BACKGROUND Conjugative spread of antibiotic resistance and virulence genes in bacteria constitutes an important threat to public health. Beyond the well-known conjugative plasmids, recent genome analyses have shown that integrative and conjugative elements (ICEs) are the most widespread conjugative elements, even if their transfer mechanism has been little studied until now. The initiator of conjugation is the relaxase, a protein catalyzing a site-specific nick on the origin of transfer (oriT) of the ICE. Besides canonical relaxases, recent studies revealed non-canonical ones, such as relaxases of the MOBT family that are related to rolling-circle replication proteins of the Rep_trans family. MOBT relaxases are encoded by ICEs of the ICESt3/ICEBs1/Tn916 superfamily, a superfamily widespread in Firmicutes, and frequently conferring antibiotic resistance. RESULTS Here, we present the first biochemical and structural characterization of a MOBT relaxase: the RelSt3 relaxase encoded by ICESt3 from Streptococcus thermophilus. We identified the oriT region of ICESt3 and demonstrated that RelSt3 is required for its conjugative transfer. The purified RelSt3 protein is a stable dimer that provides a Mn2+-dependent single-stranded endonuclease activity. Sequence comparisons of MOBT relaxases led to the identification of MOBT conserved motifs. These motifs, together with the construction of a 3D model of the relaxase domain of RelSt3, allowed us to determine conserved residues of the RelSt3 active site. The involvement of these residues in DNA nicking activity was demonstrated by targeted mutagenesis. CONCLUSIONS All together, this work argues in favor of MOBT being a full family of non-canonical relaxases. The biochemical and structural characterization of a MOBT member provides new insights on the molecular mechanism of conjugative transfer mediated by ICEs in Gram-positive bacteria. This could be a first step towards conceiving rational strategies to control gene transfer in these bacteria.

中文翻译：

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MOBT 家族松弛酶的表征，MOBT 家族是厚壁菌门中广泛分布的家族，介导 ICE 转移。


背景技术细菌中抗生素抗性和毒力基因的结合传播对公众健康构成重要威胁。除了众所周知的接合质粒之外，最近的基因组分析表明，整合和接合元件（ICE）是最广泛的接合元件，即使迄今为止其转移机制还很少被研究。缀合的引发剂是松弛酶，这是一种催化 ICE 转移起点 (oriT) 上的位点特异性切口的蛋白质。除了典型的松弛酶之外，最近的研究还揭示了非典型的松弛酶，例如与 Rep_trans 家族的滚环复制蛋白相关的 MOBT 家族的松弛酶。 MOBT 松弛酶由 ICESt3/ICEBs1/Tn916 超家族的 ICE 编码，该超家族广泛存在于厚壁菌门中，并且经常赋予抗生素耐药性。结果在这里，我们首次展示了 MOBT 松弛酶的生化和结构特征：由嗜热链球菌的 ICEST3 编码的 RelSt3 松弛酶。我们确定了 ICESt3 的 oriT 区域，并证明了 RelSt3 是其共轭转移所必需的。纯化的 RelSt3 蛋白是稳定的二聚体，提供 Mn2+ 依赖性单链核酸内切酶活性。 MOBT 松弛酶的序列比较导致了 MOBT 保守基序的鉴定。这些基序与 RelSt3 松弛酶结构域的 3D 模型一起构建，使我们能够确定 RelSt3 活性位点的保守残基。通过定向诱变证明了这些残基参与 DNA 切口活性。结论 总而言之，这项工作主张 MOBT 是一个完整的非规范松弛酶家族。 MOBT 成员的生化和结构表征为革兰氏阳性菌中 ICE 介导的接合转移的分子机制提供了新的见解。这可能是构想控制这些细菌基因转移的合理策略的第一步。