Expansion of Cultured Bacterial Diversity by Large-Scale Dilution-to-Extinction Culturing from a Single Seawater Sample.

High-throughput cultivation (HTC) based on a dilution-to-extinction method has been applied broadly to the cultivation of marine bacterial groups, which has often led to the repeated isolation of abundant lineages such as SAR11 and oligotrophic marine gammaproteobacteria (OMG). In this study, to expand the phylogenetic diversity of HTC isolates, we performed a large-scale HTC with a single surface seawater sample collected from the East Sea, the Western Pacific Ocean. Phylogenetic analyses of the 16S rRNA genes from 847 putative pure cultures demonstrated that some isolates were affiliated with not-yet-cultured clades, including the OPB35 and Puniceicoccaceae marine group of Verrucomicrobia and PS1 of Alphaproteobacteria. In addition, numerous strains were obtained from abundant clades, such as SAR11, marine Roseobacter clade, OMG (e.g., SAR92 and OM60), OM43, and SAR116, thereby increasing the size of available culture resources for representative marine bacterial groups. Comparison between the composition of HTC isolates and the bacterial community structure of the seawater sample used for HTC showed that diverse marine bacterial groups exhibited various growth capabilities under our HTC conditions. The growth response of many bacterial groups, however, was clearly different from that observed with conventional plating methods, as exemplified by numerous isolates of the SAR11 clade and Verrucomicrobia. This study showed that a large number of novel bacterial strains could be obtained by an extensive HTC from even a small number of samples.

Developing a Base Editing System for Marine Roseobacter Clade Bacteria.

The Roseobacter clade bacteria are of great significance in marine ecology and biogeochemical cycles, and they are potential microbial chassis for marine synthetic biology due to their versatile metabolic capabilities. Here, we adapted a CRISPR-Cas-based system, base editing, with the combination of nuclease-deactivated Cas9 and deaminase for Roseobacter clade bacteria. Taking the model roseobacter Roseovarius nubinhibens as an example, we achieved precise and efficient genome editing at single-nucleotide resolution without generating double-strand breaks or requesting donor DNAs. Since R. nubinhibens can metabolize aromatic compounds, we interrogated the key genes in the ß-ketoadipate pathway with our base editing system via the introduction of premature STOP codons. The essentiality of these genes was demonstrated, and for the first time, we determined PcaQ as a transcription activator experimentally. This is the first report of CRISPR-Cas-based genome editing in the entire clade of Roseobacter bacteria. We believe that our work provides a paradigm for interrogating marine ecology and biogeochemistry with direct genotype-and-phenotype linkages and potentially opens a new avenue for the synthetic biology of marine Roseobacter bacteria.