea hakodatensis sp. nov. (type strain PT3T = JCM 34607T = KCTC 82591T) were suggested.

Sea urchin is an indicator of coastal environmental changes in the global warming era, and is also a model organism in developmental biology and evolution. Due to the depletion of wild resources, new aquaculture techniques for improving stocks have been well studied. The gut microbiome shapes various aspects of a host’s physiology. However, these microbiome structures and functions on sea urchins, particularly Mesocentrotus nudus and Strongylocentrotus intermedius which are important marine bioresources commonly found in Japan, have not been fully investigated yet. Using metagenomic approaches including meta16S and shotgun metagenome sequencings, the structures, functions, and dynamics of the gut microbiome of M. nudus and S. intermedius, related to both habitat environment and host growth, were studied. Firstly, a broad meta16S analysis revealed that at the family level, Psychromonadaceae and Flavobacteriaceae reads (38–71%) dominated in these sea urchins, which is a unique feature observed in species in Japan. Flavobacteriaceae reads were more abundant in individuals after rearing in an aquarium with circulating compared to one with running water. Campylobacteraceae and Vibrionaceae abundances increased in both kinds of laboratory-reared sea urchins in both types of experiments. 2-weeks feeding experiments of M. nudus and S. intermedius transplanted from the farm to laboratory revealed that these gut microbial structures were affected by diet rather than rearing environments and host species. Secondly, further meta16S analysis of microbial reads related to M. nudus growth revealed that at least four Amplicon Sequence Variant (ASV) affiliated to Saccharicrinis fermentans, which is known to be a nitrogen (N2) fixing bacterium, showed a significant positive correlation to the body weight and test diameter. Interestingly, gut microbiome comparisons using shotgun metagenome sequencing of individuals showing higher and lower growth rates revealed a significant abundance of “Nitrate and nitrite ammonification” genes in the higher-grown individuals under the circulating water rearing. These findings provide new insights on the structure-function relationship of sea urchin gut microbiomes beyond previously reported nitrogen fixation function in sea urchin in 1950s; we discovered a nitrate reduction function into ammonium for the growth promotion of sea urchin.

Summary: Nitrous oxide (N2O) is a potent greenhouse gas and has significantly increased in the atmosphere. Deep-sea hydrothermal fields are representative environments dominated by mesophilic to thermophilic members of the class Campylobacteria that possess clade II nosZ encoding nitrous oxide reductase. Here, we report a strain HRV44T representing the first thermophilic campylobacterium capable of growth by H2 oxidation coupled to N2O reduction. On the basis of physiological and genomic properties, it is proposed that strain HRV44T (=JCM 34002 = DSM 111345) represents a novel species of the genus Nitratiruptor, Nitratiruptor labii sp. nov. The comparison of the N2O consumption ability of strain HRV44T with those of additional Nitratiruptor and other campylobacterial strains revealed the highest level in strain HRV44T and suggests the N2O-respiring metabolism might be the common physiological trait for the genus Nitratiruptor. Our findings provide insights into contributions of thermophilic Campylobacteria to the N2O sink in deep-sea hydrothermal environments.

A novel bacterium, strain EPR55-1T, was isolated from a deep-sea hydrothermal vent on the East Pacific Rise. The cells were motile rods. Growth was observed at temperatures between 50 and 60°C (optimum, 60°C), at pH values between 5.4 and 8.6 (optimum, pH 6.6) and in the presence of 2.4-3.2% (w/v) NaCl (optimum, 2.4%). The isolate used molecular hydrogen as its sole electron donor, carbon dioxide as its sole carbon source, ammonium as its sole nitrogen source, and thiosulfate, sulfite (0.01 to 0.001%, w/v) or elemental sulfur as its sole sulfur source. Nitrate, nitrous oxide (33%, v/v), thiosulfate, molecular oxygen (0.1%, v/v) or elemental sulfur could serve as the sole electron acceptor to support growth. Phylogenetic analyses based on both 16S rRNA gene sequences and whole genome sequences indicated that strain EPR55-1T belonged to the family Nitratiruptoraceae of the class "Campylobacteria", but it had the distinct phylogenetic relationship with the genus Nitratiruptor. On the basis of the physiological and molecular characteristics of the isolate, the name Nitrosophilus alvini gen. nov. sp. nov. is proposed, with EPR55-1T as the type strain (= JCM 32893T = KCTC 15925T). In addition, it is shown that "Nitratiruptor labii" should be transferred to the genus Nitrtosophilus; the name Nitrosophilus labii comb. nov. (JCM 34002T = DSM 111345T) is proposed for this organism. Furthermore, 16S rRNA gene-based and genome-based analyses showed that Cetia pacifica is phylogenetically associated with Caminibacter species. We therefore propose the reclassification of Cetia pacifica as Caminibacter pacificus comb. nov. (DSM 27783T = JCM 19563T). Additionally, AAI thresholds for genus classification and the reclassification of subordinate taxa within "Campylobacteria" are also evaluated, based on the analyses using publicly available genomes of all the campylobacterial species.

