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Abstract Investigations of the metabolic capabilities of anaerobic protists advances our understanding of the evolution of eukaryotic life on Earth and for uncovering analogous extraterrestrial complex microbial life. Certain species of foraminiferan protists live in environments analogous to early Earth conditions when eukaryotes evolved, including sulfidic, anoxic and hypoxic sediment porewaters. Foraminifera are known to form symbioses as well as to harbor organelles from other eukaryotes (chloroplasts), possibly bolstering the host’s independence from oxygen. The full extent of foraminiferal physiological capabilities is not fully understood. To date, evidence for foraminiferal anaerobiosis was gleaned from specimens first subjected to stresses associated with removal from in situ conditions. Here, we report comprehensive gene expression analysis of benthic foraminiferal populations preserved in situ on the euxinic (anoxic and sulfidic) bathyal seafloor, thus avoiding environmental alterations associated with sample recovery, including pressure reduction, sunlight exposure, warming, and oxygenation. Metatranscriptomics, metagenome-assembled genomes, and measurements of substrate uptake were used to study the kleptoplastidic foraminifer Nonionella stella inhabiting sulfur-oxidizing bacterial mats of the Santa Barbara Basin, off California. We show N. stella energy generation under dark euxinia is unusual because it orchestrates complex metabolic pathways for ATP production and carbon fixation through the Calvin cycle. These pathways include extended glycolysis, anaerobic fermentation, sulfide oxidation, and the presence of a membrane-bound inorganic pyrophosphatase, an enzyme that hydrolyzes inorganic pyrophosphate to actively pump protons across the mitochondrial membrane. 

Plasmids are mobile genetic elements known to carry secondary metabolic genes that affect the fitness and survival of microbes in the environment. Well-studied cases of plasmid-encoded secondary metabolic genes in marine habitats include toxin/antitoxin and antibiotic biosynthesis/resistance genes. Here, we examine metagenome-assembled genomes (MAGs) from the permanently-stratified water column of the Cariaco Basin for integrated plasmids that encode biosynthetic gene clusters of secondary metabolites (smBGCs). We identify 16 plasmid-borne smBGCs in MAGs associated primarily with Planctomycetota and Pseudomonadota that encode terpene-synthesizing genes, and genes for production of ribosomal and non-ribosomal peptides. These identified genes encode for secondary metabolites that are mainly antimicrobial agents, and hence, their uptake via plasmids may increase the competitive advantage of those host taxa that acquire them. The ecological and evolutionary significance of smBGCs carried by prokaryotes in oxygen-depleted water columns is yet to be fully elucidated. 

The reconstruction of complete microbial metabolic pathways using ‘omics data from environmental samples remains challenging. Computational pipelines for pathway reconstruction that utilize machine learning methods to predict the presence or absence of KEGG modules in incomplete genomes are lacking. Here, we present MetaPathPredict, a software tool that incorporates machine learning models to predict the presence of complete KEGG modules within bacterial genomic datasets. Using gene annotation data and information from the KEGG module database, MetaPathPredict employs deep learning models to predict the presence of KEGG modules in a genome. MetaPathPredict can be used as a command line tool or as a Python module, and both options are designed to be run locally or on a compute cluster. Benchmarks show that MetaPathPredict makes robust predictions of KEGG module presence within highly incomplete genomes. 

The Guaymas Basin in the Gulf of California is characterized by active seafloor spreading, the rapid deposition of organic-rich sediments, steep geothermal gradients, and abundant methane of mixed thermogenic and microbial origin. Subsurface sediment samples from eight drilling sites with distinct geochemical and thermal profiles were selected for DNA extraction and PCR amplification to explore the diversity of methane-cycling archaea in the Guaymas Basin subsurface. We performed PCR amplifications with general (mcrIRD), and ANME-1 specific primers that target the alpha (α) subunit of methyl coenzyme M reductase (mcrA). Diverse ANME-1 lineages associated with anaerobic methane oxidation were detected in seven out of the eight drilling sites, preferentially around the methane-sulfate interface, and in several cases, showed preferences for specific sampling sites. Phylogenetically, most ANME-1 sequences from the Guaymas Basin subsurface were related to marine mud volcanoes, seep sites, and the shallow marine subsurface. The most frequently recovered methanogenic phylotypes were closely affiliated with the hyperthermophilic Methanocaldococcaceae, and found at the hydrothermally influenced Ringvent site. The coolest drilling site, in the northern axial trough of Guaymas Basin, yielded the greatest diversity in methanogen lineages. Our survey indicates the potential for extensive microbial methane cycling within subsurface sediments of Guaymas Basin. 

