Development of a host-genotype-independent counterselectable marker and a high-frequency conjugative delivery system and their use in genetic analysis of Enterococcus faecalis
Development of a host-genotype-independent counterselectable marker and a high-frequency conjugative delivery system and their use in genetic analysis of Enterococcus faecalis
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DOI：
10.1016/j.plasmid.2006.08.003
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发表时间：
2007-03-01
期刊：
影响因子：
2.6
通讯作者：
Dunny, Gary M.
中科院分区：
文献类型：
--
作者：
Kristich, Christopher J.;Chandler, Josephine R.;Dunny, Gary M.
粪肠球菌是胃肠道的革兰氏阳性细菌。粪肠球菌也是一种机会致病菌，经常表现出对可用抗生素的耐药性。尽管肠球菌的临床意义，遗传分析一直受到限制固有的限制，在现有的遗传工具。为了促进粪肠球菌的遗传操作，我们开发了一种接合传递系统，用于将克隆的DNA高频引入粪肠球菌的靶菌株和用于无标记遗传交换的宿主基因型独立的反选择标记。我们使用这些工具构建了一系列粪肠球菌突变菌株，这些菌株在几个基因中携带定义的突变，包括cefA、eep、gelE、sprE和替代性sigma因子（sigH）。此外，我们将这些突变以各种排列组合，以产生粪肠球菌的双突变体、三突变体和四突变体，从而能够对信息素生物合成途径进行上位性测试。对突变体产生的cCF 10信息素的分析显示，ccfA 2和Delta eep 10突变对gelE/sprE中的突变具有上位性。据我们所知，这代表了上位性分析应用于肠球菌染色体编码的生物合成途径的第一个例子。因此，先进的工具，遗传操作的粪肠球菌在这里报告，使这些重要的病原体的有效和复杂的遗传分析。(c)2006年爱思唯尔公司All rights reserved.
Enterococcus faecalis is a Gram-positive commensal bacterium of the gastrointestinal tract. E faecalis is also an opportunistic pathogen that frequently exhibits resistance to available antibiotics. Despite the clinical significance of the enterococci, genetic analysis has been restricted by limitations inherent in the available genetic tools. To facilitate genetic manipulation of E faecalis, we developed a conjugative delivery system for high-frequency introduction of cloned DNA into target strains of E faecalis and a host-genotype-independent counterselectable marker for use in markerless genetic exchange. We used these tools to construct a collection of E faecalis mutant strains carrying defined mutations in several genes, including cefA, eep, gelE, sprE, and an alternative sigma factor (sigH). Furthermore, we combined these mutations in various permutations to create double mutants, triple mutants, and a quadruple mutant of E faecalis that enabled tests of epistasis to be conducted on the pheromone biosynthesis pathway. Analysis of cCF10 pheromone production by the mutants revealed that both the ccfA2 and Delta eep10 mutations are epistatic to mutations in gelE/sprE. To our knowledge, this represents the first example of epistasis analysis applied to a chromosomally encoded biosynthetic pathway in enterococci. Thus, the advanced tools for genetic manipulation of E faecalis reported here enable efficient and sophisticated genetic analysis of these important pathogens. (c) 2006 Elsevier Inc. All rights reserved.