School of Biotechnology, Gautam Buddha University, Yamuna Expressway, Greater Noida, Uttar Pradesh, India.
Appl Microbiol Biotechnol. 2020 Apr;104(7):3081-3095. doi: 10.1007/s00253-020-10441-1. Epub 2020 Feb 11.
Here, for the first time, we have investigated the hipBA toxin-antitoxin (TA) module from entomopathogenic bacterium Xenorhabdus nematophila. It is a type II TA module that consists of HipA toxin and HipB antitoxin protein and located in the complementary strand of chromosome under XNC1_operon 0810 locus tag. For functional analysis, hipA toxin, hipB antitoxin, and an operon having both genes were cloned in pBAD/His C vector and transformed in Escherichia coli cells. The expression profiles and endogenous toxicity assay were performed in these cells. To determine the active amino acid residues responsible for the toxicity of HipA toxin, site-directed mutagenesis (SDM) was performed. SDM results showed that amino acid residues S149, D306, and D329 in HipA toxin protein were significantly essential for its toxicity. For transcriptional analysis, the 157 bp upstream region of the hipBA TA module was identified as a promoter with bioinformatics tools. Further, the LacZ reporter construct with promoter region was prepared and LacZ assays as well as reverse transcriptase-polymerase chain reaction (RT-PCR) analysis was performed under different stress conditions. Electrophoretic mobility shift assay (EMSA) was also performed with recombinant HipA toxin, HipB antitoxin protein, and 157 bp promoter region. Results showed that the hipBA TA module is a well-regulated system in which the upregulation of gene expression was also found compulsive in different SOS conditions. KEY POINTS: •Functional characterization of hipBA TA module from Xenorhabdus nematophila. •hipBA TA module is a functional type II TA module. •Transcriptional characterization of hipBA TA module. •hipBA TA module is a well regulated TA module. Graphical abstract.
这里，我们首次研究了来自昆虫病原细菌 Xenorhabdus nematophila 的 hipBA 毒素-抗毒素（TA）模块。它是一种 II 型 TA 模块，由 HipA 毒素和 HipB 抗毒素蛋白组成，位于染色体的互补链上，位于 XNC1_operon0810 基因座标签下。为了进行功能分析，将 hipA 毒素、hipB 抗毒素和包含这两个基因的操纵子克隆到 pBAD/HisC 载体中，并转化到大肠杆菌细胞中。在这些细胞中进行了表达谱和内源性毒性测定。为了确定负责 HipA 毒素毒性的活性氨基酸残基，进行了定点突变（SDM）。SDM 结果表明，HipA 毒素蛋白中的氨基酸残基 S149、D306 和 D329 对其毒性至关重要。为了进行转录分析，使用生物信息学工具鉴定了 hipBA TA 模块的 157bp 上游区域作为启动子。进一步制备了带有启动子区域的 LacZ 报告基因构建体，并在不同应激条件下进行了 LacZ 测定以及逆转录-聚合酶链反应（RT-PCR）分析。还进行了电泳迁移率变动分析（EMSA），使用重组 HipA 毒素、HipB 抗毒素蛋白和 157bp 启动子区域。结果表明，hipBA TA 模块是一个受调控的系统，在不同的 SOS 条件下也发现了基因表达的上调。关键点：• Xenorhabdus nematophila 中 hipBA TA 模块的功能表征。•hipBA TA 模块是一种功能性的 II 型 TA 模块。•hipBA TA 模块的转录特征。•hipBA TA 模块是一个受调控的 TA 模块。
