Meta-Omics Approaches Reveal Unique Small RNAs Exhibited by the Uncultured Microorganisms Dwelling Deep-Sea Hydrothermal Sediment in Guaymas Basin

Small regulatory RNAs (sRNAs) are present in almost all investigated microbes, regarded as modulators and regulators of gene expression and also known to play their regulatory role in the environmentally significant process. It has been estimated that less than 1% of the microbes in nature are culturable in the laboratory, hindering our understanding of their physiology, and living strategies. However, recent big advancing of DNA sequencing and omics-related data analysis makes the understanding of the genetics, metabolic potentials, even ecological roles of uncultivated microbes possible. In this study, we used a metagenome and metatranscriptome based integrated approach to identifying small RNAs in the microbiome of Guaymas Basin sediments. Hundreds of environmental sRNAs comprising of 228 groups were identified based on their homology, 82% of which displayed high similarity with previously known small RNAs in Rfam database, whereas, “18%” are putative novel sRNA motifs. A putative cis-acting sRNA potentially binding to methyl coenzyme M reductase, a key enzyme in methanogenesis or anaerobic oxidation of methane (AOM), was discovered in the genome of ANaerobic MEthane oxidizing archaea group 1 (ANME-1), which were the dominate microbe in the sample. These sRNAs were actively expressed in local Guaymas Basin hydrothermal environment, suggesting important roles of sRNAs in regulating microbial activity in natural environments.

Microbial Community Response to Polysaccharide Amendment in Anoxic Hydrothermal Sediments of the Guaymas Basin

Organic-rich, hydrothermal sediments of the Guaymas Basin are inhabited by diverse microbial communities including many uncultured lineages with unknown metabolic potential. Here we investigated the short-term effect of polysaccharide amendment on a sediment microbial community to identify taxa involved in the initial stage of macromolecule degradation. We incubated anoxic sediment with cellulose, chitin, laminarin, and starch and analyzed the total and active microbial communities using bioorthogonal non-canonical amino acid tagging (BONCAT) combined with fluorescence-activated cell sorting (FACS) and 16S rRNA gene amplicon sequencing. Our results show a response of an initially minor but diverse population of Clostridia particularly after amendment with the lower molecular weight polymers starch and laminarin. Thus, Clostridia may readily become key contributors to the heterotrophic community in Guaymas Basin sediments when substrate availability and temperature range permit their metabolic activity and growth, which expands our appreciation of the potential diversity and niche differentiation of heterotrophs in hydrothermally influenced sediments. BONCAT-FACS, although challenging in its application to complex samples, detected metabolic responses prior to growth and thus can provide complementary insight into a microbial community’s metabolic potential and succession pattern. As a primary application of BONCAT-FACS on a diverse deep-sea sediment community, our study highlights important considerations and demonstrates inherent limitations associated with this experimental approach.

Environmental factors shaping bacterial, archaeal and fungal community structure in hydrothermal sediments of Guaymas Basin, Gulf of California

The flanking regions of Guaymas Basin, a young marginal rift basin located in the Gulf of California, are covered with thick sediment layers that are hydrothermally altered due to magmatic intrusions. To explore environmental controls on microbial community structure in this complex environment, we analyzed site- and depth-related patterns of microbial community composition (bacteria, archaea, and fungi) in hydrothermally influenced sediments with different thermal conditions, geochemical regimes, and extent of microbial mats. We compared communities in hot hydrothermal sediments (75-100°C at ~40 cm depth) covered by orange-pigmented Beggiatoaceae mats in the Cathedral Hill area, temperate sediments (25-30°C at ~40 cm depth) covered by yellow sulfur precipitates and filamentous sulfur oxidizers at the Aceto Balsamico location, hot sediments (>115°C at ~40 cm depth) with orange-pigmented mats surrounded by yellow and white mats at the Marker 14 location, and background, non-hydrothermal sediments (3.8°C at ~45 cm depth) overlain with ambient seawater. Whereas bacterial and archaeal communities are clearly structured by site-specific in-situ thermal gradients and geochemical conditions, fungal communities are generally structured by sediment depth. Unexpectedly, chytrid sequence biosignatures are ubiquitous in surficial sediments whereas deeper sediments contain diverse yeasts and filamentous fungi. In correlation analyses across different sites and sediment depths, fungal phylotypes correlate to each other to a much greater degree than Bacteria and Archaea do to each other or to fungi, further substantiating that site-specific in-situ thermal gradients and geochemical conditions that control bacteria and archaea do not extend to fungi.

