Bacterial artificial chromosomes (BACs) are widely used in studies of vertebrate gene regulation and function because they often closely recapitulate the expression patterns of endogenous genes. Here we report a step-by-step protocol for efficient BAC transgenesis in zebrafish using the medaka Tol2 transposon. Using recombineering in Escherichia coli, we introduce the iTol2 cassette in the BAC plasmid backbone, which contains the inverted minimal cis-sequences required for Tol2 transposition, and a reporter gene to replace a target locus in the BAC. Microinjection of the Tol2-BAC and a codon-optimized transposase mRNA into fertilized eggs results in clean integrations in the genome and transmission to the germline at a rate of ∼15%. A single person can prepare a dozen constructs within 3 weeks, and obtain transgenic fish within approximately 3-4 months. Our protocol drastically reduces the labor involved in BAC transgenesis and will greatly facilitate biological and biomedical studies in model vertebrates.

Abyssivirga alkaniphila gen. nov., sp. nov., an alkane-degrading, anaerobic bacterium from a deep-sea hydrothermal vent system, and emended descriptions of Natranaerovirga pectinivora and Natranaerovirga hydrolytica A strictly anaerobic, mesophilic, syntrophic, alkane-degrading strain, L81 T , was isolated from a biofilm sampled from a black smoker chimney at the Loki's Castle vent field. Cells were straight, rod-shaped, Gram-positive-staining and motile. Growth was observed at pH 6.2-9.5, 14-42 8C and 0.5-6 % (w/w) NaCl, with optima at pH 7.0-8.2, 37 8C and 3 % (w/w) NaCl. Proteinaceous substrates, sugars, organic acids and hydrocarbons were utilized for growth. Thiosulfate was used as an external electron acceptor during growth on crude oil. Strain L81 T was capable of syntrophic hydrocarbon degradation when co-cultured with a methanogenic archaeon, designated strain LG6, isolated from the same enrichment. Phylogenetic analysis based on the 16S rRNA gene sequence indicated that strain L81 T is affiliated with the family Lachnospiraceae, and is most closely related to the type strains of Natranaerovirga pectinivora (92 % sequence similarity) and Natranaerovirga hydrolytica (90 %). The major cellular fatty acids of strain L81 T were C 15 : 0 , anteiso-C 15 : 0 and C 16 : 0 , and the profile was distinct from those of the species of the genus Natranaerovirga. The polar lipids were phosphatidylglycerol, diphosphatidylglycerol, three unidentified phospholipids, four unidentified glycolipids and two unidentified phosphoglycolipids. The G+C content of genomic DNA was determined to be 31.7 mol%. Based on our phenotypic, phylogenetic and chemotaxonomic results, strain L81 T is considered to represent a novel species of a new genus of the family Lachnospiraceae, for which we propose the name Abyssivirga alkaniphila gen. nov., sp. nov. The type strain of Abyssivirga alkaniphila is L81 T (5DSM 29592 T 5JCM 30920 T ). We also provide emended descriptions of Natranaerovirga pectinivora and Natranaerovirga hydrolytica.

