An allelic exchange system for compliant genetic manipulation of the select agents Burkholderia pseudomallei and Burkholderia mallei.
An allelic exchange system for compliant genetic manipulation of the select agents Burkholderia pseudomallei and Burkholderia mallei.
复制标题
用于选择特工Burkholderia pseudomallei和Burkholderia Mallei的符合遗传操作的等位基因交换系统。
DOI：
10.1016/j.gene.2008.10.011
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发表时间：
2009-02-01
期刊：
影响因子：
3.5
通讯作者：
Voskuil, Martin I.
中科院分区：
文献类型：
--
作者：
Hamad, Mohamad A.;Zajdowicz, Sheryl L.;Holmes, Randall K.;Voskuil, Martin I.
类鼻疽伯克霍尔德菌和B.鼻疽是革兰氏阴性细菌病原体，分别引起人类的类鼻疽病和马的鼻疽。这两种细菌在美国都被归类为B类选择剂。由于严格的选择剂法规，批准用于这些细菌的抗生素选择标记的数量非常有限。B的批准标记。类鼻疽包括编码对卡那霉素（Km）、庆大霉素（Gm）和博来霉素（Zeo）的抗性的基因;然而，野生型B。鼻疽假单胞菌对这些抗生素具有内在的耐药性。B的选择标记。鼻疽的抗性基因仅限于Km和Zeo。此外，很少有开发良好的反选择标记用于伯克霍尔德氏菌。由于在B的基因组中存在内源sacBC基因，所以使用SacB作为反选择方法的成功有限。类鼻疽和B.鼻疽这些障碍极大地阻碍了对B的遗传操作。类鼻疽和B.鼻疽和目前存在几个用于伯克霍尔德氏菌遗传操作的可靠工具。为了扩大用于伯克霍尔德氏菌的遗传工具库，我们开发了自杀质粒pMo 130，其允许选择剂B的顺应性遗传操作。类鼻疽和B.鼻疽使用等位基因交换。pMo 130含有允许Km选择的aphA基因、允许伯克霍尔德氏菌转化体的可靠视觉检测的报告基因xylE，并且携带允许共整合体解析的修饰的sacB基因。我们采用该系统在B中产生多个未标记的和符合读码框的突变体。类鼻疽，B中有一个突变体。鼻疽该载体显著扩大了可用于B的遗传操作的选择剂兼容的可用工具的数量。类鼻疽和B.鼻疽
Burkholderia pseudomallei and B. mallei are Gram-negative bacterial pathogens that cause melioidosis in humans and glanders in horses, respectively. Both bacteria are classified as category B select agents in the United States. Due to strict select-agent regulations, the number of antibiotic selection markers approved for use in these bacteria is greatly limited. Approved markers for B. pseudomallei include genes encoding resistance to kanamycin (Km), gentamicin (Gm), and zeocin (Zeo); however, wild type B. pseudomallei is intrinsically resistant to these antibiotics. Selection markers for B. mallei are limited to Km and Zeo resistance genes. Additionally, there are few well developed counter-selection markers for use in Burkholderia. The use of SacB as a counter-selection method has been of limited success due to the presence of endogenous sacBC genes in the genomes of B. pseudomallei and B. mallei. These impediments have greatly hampered the genetic manipulation of B. pseudomallei and B. mallei and currently few reliable tools for the genetic manipulation of Burkholderia exist. To expand the repertoire of genetic tools for use in Burkholderia, we developed the suicide plasmid pMo130, which allows for the compliant genetic manipulation of the select agents B. pseudomallei and B. mallei using allelic exchange. pMo130 harbors an aphA gene which allows for Km selection, the reporter gene xylE, which allows for reliable visual detection of Burkholderia transformants, and carries a modified sacB gene that allows for the resolution of co-integrants. We employed this system to generate multiple unmarked and in-frame mutants in B. pseudomallei, and one mutant in B. mallei. This vector significantly expands the number of available tools that are select-agent compliant for the genetic manipulation of B. pseudomallei and B. mallei.
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影响因子：
11.8
作者：
Rolim, DB;Vilar, DCFL;Inglis, TJJ
通讯作者：
Inglis, TJJ
影响因子：
3.2
作者：
DeShazer, D;Brett, PJ;Woods, DE
通讯作者：
Woods, DE
影响因子：
11.8
作者：
Limmathurotsakul, Direk;Chaowagul, Wipada;Peacock, Sharon J.
通讯作者：
Peacock, Sharon J.
影响因子：
3.1
作者：
Essex-Lopresti, AE;Boddey, JA;Titball, RW
通讯作者：
Titball, RW
影响因子：
3.1
作者：
Cuccui, J.;Easton, A.;Wren, B. W.
通讯作者：
Wren, B. W.