Conspicuous Smooth and White Egg-Shaped Sulfur Structures on a Deep-Sea Hydrothermal Vent Formed by Sulfide-Oxidizing Bacteria

At the deep-sea Guaymas Basin hydrothermal vent system, sulfide-rich hydrothermal fluids mix with oxygenated seawater, thereby providing a habitat for microbial sulfur oxidation. Microbial sulfur oxidation in the deep sea involves a variety of organisms and processes and can result in the excretion of elemental sulfur.

Microbial Communities of Hydrothermal Guaymas Basin Surficial Sediment Profiled at 2 Millimeter-Scale Resolution

The surficial hydrothermal sediments of Guaymas Basin harbor complex microbial communities where oxidative and reductive nitrogen, sulfur, and carbon-cycling populations and processes overlap and coexist. Here, we resolve microbial community profiles in hydrothermal sediment cores of Guaymas Basin on a scale of 2 millimeters, using Denaturing Gradient Gel Electrophoresis (DGGE) to visualize the rapid downcore changes among dominant bacteria and archaea. DGGE analysis of bacterial 16S rRNA gene amplicons identified free-living and syntrophic deltaproteobacterial sulfate-reducing bacteria, fermentative Cytophagales, members of the Chloroflexi (Thermoflexia), Aminicenantes, and uncultured sediment clades. The DGGE pattern indicates a gradually changing downcore community structure where small changes on a 2-millimeter scale accumulate to significantly changing populations within the top 4 cm sediment layer. Functional gene DGGE analyses identified anaerobic methane-oxidizing archaea (ANME) based on methyl-coenzyme M reductase genes, and members of the Betaproteobacteria and Thaumarchaeota based on bacterial and archaeal ammonia monooxygenase genes, respectively. The co-existence and overlapping habitat range of aerobic, nitrifying, sulfate-reducing and fermentative bacteria and archaea, including thermophiles, in the surficial sediments is consistent with dynamic redox and thermal gradients that sustain highly complex microbial communities in the hydrothermal sediments of Guaymas Basin.

Degradation of biological macromolecules supports uncultured microbial populations in Guaymas Basin hydrothermal sediments

Hydrothermal sediments contain large numbers of uncultured heterotrophic microbial lineages. Here, we amended Guaymas Basin sediments with proteins, polysaccharides, nucleic acids or lipids under different redox conditions and cultivated heterotrophic thermophiles with the genomic potential for macromolecule degradation. We reconstructed 20 metagenome-assembled genomes (MAGs) of uncultured lineages affiliating with known archaeal and bacterial phyla, including endospore-forming Bacilli and candidate phylum Marinisomatota. One Marinisomatota MAG had 35 different glycoside hydrolases often in multiple copies, seven extracellular CAZymes, six polysaccharide lyases, and multiple sugar transporters. This population has the potential to degrade a broad spectrum of polysaccharides including chitin, cellulose, pectin, alginate, chondroitin, and carrageenan. We also describe thermophiles affiliating with the genera Thermosyntropha, Thermovirga, and Kosmotoga with the capability to make a living on nucleic acids, lipids, or multiple macromolecule classes, respectively. Several populations seemed to lack extracellular enzyme machinery and thus likely scavenged oligo- or monomers (e.g., MAGs affiliating with Archaeoglobus) or metabolic products like hydrogen (e.g., MAGs affiliating with Thermodesulfobacterium or Desulforudaceae). The growth of methanogens or the production of methane was not observed in any condition, indicating that the tested macromolecules are not degraded into substrates for methanogenesis in hydrothermal sediments. We provide new insights into the niches, and genomes of microorganisms that actively degrade abundant necromass macromolecules under oxic, sulfate-reducing, and fermentative thermophilic conditions. These findings improve our understanding of the carbon flow across trophic levels and indicate how primary produced biomass sustains complex and productive ecosystems.

Magma plumbing system and associated hydrothermal vents in the Guaymas Basin - geometry and implications

<p>We document the geometry of a massive sill at the root of an approximately 20-m high and 800 m-wide ring of hydrothermal formations, termed Ringvent, located 28.5 km off-axis on the northwestern flanking regions of the actively rifting Guaymas Basin (Gulf of California). Using petrophysical data collected during the IODP Expedition 385 and processed 2D seismic profiles, we present evidence on the mechanics of sill emplacement and how the related hydrothermal vent conduits were constructed. The currently active moderate-temperature hydrothermal vent field indicates that, despite being cold and crystallized, the magma plumbing system, is tapping into a deeper geothermal source of the basin. The vent system roots at the vertical end of the magma plumbing system with the top of the sill located at a depth range of 80 to 150 m below the seafloor. Our research aims at constraining how far deep the geothermal fluids are coming from, and identifying how close the hydrothermal system is from a steady-state condition, to draw implications for how frequently such a system may arise in nascent ocean basins.</p>