[This corrects the article DOI: 10.1371/journal.pone.0180053.].

A novel strain Vibrio aphrogenes sp. nov. strain CA-1004T isolated from the surface of seaweed collected on the coast of Mie Prefecture in 1994 [1] was characterized using polyphasic taxonomy including multilocus sequence analysis (MLSA) and a genome based comparison. Both phylogenetic analyses on the basis of 16S rRNA gene sequences and MLSA based on eight protein-coding genes (gapA, gyrB, ftsZ, mreB, pyrH, recA, rpoA, and topA) showed the strain could be placed in the Rumoiensis clade in the genus Vibrio. Sequence similarities of the 16S rRNA gene and the multilocus genes against the Rumoiensis clade members, V. rumoiensis, V. algivorus, V. casei, and V. litoralis, were low enough to propose V. aphrogenes sp. nov. strain CA-1004T as a separate species. The experimental DNA-DNA hybridization data also revealed that the strain CA-1004T was separate from four known Rumoiensis clade species. The G+C content of the V. aphrogenes strain was determined as 42.1% based on the genome sequence. Major traits of the strain were non-motile, halophilic, fermentative, alginolytic, and gas production. A total of 27 traits (motility, growth temperature range, amylase, alginase and lipase productions, and assimilation of 19 carbon compounds) distinguished the strain from the other species in the Rumoiensis clade. The name V. aphrogenes sp. nov. is proposed for this species in the Rumoiensis clade, with CA-1004T as the type strain (JCM 31643T = DSM 103759T).

Advances in genomic microbial taxonomy have opened the way to create a more universal and transparent concept of species but is still in a transitional stage towards becoming a defining robust criteria for describing new microbial species with minimum features obtained using both genome and classical polyphasic taxonomies. Here we performed advanced microbial taxonomies combined with both genome-based and classical approaches for new agarolytic vibrio isolates to describe not only a novel Vibrio species but also a member of a new Vibrio clade. Two novel vibrio strains (Vibrio astriarenae sp. nov. C7T and C20) showing agarolytic, halophilic and fermentative metabolic activity were isolated from a seawater sample collected in a coral reef in Okinawa. Intraspecific similarities of the isolates were identical in both sequences on the 16S rRNA and pyrH genes, but the closest relatives on the molecular phylogenetic trees on the basis of 16S rRNA and pyrH gene sequences were V. hangzhouensis JCM 15146T (97.8% similarity) and V. agarivorans CECT 5085T (97.3% similarity), respectively. Further multilocus sequence analysis (MLSA) on the basis of 8 protein coding genes (ftsZ, gapA, gyrB, mreB, pyrH, recA, rpoA, and topA) obtained by the genome sequences clearly showed the V. astriarenae strain C7T and C20 formed a distinct new clade protruded next to V. agarivorans CECT 5085T. The singleton V. agarivorans has never been included in previous MLSA of Vibrionaceae due to the lack of some gene sequences. Now the gene sequences are completed and analysis of 100 taxa in total provided a clear picture describing the association of V. agarivorans into pre-existing concatenated network tree and concluded its relationship to our vibrio strains. Experimental DNA-DNA hybridization (DDH) data showed that the strains C7T and C20 were conspecific but were separated from all of the other Vibrio species related on the basis of both 16S rRNA and pyrH gene phylogenies (e.g., V. agarivorans CECT 5085T, V. hangzhouensis JCM 15146T V. maritimus LMG 25439T, and V. variabilis LMG 25438T). In silico DDH data also supported the genomic relationship. The strains C7T also had less than 95% average amino acid identity (AAI) and average nucleotide identity (ANI) towards V. maritimus C210, V. variabilis C206, and V. mediterranei AK1T, V. brasiliensis LMG 20546T, V. orientalis ATCC 33934T, and V. sinaloensis DSM 21326. The name Vibrio astriarenae sp. nov. is proposed with C7 as the type strains. Both V. agarivorans CECT 5058T and V. astriarenae C7T are members of the newest clade of Vibrionaceae named Agarivorans.