Uncovering the Prevalence and Diversity of Integrating Conjugative Elements in Actinobacteria

Horizontal gene transfer greatly facilitates rapid genetic adaptation of bacteria to shifts in environmental conditions and colonization of new niches by allowing one-step acquisition of novel functions. Conjugation is a major mechanism of horizontal gene transfer mediated by conjugative plasmids and integrating conjugative elements (ICEs). While in most bacterial conjugative systems DNA translocation requires the assembly of a complex type IV secretion system (T4SS), in Actinobacteria a single DNA FtsK/SpoIIIE-like translocation protein is required. To date, the role and diversity of ICEs in Actinobacteria have received little attention. Putative ICEs were searched for in 275 genomes of Actinobacteria using HMM-profiles of proteins involved in ICE maintenance and transfer. These exhaustive analyses revealed 144 putative FtsK/SpoIIIE-type ICEs and 17 putative T4SS-type ICEs. Grouping of the ICEs based on the phylogenetic analyses of maintenance and transfer proteins revealed extensive exchanges between different sub-families of ICEs. 17 ICEs were found in Actinobacteria from the genus Frankia, globally important nitrogen-fixing microorganisms that establish root nodule symbioses with actinorhizal plants. Structural analysis of ICEs from Frankia revealed their unexpected diversity and a vast array of predicted adaptive functions. Frankia ICEs were found to excise by site-specific recombination from their host's chromosome in vitro and in planta suggesting that they are functional mobile elements whether Frankiae live as soil saprophytes or plant endosymbionts. Phylogenetic analyses of proteins involved in ICEs maintenance and transfer suggests that active exchange between ICEs cargo-borne and chromosomal genes took place within the Actinomycetales order. Functionality of Frankia ICEs in vitro as well as in planta lets us anticipate that conjugation and ICEs could allow the development of genetic manipulation tools for this challenging microorganism and for many other Actinobacteria.水平基因转移（Horizontal gene transfer）可通过赋予细菌一次性获得全新功能的能力，极大促进其快速适应环境条件变化并定殖新生态位。接合作用是由接合性质粒与整合接合元件（integrating conjugative elements，ICEs）介导的主要水平基因转移机制。在多数细菌的接合系统中，DNA转位依赖复杂的IV型分泌系统（type IV secretion system，T4SS）的组装，但放线菌门（Actinobacteria）仅需单个类FtsK/SpoIIIE易位蛋白即可完成该过程。迄今为止，放线菌门中整合接合元件的功能与多样性尚未得到足够关注。本研究借助参与ICE维持与转移的蛋白隐马尔可夫模型谱（HMM-profiles），在275个放线菌基因组中搜寻推定的ICEs。经全面分析，共发现144个推定的FtsK/SpoIIIE型ICEs与17个推定的T4SS型ICEs。基于维持与转移蛋白的系统发育分析对ICEs进行聚类，结果显示不同ICE亚家族间存在广泛的基因交换。在弗兰克氏菌属（Frankia）的放线菌中共发现17个ICEs，该属微生物为全球重要的固氮微生物，可与放线菌根植物建立根瘤共生关系。对弗兰克氏菌ICEs的结构分析揭示了其出人意料的多样性，以及大量预测的适应性功能。实验证实，弗兰克氏菌ICEs可通过位点特异性重组从宿主染色体上切下，且无论弗兰克氏菌以土壤腐生菌还是植物内共生体的形式生存，该过程在体外与植物体内均可发生，表明其为具有功能的可移动遗传元件。对参与ICE维持与转移的蛋白进行系统发育分析，结果表明ICE载荷携带基因与染色体基因间的活跃交换发生于放线菌目（Actinomycetales）内。弗兰克氏菌ICEs在体外与植物体内均具备功能，这提示我们可开发基于接合作用与ICEs的遗传操作工具，用于这类难以操控的微生物以及其他多种放线菌的遗传改造。