Genetic tools for allelic replacement in Burkholderia species
Genetic tools for allelic replacement in Burkholderia species
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DOI：
10.1128/aem.00531-08
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发表时间：
2008-07-01
影响因子：
4.4
通讯作者：
Hoang, Tung T.
中科院分区：
文献类型：
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作者：
Barrett, Ashley R.;Kang, Yun;Hoang, Tung T.
由于缺乏适当的反选择性和选择性标记，伯克霍尔德氏菌属中的等位基因置换一直存在问题。通常用于革兰氏阴性菌的反向选择标记sacB在许多伯克霍尔德氏菌属物种中对蔗糖无选择性。此外，美国禁止使用具有临床重要性的抗生素耐药性标记来选择两种潜在的生物恐怖主义制剂，即鼻疽伯克霍尔德氏菌和类鼻疽伯克霍尔德氏菌。在此，我们基于B设计了突变的反向选择标记。pseudomallei PheS（苯丙氨酰tRNA合成酶的α-亚基）蛋白，并在三种不同的伯克霍尔德氏菌属物种中测试其有效性。突变体PheS蛋白在0.1%对氯苯丙氨酸存在下有效地杀死100%的细菌。除了构建基于亚碲酸盐（Tel（r））和甲氧苄啶（Tp（r））抗性的可选择标记之外，我们还将突变体pheS组装在几种等位基因置换载体上，所述可选择标记可通过侧翼独特的FLP重组靶（FRT）序列切除。作为概念证明，我们利用这些基因置换载体之一（pBAKA）和Tel（r）-FRT盒在编码甜菜碱醛脱氢酶和胆碱脱氢酶的伯克霍尔德氏菌泰国亚种betBA操纵子中产生染色体突变。通过引入pFLP-AB 5（Tp（r））可以切除染色体抗性标记，pFLP-AB 5（Tp（r））是两种构建的含flp的质粒pFLP-AB 4（Tel（r））和pFLP-AB 5（Tp（r））之一。这些含有flp的质粒携带突变体pheS基因，并允许在Flp-FRT切除后在含有对氯苯丙氨酸的培养基上自固化。Delta betBA：：Tel（r）-FRT和Delta betBA：：FRT突变体的表征表明，以胆碱作为唯一碳源时生长缺陷，而这些突变体以琥珀酸和葡萄糖作为替代碳源时生长良好。
Allelic replacement in the Burkholderia genus has been problematic due to the lack of appropriate counter-selectable and selectable markers. The counter-selectable marker sacB, commonly used in gram-negative bacteria, is nonselective on sucrose in many Burkholderia species. In addition, the use of antibiotic resistance markers of clinical importance for the selection of desirable genetic traits is prohibited in the United States for two potential bioterrorism agents, Burkholderia mallei and Burkholderia pseudomallei. Here, we engineered a mutated counter-selectable marker based on the B. pseudomallei PheS (the alpha-subunit of phenylalanyl tRNA synthase) protein and tested its effectiveness in three different Burkholderia species. The mutant PheS protein effectively killed 100% of the bacteria in the presence of 0.1% p-chlorophenylalanine. We assembled the mutant pheS on several allelic replacement vectors, in addition to constructing selectable markers based on tellurite (Tel(r)) and trimethoprim (Tp(r)) resistance that are excisable by flanking unique FLP recombination target (FRT) sequences. As a proof of concept, we utilized one of these gene replacement vectors (pBAKA) and the Tel(r)-FRT cassette to produce a chromosomal mutation in the Burkholderia thailandensis betBA operon, which codes for betaine aldehyde dehydrogenase and choline dehydrogenase. Chromosomal resistance markers could be excised by the introduction of pFLP-AB5 (Tp(r)), which is one of two constructed flp-containing plasmids, pFLP-AB4 (Tel(r)) and pFLP-AB5 (Tp(r)). These flp-containing plasmids harbor the mutant pheS gene and allow self curing on media that contain p-chlorophenylalanine after Flp-FRT excision. The characterization of the Delta betBA::Tel(r)-FRT and Delta betBA::FRT mutants indicated a defect in growth with choline as a sole carbon source, while these mutants grew as well as the wild type with succinate and glucose as alternative carbon sources.