Cultivation of novel bacteria using dilution-to-extinction approach and genome analysis of groundwater OPB56 bacteria

초록

Bacteria play crucial biogeochemical roles in groundwater, a water resource important for human use. Cultivation of groundwater bacteria, is important to understand their ecophysiological and genomic features relevant to groundwater ecosystems. But, research on cultivation of diverse groundwater microbes has not been performed much. To expand the diversity of cultured bacterial groups in groundwater, we performed high-throughput dilution-to-extinction culturing (HTC) for water samples collected from two groundwater wells located in an agricultural area, South Korea, during four seasons. A total of 1,042 putative pure bacterial strains were successfully isolated. Phylogenetic analyses of 16S rRNA genes showed that these HTC isolates were affiliated with diverse phyla, such as Proteobacteria (Alpha-, Beta- and Gammaproteobacteria), Bacteroidetes, and Actinobacteria. Notably, isolates belonging to abundant but not-yet or rarely-cultured bacterial groups were obtained, including the OPB56, env.OPS_17, SJA-28, KD4-96 and OM190 clade. Genome sequencing and analyses were performed for three isolates of the OPB56 clade (c_Kapabacteria; GTDB), a bacterial group found to be widespread in various habitats but never been cultured in pure to date. The OPB56 genomes had complete gene sets for the EMP pathway, TCA cycle, and aerobic electron transport chain, suggesting aerobic heterotrophic lifestyle. But, biosynthetic pathways for quinone and heme, essential electron carriers in aerobic respiration, were lacking, implicating either auxotrophy or facultatively anaerobic lifestyle. This culture collection of diverse groundwater bacteria would be a valuable resource for better understanding of ecophysiological and genomic characteristics of groundwater microbes relevant to their habitats.