Dedication: To Katrina Edwards 18 19 Originality-Significance Statement: This work provides insights into the metabolism and 20 adaptations of elusive Atribacteria (JS-1 clade) that are ubiquitous and abundant in methane-rich 21 ecosystems. We show that JS-1 (Genus 1) from methane hydrate stability zones contain 22 metabolisms and stress survival strategies similar to hyperthermophilic archaea. 23 35 were downstream from a novel helix-turn-helix transcriptional regulator, AtiR, which was not 36 present in Atribacteria from other sites. Overall, Atribacteria appear to be endowed with unique 37 strategies that may contribute to its dominance in methane-hydrate bearing sediments. Active 38 microbial transport of amino and carboxylic acids in the gas hydrate stability zone may influence 39 gas hydrate stability. 40 41 131 132 JS-1 Genus-1 partial genome. To gain insight into the function of JS-1 Atribacteria in the 133 GHSZ, we analyzed a 4-Mbp metagenome-assembled genome (MAG) from sample E10-H5 134 (Table S3). This MAG, hereafter designated "B2", was chosen for its relatively high 135 completeness (69%) and low contamination (2%). B2 lacked a 16S rRNA gene, but contained a 136 rpoB gene with 94% similarity to Atribacteria bacterium 34_128 from an oil reservoir (Hu et al., 137 2016). B2 had 35% GC content, similar to other Atribacteria (Carr et al., 2015). Phylogenetic 138 placement based on 69 concatenated single-copy genes confirmed that B2 belonged to JS1-Genus 139 195 heterodisulfide reductase (HdrA)-methyl viologen hydrogenase (MvhAGD) complex (Fig. 4). A 196 redox-sensing transcriptional repressor gene (hunR) immediately upstream of the hun operon 197 J

Summary Gas hydrates harbour gigatons of natural gas, yet their microbiomes remain understudied. We bioprospected 16S rRNA amplicons, metagenomes, and metaproteomes from methane hydrate‐bearing sediments under Hydrate Ridge (offshore Oregon, USA, ODP Site 1244, 2–69 mbsf) for novel microbial metabolic and biosynthetic potential. Atribacteria sequences generally increased in relative sequence abundance with increasing sediment depth. Most Atribacteria ASVs belonged to JS‐1‐Genus 1 and clustered with other sequences from gas hydrate‐bearing sediments. We recovered 21 metagenome‐assembled genomic bins spanning three geochemical zones in the sediment core: the sulfate–methane transition zone, the metal (iron/manganese) reduction zone, and the gas hydrate stability zone. We found evidence for bacterial fermentation as a source of acetate for aceticlastic methanogenesis and as a driver of iron reduction in the metal reduction zone. In multiple zones, we identified a Ni‐Fe hydrogenase‐Na+/H+ antiporter supercomplex (Hun) in Atribacteria and Firmicutes bins and in other deep subsurface bacteria and cultured hyperthermophiles from the Thermotogae phylum. Atribacteria expressed tripartite ATP‐independent transporters downstream from a novel regulator (AtiR). Atribacteria also possessed adaptations to survive extreme conditions (e.g. high salt brines, high pressure and cold temperatures) including the ability to synthesize the osmolyte di‐myo‐inositol‐phosphate as well as expression of K+‐stimulated pyrophosphatase and capsule proteins.

Gas clathrates are both a resource and a hindrance. They store massive quantities of natural gas but also can clog natural gas pipelines, with disastrous consequences. Eco-friendly technologies for controlling and modulating gas clathrate growth are needed. Type I Antifreeze Proteins (AFPs) from cold-water fish have been shown to bind to gas clathrates via repeating motifs of threonine and alanine. We tested whether proteins encoded in the genomes of bacteria native to natural gas clathrates bind to and alter clathrate morphology. We identified putative clathrate-binding proteins (CBPs) with multiple threonine/alanine motifs in a putative operon (cbp) in metagenomes from natural clathrate deposits. We recombinantly expressed and purified five CbpA proteins, four of which were stable, and experimentally confirmed that CbpAs bound to tetrahydrofuran (THF) clathrate, a low-pressure analogue for structure II gas clathrate. When grown in the presence of CbpAs, the THF clathrate was polycrystalline and platelike instead of forming single, octahedral crystals. Two CbpAs yielded branching clathrate crystals, similar to the effect of Type I AFP, while the other two produced hexagonal crystals parallel to the [1 1 1] plane, suggesting two distinct binding modes. Bacterial CBPs may find future utility in industry, such as maintaining a platelike structure during gas clathrate transportation.

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Gas clathrates are both a resource and a hindrance. They store massive quantities of natural gas but also can clog natural gas pipelines, with disastrous consequences. Eco-friendly technologies for controlling and modulating gas clathrate growth are needed. Type I Antifreeze Proteins (AFPs) from cold-water fish have been shown to bind to gas clathrates via repeating motifs of threonine and alanine. We tested whether proteins encoded in the genomes of bacteria native to natural gas clathrates bind to and alter clathrate morphology. We identified putative clathrate-binding proteins (CBPs) with multiple threonine/alanine motifs in a putative operon (cbp) in metagenomes from natural clathrate deposits. We recombinantly expressed and purified five CbpA proteins, four of which were stable, and experimentally confirmed that CbpAs bound to tetrahydrofuran (THF) clathrate, a lowpressure analog for structure II gas clathrate. When grown in the presence of CbpAs, THF clathrate was polycrystalline and platelike instead of forming single, octahedral crystals. Two CbpAs yielded branching clathrate crystals, similar to the effect of Type I AFP, while the other two produced hexagonal crystals parallel to the [1 1 1] plane, suggesting two distinct binding modes. Bacterial CBPs may find future utility in industry, such as maintaining a plate-like structure during gas clathrate transportation. ACKNOWLEDGMENTWe thank William Waite (USGS) for helpful suggestions. ABBREVIATIONSCbp: clathrate-binding protein; eGFP: enhanced green fluorescent protein; THF, tetrahydrofuran.

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In this paper we propose a methodology for evaluating the accuracy of an automatic license plate reading system. The proposed methodology estimates accuracy of state (country) recognition and the license plate characters. In addition, we examine the influence of external parameters (time of day, weather) on the accuracy. A case study of a license plate reader at a university testbed is performed.