An efficient system for the generation of marked genetic mutants in members of the genus Burkholderia.
An efficient system for the generation of marked genetic mutants in members of the genus Burkholderia.
复制标题
DOI：
10.1016/j.plasmid.2016.11.002
复制
发表时间：
2017-01
期刊：
影响因子：
2.6
通讯作者：
Thomas MS
中科院分区：
文献类型：
--
作者：
Shastri S;Spiewak HL;Sofoluwe A;Eidsvaag VA;Asghar AH;Pereira T;Bull EH;Butt AT;Thomas MS
为了阐明细菌中基因的功能，能够实现靶向基因失活(等位基因替换)是至关重要的。等位基因替换通常是通过插入抗生素抗性标记来破坏目标基因，然后将突变的等位基因转移到宿主生物体的基因组中来代替野生型基因。然而，由于伯克霍尔德氏菌对许多抗生素的内在耐药性，只有选定的抗生素耐药性标记可以用于伯克霍尔德氏菌属的成员，包括洋葱伯克霍尔德氏菌复合体(BCC)。在这里，我们描述了改进的抗药性试剂盒的构建，该试剂盒可以有效地在基底细胞癌和相关物种中指定对卡那霉素、氯霉素或甲氧普林的耐药性。然后将其与一系列增强型自杀载体pSHAFT2、pSHAFT3和pSHAFT-GFP结合使用和/或构建一系列增强型自杀载体，以促进BCC中有效的等位基因替换。这些改进的自杀载体的有效性被证明是通过在体细胞癌物种Burkholderia cenocepacia和B.lata以及在非体细胞体细胞肿瘤物种B.thailandensis中选择的基因的遗传失活来证明的。我们已经构建了耐药盒和自杀载体，用于伯克霍尔德氏菌和相关物种。这些载体通过用抗生素耐药标记破坏基因来促进突变体的构建。我们已经验证了这些载体在伯克霍尔德氏菌属成员中用于标记基因失活的实用性。
To elucidate the function of a gene in bacteria it is vital that targeted gene inactivation (allelic replacement) can be achieved. Allelic replacement is often carried out by disruption of the gene of interest by insertion of an antibiotic-resistance marker followed by subsequent transfer of the mutant allele to the genome of the host organism in place of the wild-type gene. However, due to their intrinsic resistance to many antibiotics only selected antibiotic-resistance markers can be used in members of the genus Burkholderia, including the Burkholderia cepacia complex (Bcc). Here we describe the construction of improved antibiotic-resistance cassettes that specify resistance to kanamycin, chloramphenicol or trimethoprim effectively in the Bcc and related species. These were then used in combination with and/or to construct a series enhanced suicide vectors, pSHAFT2, pSHAFT3 and pSHAFT-GFP to facilitate effective allelic replacement in the Bcc. Validation of these improved suicide vectors was demonstrated by the genetic inactivation of selected genes in the Bcc species Burkholderia cenocepacia and B. lata, and in the non-Bcc species, B. thailandensis. We have constructed antibiotic-resistance cassettes and suicide vectors for use in Burkholderia and related species. These vectors facilitate construction of mutants by gene disruption with antibiotic-resistance markers. We have validated the utility of the vectors for marked genetic inactivation in members of the genus Burkholderia.