" Nealsonbacteria" Are Likely Biomass Recycling Ectosymbionts of Methanogenic Archaea in a Stable Benzene-Degrading Enrichment Culture.

The Candidate Phyla Radiation (CPR), also referred to as superphylum , is a very large group of bacteria with no pure culture representatives discovered by 16S rRNA sequencing or genome-resolved metagenomic analyses of environmental samples. Within the CPR, candidate phylum , previously referred to as OD1, is prevalent in anoxic sediments and groundwater. Previously, we had identified a specific member of the (referred to as DGGOD1a) as an important member of a methanogenic benzene-degrading consortium. Phylogenetic analyses herein place DGGOD1a within the clade " Nealsonbacteria." Because of its persistence over many years, we hypothesized that " Nealsonbacteria" DGGOD1a must play an important role in sustaining anaerobic benzene metabolism in the consortium. To try to identify its growth substrate, we amended the culture with a variety of defined compounds (pyruvate, acetate, hydrogen, DNA, and phospholipid), as well as crude culture lysate and three subfractions thereof. We observed the greatest (10-fold) increase in the absolute abundance of " Nealsonbacteria" DGGOD1a only when the consortium was amended with crude cell lysate. These results implicate " Nealsonbacteria" in biomass recycling. Fluorescence hybridization and cryogenic transmission electron microscope images revealed that " Nealsonbacteria" DGGOD1a cells were attached to larger archaeal cells. This apparent epibiont lifestyle was supported by metabolic predictions from a manually curated complete genome. This is one of the first examples of bacterial-archaeal episymbiosis and may be a feature of other " Nealsonbacteria" found in anoxic environments. An anaerobic microbial enrichment culture was used to study members of candidate phyla that are difficult to grow in the lab. We were able to visualize tiny " Nealsonbacteria" cells attached to a large cell, revealing a novel episymbiosis.