Hydrostatic Pressure Helps to Cultivate an Original Anaerobic Bacterium From the Atlantis Massif Subseafloor (IODP Expedition 357): Petrocella atlantisensis gen. nov. sp. nov.

Abstract

Rock-hosted subseafloor habitats are very challenging for life, and current knowledge about microorganisms inhabiting such lithic environments is still limited. This study explored the cultivable microbial diversity in anaerobic enrichment cultures from cores recovered during the International Ocean Discovery Program (IODP) Expedition 357 from the Atlantis Massif (Mid-Atlantic Ridge, 30°N). 16S rRNA gene survey of enrichment cultures grown at 10–25°C and pH 8.5 showed that Firmicutes and Proteobacteria were generally dominant. However, cultivable microbial diversity significantly differed depending on incubation at atmospheric pressure (0.1 MPa), or hydrostatic pressures (HP) mimicking the in situ pressure conditions (8.2 or 14.0 MPa). An original, strictly anaerobic bacterium designated 70B-A T was isolated from core M0070C-3R1 (1150 meter below sea level; 3.5 m below seafloor) only from cultures performed at 14.0 MPa. This strain named Petrocella atlantisensis is a novel species of a new genus within the newly described family Vallitaleaceae (order Clostridiales , phylum Firmicutes ). It is a mesophilic, moderately halotolerant and piezophilic chemoorganotroph, able to grow by fermentation of carbohydrates and proteinaceous compounds. Its 3.5 Mb genome contains numerous genes for ABC transporters of sugars and amino acids, and pathways for fermentation of mono- and di-saccharides and amino acids were identified. Genes encoding multimeric [FeFe] hydrogenases and a Rnf complex form the basis to explain hydrogen and energy production in strain 70B-A T . This study outlines the importance of using hydrostatic pressure in culture experiments for isolation and characterization of autochthonous piezophilic microorganisms from subseafloor rocks.

Microbial Diversity and Function in Shallow Subsurface Sediment and Oceanic Lithosphere of the Atlantis Massif

“…No essential genes for methane metabolisms, such as methane monooxygenase ( mmoX , pmoA ) or methyl coenzyme M reductase associated with methane oxidation/methanogenesis, were found in any MAGs or SAGs generated in this study, suggesting that methane cycling is not a dominant metabolism in the shallow serpentinite subsurface of the Atlantis Massif. This is in contrast to methane cycling observed to support LCHF venting fluids and chimney microbiota ( 17 , 21 , 22 , 58 ) and the observation of elevated methane in Atlantis Massif samples during drilling (2 to 48 nM in samples compared to below detection limit [0.7 nM] in bottom water) ( 14 ), though consistent with cultivation attempts in parallel samples, which did not identify any methanogens ( 59 ). Acetyl-CoA decarbonylase/synthase complex genes associated with methanogenesis via acetate (via the WL pathway), and trimethylamine methyltransferase associated with methane production via trimethylamine, were found in the deepest crustal metagenome from sample 357-69A-9R2-14.61 mbsf ( Fig.…”

Expedition 399 Scientific Prospectus: Building Blocks of Life, Atlantis Massif

“…Distinguishing endemic microbial taxa from those introduced from seawater or contamination is a major challenge requiring multiple strategies to overcome (Kallmeyer et al, 2017;Sylvan et al, 2021;Pendleton et al, 2021). Nonetheless, the signatures of endolithic commu- nities have been successfully identified from the subseafloor of the Atlantis Massif during both Expeditions 304 (Mason et al, 2010) and 357 (Motamedi et al, 2020;Goordial et al, 2021;Quéméneur et al, 2019) (Figure F11). Despite the widespread dominance of communities that cycle H 2 and CH 4 in the Lost City hydrothermal chimneys (Schrenk et al, 2004(Schrenk et al, , 2013Brazelton et al, 2006), genes associated with H 2 and CH 4 metabolisms were rare or absent in the Atlantis Massif subseafloor (Goordial et al, 2021).…”

Alkalicella caledoniensis gen. nov., sp. nov., a novel alkaliphilic anaerobic bacterium isolated from ‘La Crouen’ alkaline thermal spring, New Caledonia

“…Cellular morphology and purity of the strain and the presence of spores were assessed by observation using a phase-contrast microscope (Optiphot, Nikon). For transmission electron microscopy (using an MET FEI Tecnai G2), exponentially grown cells were negatively stained with sodium phosphotungstate (to observe flagella) or fixed with glutaraldehyde and osmium tetraoxide, epon-embedded and stained with uranyl acetate and lead citrate (to watch the cell wall), as described by Quéméneur et al [25].…”

“…Whole-genome analysis was performed from high molecular weight DNA of the isolate, extracted using a phenol–chloroform method, then sequenced at the GeT-PlaGe (INRAe Toulouse, France) by combining long reads (Oxford Nanopore) and short-reads (Illumina MiSeq) technologies, as described by Quéméneur et al [25] with some modifications because it was sequenced by multiplex sequencing according to the manufacturer’s instructions ‘1D Native barcoding genomic DNA’ (EXP-NBD104 and SQK-LSK109). Base-calling was carried out by MinKNOW-Live-Base-calling software version 3.6.0 (Oxford Nanopore Technologies).…”