USER Friendly Cloning Coupled with Chitin-Based Natural Transformation Enables Rapid Mutagenesis of Vibrio vulnificus
USER Friendly Cloning Coupled with Chitin-Based Natural Transformation Enables Rapid Mutagenesis of Vibrio vulnificus
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DOI：
10.1128/aem.02564-08
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发表时间：
2009-08-01
影响因子：
4.4
通讯作者：
Brown, Roslyn N.
中科院分区：
文献类型：
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作者：
Gulig, Paul A.;Tucker, Matthew S.;Brown, Roslyn N.
创伤弧菌是贝类的一种细菌污染物，可引起高度致命的败血症和破坏性伤口感染。由于对这种机会致病菌进行分子遗传学分析的困难，使用科赫氏假设的分子版本对毒力因子进行确定性鉴定受到阻碍。例如，需要接合来引入质粒DNA，并且依赖于蔗糖敏感性进行反选择的等位基因交换自杀载体是无效的。因此，我们将用户友好的克隆技术整合到基于pCVD 442的等位基因交换自杀载体和其他表达载体中，以使PCR扩增子的快速有效捕获成为可能。在单个步骤中将靶向缺失的基因侧翼的上游和下游DNA序列克隆在一起。基于来自霍乱弧菌的结果，我们确定创伤弧菌在蟹壳形式的几丁质上生长期间可自然转化线性DNA。通过将用户友好克隆和基于几丁质的转化相结合，我们快速有效地在创伤弧菌中产生靶向缺失，绕过了两步自杀载体介导的等位基因交换的需要。这些方法被用来检查两个鞭毛蛋白位点（flaCDE和flaFBA），motAB基因，和cheY-3基因在运动中的作用，并创建缺失的rtxC，rtxA 1，和fadR。此外，基于几丁质的转化可用于通过简单地在蟹壳上共培养创伤弧菌菌株来移动抗生素抗性标记的突变。我们开发的方法和遗传工具应该对那些进行分子遗传分析和操纵其他革兰氏阴性菌的人具有普遍意义。
Vibrio vulnificus is a bacterial contaminant of shellfish and causes highly lethal sepsis and destructive wound infections. A definitive identification of virulence factors using the molecular version of Koch's postulates has been hindered because of difficulties in performing molecular genetic analysis of this opportunistic pathogen. For example, conjugation is required to introduce plasmid DNA, and allelic exchange suicide vectors that rely on sucrose sensitivity for counterselection are not efficient. We therefore incorporated USER friendly cloning techniques into pCVD442-based allelic exchange suicide vectors and other expression vectors to enable the rapid and efficient capture of PCR amplicons. Upstream and downstream DNA sequences flanking genes targeted for deletion were cloned together in a single step. Based on results from Vibrio cholerae, we determined that V. vulnificus becomes naturally transformable with linear DNA during growth on chitin in the form of crab shells. By combining USER friendly cloning and chitin-based transformation, we rapidly and efficiently produced targeted deletions in V. vulnificus, bypassing the need for two-step, suicide vector-mediated allelic exchange. These methods were used to examine the roles of two flagellin loci (flaCDE and flaFBA), the motAB genes, and the cheY-3 gene in motility and to create deletions of rtxC, rtxA1, and fadR. Additionally, chitin-based transformation was useful in moving antibiotic resistance-labeled mutations between V. vulnificus strains by simply coculturing the strains on crab shells. The methods and genetic tools that we developed should be of general use to those performing molecular genetic analysis and manipulation of other gram-negative bacteria.