Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America.
PLoS Genet. 2013;9(1):e1003198. doi: 10.1371/journal.pgen.1003198. Epub 2013 Jan 10.
Integrative and conjugative elements (ICEs) are agents of horizontal gene transfer and have major roles in evolution and acquisition of new traits, including antibiotic resistances. ICEs are found integrated in a host chromosome and can excise and transfer to recipient bacteria via conjugation. Conjugation involves nicking of the ICE origin of transfer (oriT) by the ICE-encoded relaxase and transfer of the nicked single strand of ICE DNA. For ICEBs1 of Bacillus subtilis, nicking of oriT by the ICEBs1 relaxase NicK also initiates rolling circle replication. This autonomous replication of ICEBs1 is critical for stability of the excised element in growing cells. We found a conserved and previously uncharacterized ICE gene that is required for conjugation and replication of ICEBs1. Our results indicate that this gene, helP (formerly ydcP), encodes a helicase processivity factor that enables the host-encoded helicase PcrA to unwind the double-stranded ICEBs1 DNA. HelP was required for both conjugation and replication of ICEBs1, and HelP and NicK were the only ICEBs1 proteins needed for replication from ICEBs1 oriT. Using chromatin immunoprecipitation, we measured association of HelP, NicK, PcrA, and the host-encoded single-strand DNA binding protein Ssb with ICEBs1. We found that NicK was required for association of HelP and PcrA with ICEBs1 DNA. HelP was required for association of PcrA and Ssb with ICEBs1 regions distal, but not proximal, to oriT, indicating that PcrA needs HelP to progress beyond nicked oriT and unwind ICEBs1. In vitro, HelP directly stimulated the helicase activity of the PcrA homologue UvrD. Our findings demonstrate that HelP is a helicase processivity factor needed for efficient unwinding of ICEBs1 for conjugation and replication. Homologues of HelP and PcrA-type helicases are encoded on many known and putative ICEs. We propose that these factors are essential for ICE conjugation, replication, and genetic stability.
整合子-接合元件（ICEs）是水平基因转移的媒介，在进化和新性状的获得中起着重要作用，包括抗生素抗性。ICEs 整合在宿主染色体中，可以通过接合切除并转移到受体细菌。接合涉及 ICE 编码的松弛酶对 ICE 转移起始位点（oriT）的切口，以及 ICE DNA 单链的转移。对于枯草芽孢杆菌的 ICEBs1，ICEBs1 松弛酶 NicK 对 oriT 的切口也启动了滚环复制。ICEBs1 的这种自主复制对于切除元件在生长细胞中的稳定性至关重要。我们发现了一个保守的、以前未被描述的 ICE 基因，它是 ICEBs1 接合和复制所必需的。我们的结果表明，这个基因，helP（以前称为 ydcP），编码一个解旋酶持续因子，使宿主编码的解旋酶 PcrA 能够解开双链的 ICEBs1 DNA。HelP 对于 ICEBs1 的接合和复制都是必需的，并且 HelP 和 NicK 是从 ICEBs1 oriT 复制 ICEBs1 所需的唯一 ICEBs1 蛋白。通过染色质免疫沉淀，我们测量了 HelP、NicK、PcrA 和宿主编码的单链 DNA 结合蛋白 Ssb 与 ICEBs1 的结合。我们发现 NicK 对于 HelP 和 PcrA 与 ICEBs1 DNA 的结合是必需的。HelP 对于 PcrA 和 Ssb 与 ICEBs1 远离 oriT 但不靠近 oriT 的区域的结合是必需的，这表明 PcrA 需要 HelP 来超越切口 oriT 并解开 ICEBs1。在体外，HelP 直接刺激了 PcrA 同源物 UvrD 的解旋酶活性。我们的发现表明，HelP 是一种解旋酶持续因子，对于 ICEBs1 的接合和复制的有效解旋是必需的。HelP 和 PcrA 型解旋酶的同源物编码在许多已知和假定的 ICEs 上。我们提出这些因素对于 ICE 接合、复制和遗传稳定性是必不可少的。
