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

Molecular Ecology Resources, vol. 21, pp. 110–121

Abstract

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.

Authors 7

1. Vanessa Oliveira corresponding

   Centro de Estudos do Ambiente e do Mar · University of Aveiro

   Affiliation as printed

   Department of Biology and Centre for Environmental and Marine Studies (CESAM) University of Aveiro Aveiro Portugal

   Department of Biology and Centre for Environmental and Marine Studies (CESAM), University of Aveiro, Aveiro, Portugal

2. Ana R. M. Polónia

   Centro de Estudos do Ambiente e do Mar · University of Aveiro

   Affiliation as printed

   Department of Biology and Centre for Environmental and Marine Studies (CESAM) University of Aveiro Aveiro Portugal

   Department of Biology and Centre for Environmental and Marine Studies (CESAM), University of Aveiro, Aveiro, Portugal

3. Daniel F. R. Cleary

   Centro de Estudos do Ambiente e do Mar · University of Aveiro

   Affiliation as printed

   Department of Biology and Centre for Environmental and Marine Studies (CESAM) University of Aveiro Aveiro Portugal

   Department of Biology and Centre for Environmental and Marine Studies (CESAM), University of Aveiro, Aveiro, Portugal

4. Yusheng M. Huang

   National Penghu University of Science and Technology

   Affiliation as printed

   Department of Marine Recreation National Penghu University of Science and Technology Magong City Penghu Taiwan

   Tropical Island Sustainable Development Research Center National Penghu University of Science and Technology Magong City Penghu Taiwan

   Department of Marine Recreation, National Penghu University of Science and Technology, Magong City Penghu, Taiwan

   Tropical Island Sustainable Development Research Center, National Penghu University of Science and Technology, Magong City Penghu, Taiwan

5. Nicole Joy de Voogd Aachen Institute of Environmental Sciences (CML) Leiden University Leiden The Netherlands Marine Biodiversity Naturalis Biodiversity Center Leiden The Netherlands Institute of Environmental Sciences (CML) Marine Biodiversity Naturalis Biodiversity Center

   Leiden University · Naturalis Biodiversity Center

   Affiliation as printed

   Institute of Environmental Sciences (CML) Leiden University Leiden The Netherlands

   Marine Biodiversity Naturalis Biodiversity Center Leiden The Netherlands

   Institute of Environmental Sciences (CML), Leiden University, Leiden, The Netherlands

   Marine Biodiversity, Naturalis Biodiversity Center, Leiden, The Netherlands

6. Ulisses Nunes da Rocha

   Helmholtz Centre for Environmental Research

   Affiliation as printed

   Department of Environmental Microbiology Helmholtz Centre for Environmental Research—UFZ Leipzig Germany

7. Newton C. M. Gomes corresponding

   Centro de Estudos do Ambiente e do Mar · University of Aveiro

   Affiliation as printed

   Department of Biology and Centre for Environmental and Marine Studies (CESAM) University of Aveiro Aveiro Portugal

   Department of Biology and Centre for Environmental and Marine Studies (CESAM), University of Aveiro, Aveiro, Portugal