Sample collection: Borehole observatories accessing subseafloor crustal fluids were installed on the eastern flank of the JdFR in 2004 and 2010 during IODP Expeditions 301 and 327, respectively (Fisher et al. 2011; Fisher et al. 2005). These observatories penetrate ~ 240 m of sediment and ~110-290 m of underlying basement to enable crustal fluid collection from 8-190 m below the sediment-basement interface. Crustal fluids in this region are consistently 62-64°C, anoxic, and sulfate-replete (~18 mmol sulfate per kg water; Wheat et al. 2013). New crustal fluid sampling occurred in May 2019 during expedition AT42-11 on R/V Atlantis with ROV Jason II, both operated by Woods Hole Oceanographic Institution. Single cell genomic data was generated from cells collected from borehole observatory U1362B using established syringe sampling approaches (Carr et al. 2019; Wheat et al. 2011). Briefly, the top plug on the wellhead of U1362B was removed to freely vent crustal fluid from the top of the observatory (Fisher et al. 2011). After the borehole dead volume had been flushed, crustal fluid was collected using a sterilized syringe (Wheat et al. 2011). Once recovered, the fluid was distributed in a nitrogen-flushed glove bag into cryovials, amended with 5% glycerol and 1X-Tris-EDTA buffer (final concentrations), and immediately flash-frozen with liquid nitrogen before long term storage at -70°C . In addition to the new sample, we examined existing single cell genomic and metagenomic data generated from samples collected from borehole observatories in International Ocean Discovery Program (IODP) Holes U1301A, U1362A, and U1362B in 2011 (AT18-07) using methods described elsewhere (Carr et al. 2019; Jungbluth et al. 2016; Fisher et al. 2012; Jungbluth et al. 2017).
 

Genomic sequencing, assembly, and phylogenomic analysis: Nineteen JdFR Aminicenantia metagenome assembled genomes (MAGs) and single-cell amplified genomes (SAGs) were identified across three independent sampling expeditions that occurred over a nine-year period. Two new metagenome assembled genomes (MAGs 3300037598_45, 3300037558_26) were sequenced, assembled, and binned from 2010 and 2011 metagenomes by the Joint Genome Institute (Jungbluth et al. 2013; Clum et al. 2021). Four MAGs from 2011 sampling were produced previously (Jungbluth et al. 2016; Jungbluth et al. 2017). Single cell amplified genomes (SAGs) from 2011 sampling were created as previously described from Holes U1362A and U1362B (Jungbluth et al. 2016; Carr et al. 2019). This study examines ten of these 2011 SAGs from U1362B (IDs with AC-334- and AC-335-) and two SAGs from U1362A (AC-708-). One new SAG was generated from the 2019 Hole U1362B fluid sampling (AH-873) following the same cell sorting, lysis, amplification, sequencing, and assembly approach as described elsewhere (Carr et al. 2019), and based on the standard workflow of the Single Cell Genomics Center at Bigelow Laboratory for Ocean Sciences (East Boothbay, Maine, USA; Stepanauskas et al. 2017). Nine SAGs within the Aminicenantia order JdFR-78 are nearly identical (average pairwise nucleotide identity, ANI, >99%), prompting the generation of a nearly complete composite genome assembly from these SAGs named JDF1.

Genome annotation and assessment of viral interactions: JdFR Aminicenantia SAGs and MAGs were annotated with KOFAMSCAN using KEGG Orthology Hidden Markov Model (KO HMM) version 4.3, -mapper option using default settings (Kanehisa and Sato, 2020). Single KEGG Orthology IDs were reported for each open reading frame (ORF) that met default thresholds (Aramaki et al. 2020). Annotations were input into KEGGDecoder and KEGG Mapper - Reconstruct Pathway visualization tools (Kanehisa and Sato; Graham et al., 2018).

Genome annotation and assessment of viral interactions: JdFR Aminicenantia SAGs and MAGs were annotated with KOFAMSCAN using KEGG Orthology Hidden Markov Model (KO HMM) version 4.3, -mapper option using default settings. Single KEGG Orthology IDs were reported for each open reading frame (ORF) that met default thresholds. Annotations were input into KEGGDecoder and KEGG Mapper - Reconstruct Pathway visualization tools.