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.

中文翻译：

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使用选择性多重引物滚环扩增表征海绵相关细菌群落中的假定环状质粒。

细菌种群之间的质粒转移可以直接影响这些种群的生态适应及其与宿主物种和环境的相互作用。在这项研究中，我们开发了一种选择性多重引物滚环扩增（smRCA）方法，通过高通量测序（HTS）从海绵微生物共生体中富集和表征环状质粒 DNA。从海绵（Cinachyrella sp.）样本和细菌共生体（弧菌）中获得的 DNA（质粒和总群落 DNA）sp. 从相同的海绵物种（携带未知质粒）中分离出的 CyArs1）用于开发和验证我们的方法。smRCA 在 16 小时内使用 141 种质粒特异性引物进行，涵盖所有已知的环状质粒组。扩增产物经过纯化并使用随机六聚体引物进行再扩增（2 小时），然后使用 Illumina MiSeq 进行测序。开发的方法导致海绵质粒组的成功扩增和表征，并使我们能够检测与细菌共生体弧菌相关的质粒sp. 海绵宿主中的 CyArs1。除此之外，还在海绵样品中扩增了大量小（<2 kbp）和隐匿质粒。功能分析鉴定了参与控制质粒分配、维持和复制的蛋白质。然而，大多数质粒含有未知基因，这可能作为未知遗传信息和新复制系统的资源。总的来说，我们的结果表明，这里开发的 smRCA-HTS 方法能够选择性地富集和表征来自细菌分离物和海绵宿主微生物群落的质粒，包括大于 20 kbp 的质粒。