Abstract In Guaymas Basin, organic-rich hydrothermal sediments produce complex hydrocarbon mixtures including saturated, aromatic and alkylated aromatic compounds. We examined sediments from push cores from Guyamas sites with distinct temperature and geochemistry profiles to gain a better understanding on abiotic and biological hydrocarbon alteration. Here we provide evidence for biodegradation of hopanoids, producing saturated hydrocarbons like drimane and homodrimane as intermediate products. These sesquiterpene by-products are present throughout cooler sediments, but their relative abundance is drastically reduced within hotter hydrothermal sediments, likely due to hydrothermal mobilization. Within the sterane pool we detect a trend toward aromatization of steroidal compounds within hotter sediments. The changes in hopane and sterane biomarker composition at different sites reflect temperature-related differences in geochemical and microbial hydrocarbon alterations. In contrast to traditionally observed microbial biodegradation patterns that may extend over hundreds of meters in subsurface oil reservoirs, Guaymas Basin shows highly compressed changes in surficial sediments. 

Certain benthic foraminifera thrive in marine sediments with low or undetectable oxygen. Potential survival avenues used by these supposedly aerobic protists include fermentation and anaerobic respiration, although details on their adaptive mechanisms remain elusive. To better understand the metabolic versatility of foraminifera, we studied two benthic species that thrive in oxygen-depleted marine sediments. Here we detail, via transcriptomics and metatranscriptomics, differential gene expression of Nonionella stella and Bolivina argentea , collected from Santa Barbara Basin, California, USA, in response to varied oxygenation and chemical amendments. Organelle-specific metabolic reconstructions revealed these two species utilize adaptable mitochondrial and peroxisomal metabolism. N. stella , most abundant in anoxia and characterized by lack of food vacuoles and abundance of intracellular lipid droplets, was predicted to couple the putative peroxisomal beta-oxidation and glyoxylate cycle with a versatile electron transport system and a partial TCA cycle. In contrast, B. argentea , most abundant in hypoxia and contains food vacuoles, was predicted to utilize the putative peroxisomal gluconeogenesis and a full TCA cycle but lacks the expression of key beta-oxidation and glyoxylate cycle genes. These metabolic adaptations likely confer ecological success while encountering deoxygenation and expand our understanding of metabolic modifications and interactions between mitochondria and peroxisomes in protists. 

ABSTRACT International Ocean Discovery Program Expedition 360 drilled Hole U1473A at Atlantis Bank, an oceanic core complex on the Southwest Indian Ridge, with the aim of recovering representative samples of the lower oceanic crust. Recovered cores were primarily gabbro and olivine gabbro. These mineralogies may host serpentinization reactions that have the potential to support microbial life within the recovered rocks or at greater depths beneath Atlantis Bank. We quantified prokaryotic cells and analyzed microbial community composition for rock samples obtained from Hole U1473A and conducted nutrient addition experiments to assess if nutrient supply influences the composition of microbial communities. Microbial abundance was low (≤10 4 cells cm −3 ) but positively correlated with the presence of veins in rocks within some depth ranges. Due to the heterogeneous nature of the rocks downhole (alternating stretches of relatively unaltered gabbros and more significantly altered and fractured rocks), the strength of the positive correlations between rock characteristics and microbial abundances was weaker when all depths were considered. Microbial community diversity varied at each depth analyzed. Surprisingly, addition of simple organic acids, ammonium, phosphate, or ammonium plus phosphate in nutrient addition experiments did not affect microbial diversity or methane production in nutrient addition incubation cultures over 60 weeks. The work presented here from Site U1473A, which is representative of basement rock samples at ultraslow spreading ridges and the usually inaccessible lower oceanic crust, increases our understanding of microbial life present in this rarely studied environment and provides an analog for basement below ocean world systems such as Enceladus. IMPORTANCE The lower oceanic crust below the seafloor is one of the most poorly explored habitats on Earth. The rocks from the Southwest Indian Ridge (SWIR) are similar to rock environments on other ocean-bearing planets and moons. Studying this environment helps us increase our understanding of life in other subsurface rocky environments in our solar system that we do not yet have the capability to access. During an expedition to the SWIR, we drilled 780 m into lower oceanic crust and collected over 50 rock samples to count the number of resident microbes and determine who they are. We also selected some of these rocks for an experiment where we provided them with different nutrients to explore energy and carbon sources preferred for growth. We found that the number of resident microbes and community structure varied with depth. Additionally, added nutrients did not shape the microbial diversity in a predictable manner. 

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.