Comprehensive analysis of IncC plasmid conjugation identifies a crucial role for the transcriptional regulator AcaB
Comprehensive analysis of IncC plasmid conjugation identifies a crucial role for the transcriptional regulator AcaB
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DOI：
10.1038/s41564-020-0775-0
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发表时间：
2020-08-17
影响因子：
28.3
通讯作者：
Schembri, Mark A.
中科院分区：
文献类型：
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作者：
Hancock, Steven J.;Minh-Duy Phan;Schembri, Mark A.
多药质粒IncC接合所需的整套基因的鉴定将为防止质粒传播的努力提供信息。广泛宿主范围质粒的IncC家族使抗生素抗性基因能够在人类肠道病原体中传播（1-3）。虽然IncC质粒接合的方面已经被充分研究（4-9），但是接合基因的许多作用已经仅仅基于序列相似性来分配。我们应用过饱和转座子诱变和转座子定向插入位点测序来确定IncC缀合所需的基因组。我们确定了27个接合基因，包括19个先前确定的（包括两个调控基因，acaDC）和8个先前没有与接合。我们发现，一个以前未知的基因，acaB，编码的转录调节因子，具有至关重要的作用，在调节IncC共轭。cDNAB结合在acaDC启动子的上游以增加acaDC的转录;反过来，cDNADC激活IncC结合基因的转录。我们以2.9埃的分辨率解决了BMPB的晶体结构，并利用它来指导揭示BMPB如何与DNA结合的功能分析。这种对IncC结合的更好理解为开发新方法以减少这些多药耐药质粒的传播提供了基础。
Identification of the entire set of genes needed for multi-drug plasmid IncC conjugation will inform efforts to prevent plasmid spread.The IncC family of broad-host-range plasmids enables the spread of antibiotic resistance genes among human enteric pathogens(1-3). Although aspects of IncC plasmid conjugation have been well studied(4-9), many roles of conjugation genes have been assigned based solely on sequence similarity. We applied hypersaturated transposon mutagenesis and transposon-directed insertion-site sequencing to determine the set of genes required for IncC conjugation. We identified 27 conjugation genes, comprising 19 that were previously identified (including two regulatory genes,acaDC) and eight not previously associated with conjugation. We show that one previously unknown gene,acaB, encodes a transcriptional regulator that has a crucial role in the regulation of IncC conjugation. AcaB binds upstream of theacaDCpromoter to increaseacaDCtranscription; in turn, AcaDC activates the transcription of IncC conjugation genes. We solved the crystal structure of AcaB at 2.9-angstrom resolution and used this to guide functional analyses that reveal how AcaB binds to DNA. This improved understanding of IncC conjugation provides a basis for the development of new approaches to reduce the spread of these multi-drug-resistance plasmids.