Mitigation of Stress Responses By Protein Transfer Through Conjugation Machines

项目总结 接合系统因其在传播可移动遗传元件(MGES)和 在许多种类的细菌中携带抗生素耐药性、适应性和毒力基因。巨大的 与MGE传播的抗生素耐药性相关的医疗问题因以下事实而加剧 结合系统还编码促进细胞间接触的表面粘附素或结合菌毛 有利于传播和建立强大的、对抗生素具有抗药性的生物膜群落。此外， 各种环境压力，包括抗生素，已被证明可以刺激MGE的传播 相反，MGE转移诱导SOS反应并激活整合子的运动，这 是抗生素耐药性基因的储存库。增加了共轭可以影响的无数方式 抗生素耐药性的发展和基因组的进化，我们已经证明了 接合诱导的SOS是受体细胞中突变积累的增加。为了缓解 交配诱导突变的有害后果，这里称为MIM，许多结合元件携带 例如psiB和SSB基因，其产物抑制SOS反应。在我们对模型F质粒的研究中， PED208中，我们发现PsiB和SSB是通过共轭通道传递的，也称为 IV型分泌系统(T4SS)，至受体细胞。此外，移位的psiB和SSB都抑制了 接合子的SOS反应和MIM。SSB和psiB基因由维护/领导携带 F质粒和许多其他结合质粒的区域(MLR)，以及其他几个基因，其 产品预计将促进建立MGE后，通过使用一种 可靠和定量的Cre重组酶易位分析(CRATE)，我们已经证明了pED208 接合转移至少9个MLR编码的蛋白质以及7个染色体编码的蛋白质 预测或已知在DNA代谢和缓解压力中的作用。在目标1中，我们将定义泛函 PsiB、SSB和第三种蛋白PSIA在调节供体SOS反应和MIM中的相互作用 接合过程中的受体细胞。我们还将评估PsiB和SSB的更广泛的生物学作用 通过研究其他模式接合系统以及种内和种间交配，以及通过 评估外源压力对MGE转移和PsiB/SSB缓解应激反应的影响 突变。在目标2中，我们将鉴定和表征其他转位的MLR蛋白的生物学功能。 由模型F质粒pED208编码。我们将定义染色体编码的曲目 转位的蛋白质，并开始探索生物功能的研究，最初专注于验证假说 这种交配会诱导氧化应激反应，而易位的苏打水可以减轻这种应激反应。 这些研究的结果将极大地扩展我们对接合系统如何有助于 细菌通讯、压力调节、抗生素耐药性和基因组进化。

PROJECT SUMMARY Conjugation systems are widely known for their roles in disseminating mobile genetic elements (MGEs) and cargoes of antibiotic resistance, fitness, and virulence genes among many species of bacteria. The enormous medical problem associated with MGE-transmitted antibiotic resistance is enhanced by the fact that conjugation systems also code for surface adhesins or conjugative pili that promote intercellular contacts favoring transmission and establishment of robust, antibiotic-resistant biofilm communities. Furthermore, various environmental stresses, including antibiotics, have been shown to stimulate MGE transmission whereas, conversely, MGE transfer induces the SOS response and activates movement of integrons, which are reservoirs of antibiotic resistance genes. Adding to the myriad of ways conjugation can impact development of antibiotic resistance and genome evolution, we have shown that a consequence of conjugation-induced SOS is the elevated accumulation of mutations in recipient cells. To mitigate the deleterious consequences of mating-induced mutation, here termed MIM, many conjugative elements carry genes such as psiB and ssb whose products suppress the SOS response. In our studies of a model F plasmid, pED208, we discovered that PsiB and SSB are delivered through the conjugation channel, also known as a type IV secretion system (T4SS), to recipient cells. Furthermore, translocated PsiB and SSB suppress both the SOS response and MIM in transconjugants. ssb and psiB genes are carried by the maintenance/leading regions (MLRs) of F plasmids and many other conjugative plasmids, as are several other genes whose products are predicted to promote establishment of the MGE following transfer to recipient cells By use of a robust and quantitative Cre Recombinase Assay for Translocation (CRAfT), we have shown that pED208 conjugatively transfers at least 9 MLR-encoded proteins, as well as 7 chromosomally-encoded proteins with predicted or known functions in DNA metabolism and stress mitigation. In Aim 1, we will define the functional interplay of PsiB, SSB, and a third protein, PsiA, in modulating the SOS response and MIM in donor and recipient cells during conjugation. We will also evaluate the broader biological roles of PsiB and SSB translocation through studies of other model conjugation systems and intra- and interspecies matings, and by assessing effects of exogenous stresses on MGE transfer and PsiB/SSB mitigation of stress responses and mutation. In Aim 2, we will identify and characterize the biological functions of other translocated MLR proteins encoded by the model F plasmid, pED208. We will define the repertoire of chromosomally-encoded translocated proteins, and initiate studies exploring biological functions focusing initially on testing a hypothesis that mating induces the oxidative stress response and translocated SodA mitigates this stress response. Results of these studies will significantly expand our understanding of how conjugation systems contribute to bacterial communication, stress modulation, antibiotic resistance, and genome evolution.