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首页> 外文期刊>Plasmid: An International Journal Devoted to Extrachromosomal Gene Systems >Conjugative type IV secretion in Gram-positive pathogens: TraG, a lytic transglycosylase and endopeptidase, interacts with translocation channel protein TraM
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Conjugative type IV secretion in Gram-positive pathogens: TraG, a lytic transglycosylase and endopeptidase, interacts with translocation channel protein TraM

机译：克阳性病原体中的共轭IV分泌物：曲折，裂解血糖糖基酶和内肽酶，与易位信道蛋白电车相互作用

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摘要

Conjugative transfer plays a major role in the transmission of antibiotic resistance in bacteria. pIP501 is a Grampositive conjugative model plasmid with the broadest transfer host-range known so far and is frequently found in Enterococcus faecalis and Enterococcus faecium clinical isolates. The pIP501 type IV secretion system is encoded by 15 transfer genes. In this work, we focus on the VirB1-like protein TraG, a modular peptidoglycan metabolizing enzyme, and the VirB8-homolog TraM, a potential member of the translocation channel. By providing full-length traG in trans, but not with a truncated variant, we achieved full recovery of wild type transfer efficiency in the traG-knockout mutant E. faecalis pIP501AtraG. With peptidoglycan digestion experiments and tandem mass spectrometry we could assign lytic transglycosylase and endopeptidase activity to TraG, with the CHAP domain alone displaying endopeptidase activity. We identified a novel interaction between TraG and TraM in a bacterial 2-hybrid assay. In addition we found that both proteins localize in focal spots at the E. faecalis cell membrane using immunostaining and fluorescence microscopy. Extracellular protease digestion to evaluate protein cell surface exposure revealed that correct membrane localization of TraM requires the transmembrane helix of TraG. Thus, we suggest an essential role for TraG in the assembly of the pIP501 type IV secretion system. (C) 2017 Elsevier Inc. All rights reserved.
机译：共轭转移在细菌中的抗生素抗性传播中起主要作用。 PIP501是迄今已知最广泛的转移宿主范围的研制缀合性模型质粒，并且经常在肠球菌粪便和肠球菌疾病临床分离株中发现。 PIP501型IV型分泌系统由15种转移基因编码。在这项工作中，我们专注于virb1样蛋白质曲折，模块化肽聚糖代谢酶，以及易位通道的电位构件。通过在Trans中提供全长曲折，但不是截断变体，我们在曲折敲除突变体E. FAECALIS PIP501ATRAG中完全恢复了野生型转移效率。用肽聚糖消化实验和串联质谱，我们可以将裂解的转糖基酶和内肽酶活性分配给曲调，单独使用章节域显示内肽酶活性。我们在细菌2杂交测定中鉴定了曲折和电车之间的新互动。此外，我们发现，两种蛋白质使用免疫染色和荧光显微镜的E.粪群细胞膜的焦斑定位。细胞外蛋白酶消化以评估蛋白质细胞表面暴露揭示了电车的正确膜定位需要扭曲的跨膜螺旋。因此，我们向PIP501型分泌系统组装中的组装中提出了一种基本作用。 （c）2017年Elsevier Inc.保留所有权利。

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