Discovery and assembly of plasmids in the fish pathogen Tenacibaculum

Members of the marine bacterial genus Tenacibaculum cause disease in finfish and outbreaks result in significant animal harm and losses in aquaculture around the globe. Plasmids have not been previously identified in Tenacibaculum, but long-read DNA sequencing of genomes from disease-associated Tenacibaculum isolates collected between 2017 and 2020 in British Columbia, Canada, revealed circular putative plasmids in three Tenacibaculum species. In addition to high-quality circular assembly, the putative plasmids contained genes encoding plasmid replication, mobility, and partitioning proteins. Genes for type B conjugation machinery and type 6_iii secretion system components were also identified on each of the two largest plasmid sequences. Several protocols were tested to visualize and enrich Tenacibaculum plasmid DNA. Rolling-circle replication with Phi29 DNA polymerase amplified putative plasmids smaller than 100 kb. Alkaline lysis extraction provided weak enrichment of putative plasmid DNA, but plasmids could not be confidently resolved by Eckhardt extraction and electrophoresis in agarose gels. The newly assembled plasmids matched previously sequenced Tenacibaculum contigs, suggesting that publicly available Tenacibaculum genomes contain unrecognized plasmids. The discovery of putative plasmids in Tenacibaculum is significant because plasmids often confer important functions to host cells and serve as vehicles for horizontal gene transfer within and beyond the host bacterial species.