Barrett Ashley R, Kang Yun, Inamasu Ken S, Son Mike S, Vukovich Joseph M, Hoang Tung T
Department of Microbiology, College of Natural Sciences, University of Hawaii at Manoa, Honolulu, HI 96822, USA.
Appl Environ Microbiol. 2008 Jul;74(14):4498-508. doi: 10.1128/AEM.00531-08. Epub 2008 May 23.
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.
由于缺乏合适的反选择和选择标记，伯克霍尔德菌属中的等位基因替换一直存在问题。革兰氏阴性菌中常用的反选择标记sacB，在许多伯克霍尔德菌属物种中对蔗糖无选择性。此外，在美国，对于两种潜在的生物恐怖主义病原体——鼻疽伯克霍尔德菌和类鼻疽伯克霍尔德菌，禁止使用具有临床重要性的抗生素抗性标记来选择理想的遗传性状。在此，我们基于类鼻疽伯克霍尔德菌苯丙氨酰tRNA合成酶（PheS，苯丙氨酸的α亚基）蛋白构建了一个突变的反选择标记，并在三种不同的伯克霍尔德菌属物种中测试了其有效性。在存在0.1%对氯苯丙氨酸的情况下，突变的PheS蛋白能有效杀死100%的细菌。除了构建基于亚碲酸盐（Tel(r)）和甲氧苄啶（Tp(r)）抗性的可通过侧翼独特的FLP重组靶点（FRT）序列切除的选择标记外，我们还将突变的pheS组装到了几个等位基因替换载体上。作为概念验证，我们利用其中一个基因替换载体（pBAKA）和Tel(r)-FRT盒在泰国伯克霍尔德菌的betBA操纵子中产生了一个染色体突变，该操纵子编码甜菜碱醛脱氢酶和胆碱脱氢酶。通过引入pFLP-AB5（Tp(r)）可切除染色体抗性标记，pFLP-AB5是构建的两个含flp质粒之一，另一个是pFLP-AB4（Tel(r)）。这些含flp质粒携带突变的pheS基因，并在Flp-FRT切除后在含有对氯苯丙氨酸的培养基上实现自我消除。对ΔbetBA::Tel(r)-FRT和ΔbetBA::FRT突变体的表征表明，以胆碱作为唯一碳源时生长存在缺陷，而这些突变体以琥珀酸盐和葡萄糖作为替代碳源时生长情况与野生型一样好。
