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Two Systems for Targeted Gene Deletion in Coxiella burnetii

机译：伯氏柯氏杆菌靶向基因缺失的两个系统

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Coxiella burnetii is a ubiquitous zoonotic bacterial pathogen and the cause of human acute Q fever, a disabling influenza-like illness. C. burnetii 's former obligate intracellular nature significantly impeded the genetic characterization of putative virulence factors. However, recent host cell-free (axenic) growth of the organism has enabled development of shuttle vector, transposon, and inducible gene expression technologies, with targeted gene inactivation remaining an important challenge. In the present study, we describe two methods for generating targeted gene deletions in C. burnetii that exploit pUC/ColE1 ori -based suicide plasmids encoding sacB for positive selection of mutants. As proof of concept, C. burnetii dotA and dotB , encoding structural components of the type IVB secretion system (T4BSS), were selected for deletion. The first method exploited Cre- lox- mediated recombination. Two suicide plasmids carrying different antibiotic resistance markers and a loxP site were integrated into 5′ and 3′ flanking regions of dotA . Transformation of this strain with a third suicide plasmid encoding Cre recombinase resulted in the deletion of dotA under sucrose counterselection. The second method utilized a loop-in/loop-out strategy to delete dotA and dotB. A single suicide plasmid was first integrated into 5′ or 3′ target gene flanking regions. Resolution of the plasmid cointegrant by a second crossover event under sucrose counterselection resulted in gene deletion that was confirmed by PCR and Southern blot. Δ dotA and Δ dotB mutants failed to secrete T4BSS substrates and to productively infect host cells. The repertoire of C. burnetii genetic tools now allows ready fulfillment of molecular Koch's postulates for suspected virulence genes.
机译：伯氏柯氏杆菌是一种普遍存在的人畜共患细菌病原体，是人类急性Q发热（一种致残的流感样疾病）的病因。伯氏梭菌的专性细胞内特性显着阻碍了假定毒力因子的遗传特征。然而，近来该生物的无宿主细胞生长（树突状）使穿梭载体，转座子和诱导型基因表达技术得以发展，而靶向基因失活仍然是一个重要的挑战。在本研究中，我们描述了两种在伯氏梭菌中产生靶向基因缺失的方法，这些方法利用了编码sacB的基于pUC / ColE1 ori的自杀质粒来积极选择突变体。作为概念上的证明，选择了编码IVB分泌系统（T4BSS）的结构成分的伯氏梭菌dotA和dotB进行删除。第一种方法利用了Crelox介导的重组。将两个带有不同抗生素抗性标记和一个loxP位点的自杀质粒整合到dotA的5'和3'侧翼区域。用编码Cre重组酶的第三种自杀质粒转化该菌株导致蔗糖反选择下dotA的缺失。第二种方法利用循环/循环策略删除dotA和dotB。首先将单个自杀质粒整合到5'或3'靶基因侧翼区域中。在蔗糖反选择下通过第二次交换事件对质粒共积分的解析导致基因缺失，该缺失通过PCR和Southern印迹证实。 ΔdotA和ΔdotB突变体无法分泌T4BSS底物并无法有效感染宿主细胞。伯氏梭状芽胞杆菌遗传工具的全部资料现在可以使可疑毒力基因的分子Koch假设立即得到履行。

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