Genetic transplantation:: Salmonella enterica serovar typhimurium as a host to study sigma factor and anti-sigma factor interactions in genetically intractable systems
Genetic transplantation:: Salmonella enterica serovar typhimurium as a host to study sigma factor and anti-sigma factor interactions in genetically intractable systems
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DOI：
10.1128/jb.188.1.103-114.2006
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发表时间：
2006-01-01
影响因子：
3.2
通讯作者：
Hughes, KT
中科院分区：
文献类型：
--
作者：
Karlinsey, JE;Hughes, KT
在肠炎沙门氏菌血清型鼠伤寒菌中，sigma（28）和抗sigma因子FlgM是鞭毛生物发生和运动的关键调节蛋白。在本研究中，我们使用肠炎沙门氏菌血清型鼠伤寒杆菌作为体内异源系统，研究了由于特殊生长需求而导致遗传操作面临重大挑战的生物体中的sigma（28）和抗sigma（28）相互作用。利用x - red介导的重组，有针对性地替换fliA基因位置上的一个tetRA元件，将肠沙门氏菌血清型鼠伤寒菌sigma（28）结构基因fliA的染色体拷贝与极端嗜热动物Aquifex aeolicus（一种极端嗜热动物）和沙眼衣原体（一种专一性细胞内病原体）的同源物进行交换。小肠沙门氏菌血清型鼠伤寒菌杂种菌株表现出sigma（28）依赖基因表达，表明不同物种的sigma（28）活性在异源宿主系统中得以保存。在肠炎沙门氏菌血清型鼠伤寒菌中也分离到了sigma(28)/FlgM相互作用缺陷的风单胞菌突变体。这些研究强调了在物种中分析蛋白质功能的一般策略，否则遗传上是难以解决的，以及使用lambda- red介导的重组进行染色体重组的直接方法。
In Salmonella enterica serovar Typhimurium, sigma(28) and anti-sigma factor FlgM are regulatory proteins crucial for flagellar biogenesis and motility. In this study, we used S. enterica serovar Typhimurium as an in vivo heterologous system to study sigma(28) and anti-sigma(28) interactions in organisms where genetic manipulation poses a significant challenge due to special growth requirements. The chromosomal copy of the S. enterica serovar Typhimurium sigma(28) structural gene fliA was exchanged with homologs of Aquifex aeolicus (an extreme thermophile) and Chlamydia trachmatis (an obligate intracellular pathogen) by targeted replacement of a tetRA element in the fliA gene location using X-Red-mediated recombination. The S. enterica serovar Typhimurium hybrid strains showed sigma(28)-dependent gene expression, suggesting that sigma(28) activities from diverse species are preserved in the heterologous host system. A. aeolicus mutants defective for sigma(28)/FlgM interactions were also isolated in S. enterica serovar Typhimurium. These studies highlight a general strategy for analysis of protein function in species that are otherwise genetically intractable and a straightforward method of chromosome restructuring using lambda-Red-mediated recombination.