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首页> 外文期刊>Journal of bacteriology >Enterococcus faecalis PrgJ, a VirB4-Like ATPase, Mediates pCF10 Conjugative Transfer through Substrate Binding
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Enterococcus faecalis PrgJ, a VirB4-Like ATPase, Mediates pCF10 Conjugative Transfer through Substrate Binding

机译：粪肠球菌PrgJ，一种类似于VirB4的ATPase，通过底物结合介导pCF10偶联转移。

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The Enterococcus faecalis prg and pcf genes of plasmid pCF10 encode a type IV secretion system (T4SS) required for conjugative transfer. PrgJ is a member of the VirB4 family of ATPases that are universally associated with T4SSs. Here, we report that purified PrgJ dimers displayed ATP binding and hydrolysis activities. A PrgJ nucleoside triphosphate (NTP) binding site mutation (K471E) slightly diminished ATP binding but abolished ATP hydrolysis in vitro and blocked pCF10 transfer in vivo. As shown with affinity pulldown assays, PrgJ and the K471E mutant protein interacted with the substrate receptor PcfC and with relaxase PcfG and accessory factor PcfF, which together form the relaxosome at the oriT sequence to initiate plasmid processing. The purified PrgJ and K471E proteins also bound single- and double-stranded DNA substrates without sequence specificity in vitro, and both PrgJ derivatives bound pCF10 in vivo by a mechanism dependent on an intact oriT sequence and cosynthesis of PcfC, PcfF, and PcfG, as shown by a formaldehyde-cross-linking assay. Our findings support a model in which the PcfC receptor coordinates with the PrgJ ATPase to drive early steps of pCF10 processing/transfer: (i) PcfC first binds the pCF10 relaxosome through contacts with PcfF, PcfG, and DNA; (ii) PcfC delivers the plasmid substrate to PrgJ; and (iii) PrgJ catalyzes substrate transfer to the membrane translocase. Substrate engagement with a VirB4-like subunit has not been previously described; consequently, our studies point to a novel function for these signature T4SS ATPases in mediating early steps of type IV secretion.
机译：质粒pCF10的粪肠球菌prg和 pcf 基因编码共轭转移所需的IV型分泌系统（T4SS）。 PrgJ是与T4SS普遍关联的ATPase VirB4家族的成员。在这里，我们报告纯化的PrgJ二聚体显示ATP结合和水解活性。 PrgJ核苷三磷酸（NTP）结合位点突变（K471E）略微减少了ATP的结合，但取消了体外的ATP水解，并阻止了pCF10体内的 转移。如亲和力下拉试验所示，PrgJ和K471E突变蛋白与底物受体PcfC相互作用，并与松弛酶PcfG和辅助因子PcfF相互作用，后者在 oriT 序列处共同形成松弛体，从而启动质粒加工。纯化的PrgJ和K471E蛋白还结合了单链和双链DNA底物，而在体外没有序列特异性，并且两个PrgJ衍生物都通过依赖于机制的机制在体内结合了pCF10。完整的 oriT 序列和PcfC，PcfF和PcfG的合成，如甲醛交联测定所示。我们的发现支持了一种模型，其中PcfC受体与PrgJ ATPase协同作用以驱动pCF10加工/转移的早期步骤：（i）PcfC首先通过与PcfF，PcfG和DNA接触结合pCF10弛豫体; （ii）PcfC将质粒底物递送至PrgJ; （iii）PrgJ催化底物转移至膜转座酶。以前没有描述过与VirB4样亚基的底物结合。因此，我们的研究指出了这些签名的T4SS ATPase在介导IV型分泌的早期步骤中的新功能。

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