Whitaker Neal, Berry Trista M, Rosenthal Nathan, Gordon Jay E, Gonzalez-Rivera Christian, Sheehan Kathy B, Truchan Hilary K, VieBrock Lauren, Newton Irene L G, Carlyon Jason A, Christie Peter J
Department of Microbiology and Molecular Genetics, McGovern Medical School, Houston, Texas, USA.
Department of Biology, Indiana University, Bloomington, Indiana, USA.
J Bacteriol. 2016 Sep 9;198(19):2701-18. doi: 10.1128/JB.00378-16. Print 2016 Oct 1.
Bacterial type IV secretion systems (T4SSs) are composed of two major subfamilies, conjugation machines dedicated to DNA transfer and effector translocators for protein transfer. We show here that the Escherichia coli pKM101-encoded conjugation system, coupled with chimeric substrate receptors, can be repurposed for transfer of heterologous effector proteins. The chimeric receptors were composed of the N-terminal transmembrane domain of pKM101-encoded TraJ fused to soluble domains of VirD4 homologs functioning in Agrobacterium tumefaciens, Anaplasma phagocytophilum, or Wolbachia pipientis A chimeric receptor assembled from A. tumefaciens VirD4 (VirD4At) mediated transfer of a MOBQ plasmid (pML122) and A. tumefaciens effector proteins (VirE2, VirE3, and VirF) through the pKM101 transfer channel. Equivalent chimeric receptors assembled from the rickettsial VirD4 homologs similarly supported the transfer of known or candidate effectors from rickettsial species. These findings establish a proof of principle for use of the dedicated pKM101 conjugation channel, coupled with chimeric substrate receptors, to screen for translocation competency of protein effectors from recalcitrant species. Many T4SS receptors carry sequence-variable C-terminal domains (CTDs) with unknown function. While VirD4At and the TraJ/VirD4At chimera with their CTDs deleted supported pML122 transfer at wild-type levels, ΔCTD variants supported transfer of protein substrates at strongly diminished or elevated levels. We were unable to detect binding of VirD4At's CTD to the VirE2 effector, although other VirD4At domains bound this substrate in vitro We propose that CTDs evolved to govern the dynamics of substrate presentation to the T4SS either through transient substrate contacts or by controlling substrate access to other receptor domains.
Bacterial type IV secretion systems (T4SSs) display striking versatility in their capacity to translocate DNA and protein substrates to prokaryotic and eukaryotic target cells. A hexameric ATPase, the type IV coupling protein (T4CP), functions as a substrate receptor for nearly all T4SSs. Here, we report that chimeric T4CPs mediate transfer of effector proteins through the Escherichia coli pKM101-encoded conjugation system. Studies with these repurposed conjugation systems established a role for acidic C-terminal domains of T4CPs in regulating substrate translocation. Our findings advance a mechanistic understanding of T4CP receptor activity and, further, support a model in which T4SS channels function as passive conduits for any DNA or protein substrates that successfully engage with and pass through the T4CP specificity checkpoint.
细菌IV型分泌系统（T4SSs）由两个主要亚家族组成，即负责DNA转移的接合机器和用于蛋白质转移的效应子转运体。我们在此表明，大肠杆菌pKM101编码的接合系统与嵌合底物受体相结合，可用于转移异源效应蛋白。嵌合受体由pKM101编码的TraJ的N端跨膜结构域与在根癌土壤杆菌、嗜吞噬细胞无形体或嗜皮菌属中起作用的VirD4同源物的可溶性结构域融合而成。由根癌土壤杆菌VirD4（VirD4At）组装的嵌合受体通过pKM101转移通道介导了MOBQ质粒（pML122）和根癌土壤杆菌效应蛋白（VirE2、VirE3和VirF）的转移。由立克次氏体VirD4同源物组装的等效嵌合受体同样支持立克次氏体物种中已知或候选效应子的转移。这些发现为使用专用的pKM101接合通道与嵌合底物受体来筛选顽固物种中蛋白质效应子的转运能力建立了原理证明。许多T4SS受体携带功能未知的序列可变C端结构域（CTD）。虽然删除了CTD的VirD4At和TraJ/VirD4At嵌合体在野生型水平上支持pML122转移，但ΔCTD变体在蛋白质底物转移水平上显著降低或升高。尽管VirD4At的其他结构域在体外与该底物结合，但我们无法检测到VirD4At的CTD与VirE2效应子的结合。我们提出，CTD通过短暂的底物接触或通过控制底物进入其他受体结构域，进化为控制底物向T4SS呈现的动力学。
细菌IV型分泌系统（T4SSs）在将DNA和蛋白质底物转运到原核和真核靶细胞的能力方面表现出惊人的多功能性。一种六聚体ATP酶，即IV型偶联蛋白（T4CP），几乎作为所有T4SSs的底物受体发挥作用。在此，我们报告嵌合T4CPs通过大肠杆菌pKM101编码的接合系统介导效应蛋白的转移。对这些重新利用的接合系统的研究确定了T4CPs酸性C端结构域在调节底物转运中的作用。我们的发现推进了对T4CP受体活性的机制理解，并且进一步支持了一种模型，即T4SS通道作为任何成功与T4CP特异性检查点结合并通过的DNA或蛋白质底物的被动管道发挥作用。