Submarine hydrothermal activity is responsible for heat and chemical exchanges through the seafloor. Shallow-water hydrothermal systems (SWHS), while identified around the globe, are often studied in a way that is less comprehensive than their deep-ocean counterparts (e.g., along ridges), where systematic optical and acoustic mapping is more prevalent and coupled to in situ observations and sampling. Using aerial drones, an AUV, and temperature measurements at 10-40 cm subseafloor, we investigated in 2019 one of the most extensive SWHS known to date, in Paleochori (south of Milos, Greece). Hydrothermal venting, found from the shore to water depths of almost 500 m, shows emissions of gases and high-temperature fluids, often associated with bacterial mats and/or hydrothermal mineral precipitates. This study provides extensive drone mapping coupled with local AUV surveys for seafloor characterization and ground-truthing from the shore to ~20 m water depth. Seafloor photomosaics also provide a detailed context to samples, measurements and observations carried in situ. We interpret the photomosaics to define distinct seafloor types, linked to this hydrothermal activity. White hydrothermal patches (WHPs) often show a clear polygonal organization, together with outflow areas that are both more dispersed and distributed. Polygonal patterns likely result from fluid convection in a sandy porous medium heated from below. These WHPs display elevated subseafloor temperatures, typically >50 °C, with maximum values of ~75 °C. Photomosaics also display textures of biological origin, including seagrass and bioturbation patterns. Widespread bioturbation by burrowing shrimps is often associated with WHPs, bounding them, but also occurs on sandy seafloor away from hydrothermal patterns. Subseafloor temperatures at these bioturbated areas are of ~30-40 °C, and are thus transitional between hot WHPs and sedimented seafloor unaffected by hydrothermal activity (~24 °C). In addition to linking subseafloor temperature data and interpreted seafloor 4 photomosaics, our results provide a comprehensive general overview of this SWHS, of the organization of its hydrothermal outflow through the seafloor, and of the underlying subseafloor fluid circulation. This paper also gives the first perspectives on the heat fluxes of the system, and constitutes a background for other studies on the nature and distribution of microbial communities, controlled by this hydrothermal activity.

Abyssivirga alkaniphila strain L81T, recently isolated from a black smoker biofilm at the Loki’s Castle hydrothermal vent field, was previously described as a mesophilic, obligately anaerobic heterotroph able to ferment carbohydrates, peptides, and aliphatic hydrocarbons. The strain was classified as a new genus within the family Lachnospiraceae. Herein, its genome is analyzed and A. alkaniphila is reassigned to the genus Vallitalea as a new strain of V. guaymasensis, designated V. guaymasensis strain L81. The 6.4 Mbp genome contained 5651 protein encoding genes, whereof 4043 were given a functional prediction. Pathways for fermentation of mono-saccharides, di-saccharides, peptides, and amino acids were identified whereas a complete pathway for the fermentation of n-alkanes was not found. Growth on carbohydrates and proteinous compounds supported methane production in co-cultures with Methanoplanus limicola. Multiple confurcating hydrogen-producing hydrogenases, a putative bifurcating electron-transferring flavoprotein—butyryl-CoA dehydrogenase complex, and a Rnf-complex form a basis for the observed hydrogen-production and a putative reverse electron-transport in V. guaymasensis strain L81. Combined with the observation that n-alkanes did not support growth in co-cultures with M. limicola, it seemed more plausible that the previously observed degradation patterns of crude-oil in strain L81 are explained by unspecific activation and may represent a detoxification mechanism, representing an interesting ecological function. Genes encoding a capacity for polyketide synthesis, prophages, and resistance to antibiotics shows interactions with the co-occurring microorganisms. This study enlightens the function of the fermentative microorganisms from hydrothermal vents systems and adds valuable information on the bioprospecting potential emerging in deep-sea hydrothermal systems.

IntroductionShallow hydrothermal systems share many characteristics with their deep-sea counterparts, but their accessibility facilitates their study. One of the most studied shallow hydrothermal vent fields lies at Paleochori Bay off the coast of Milos in the Aegean Sea (Greece). It has been studied through extensive mapping and its physical and chemical processes have been characterized over the past decades. However, a thorough description of the microbial communities inhabiting the bay is still missing.MethodsWe present the first in-depth characterization of the prokaryotic communities of Paleochori Bay by sampling eight different seafloor types that are distributed along the entire gradient of hydrothermal influence. We used deep sequencing of the 16S rRNA marker gene and complemented the analysis with qPCR quantification of the 16S rRNA gene and several functional genes to gain insights into the metabolic potential of the communities.ResultsWe found that the microbiome of the bay is strongly influenced by the hydrothermal venting, with a succession of various groups dominating the sediments from the coldest to the warmest zones. Prokaryotic diversity and abundance decrease with increasing temperature, and thermophilic archaea overtake the community.DiscussionRelevant geochemical cycles of the Bay are discussed. This study expands our limited understanding of subsurface microbial communities in acidic shallow-sea hydrothermal systems and the contribution of their microbial activity to biogeochemical cycling.