Characterization of putative circular plasmids in sponge-associated bacterial communities using a selective multiply-primed rolling circle amplification.

Plasmid transfers among bacterial populations can directly influence the ecological adaptation of these populations and their interactions with host species and environment. In this study, we developed a selective multiply-primed rolling circle amplification (smRCA) approach to enrich and characterize circular plasmid DNA from sponge microbial symbionts via high-throughput sequencing (HTS). DNA (plasmid and total community DNA) obtained from sponge (Cinachyrella sp.) samples and a bacterial symbiont (Vibrio sp. CyArs1) isolated from the same sponge species (carrying unknown plasmids) were used to develop and validate our methodology. The smRCA was performed during 16 hr with 141 plasmid-specific primers covering all known circular plasmid groups. The amplified products were purified and subjected to a reamplification with random hexamer primers (2 hr) and then sequenced using Illumina MiSeq. The developed method resulted in the successful amplification and characterization of the sponge plasmidome and allowed us to detect plasmids associated with the bacterial symbiont Vibrio sp. CyArs1 in the sponge host. In addition to this, a large number of small (<2 kbp) and cryptic plasmids were also amplified in sponge samples. Functional analysis identified proteins involved in the control of plasmid partitioning, maintenance and replication. However, most plasmids contained unknown genes, which could potentially serve as a resource of unknown genetic information and novel replication systems. Overall, our results indicate that the smRCA-HTS approach developed here was able to selectively enrich and characterize plasmids from bacterial isolates and sponge host microbial communities, including plasmids larger than 20 kbp.