Engineering of Tellurite-Resistant Genetic Tools for Single-Copy Chromosomal Analysis of Burkholderia spp. and Characterization of the Burkholderia thailandensis betBA Operon
Engineering of Tellurite-Resistant Genetic Tools for Single-Copy Chromosomal Analysis of Burkholderia spp. and Characterization of the Burkholderia thailandensis betBA Operon
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DOI：
10.1128/aem.02733-08
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发表时间：
2009-06-15
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：
Kang, Yun;Norris, Michael H.;Hoang, Tung T.
对于大多数伯克霍尔德氏菌属物种来说，几乎没有合适的单拷贝遗传工具，并且该属的高水平抗生素耐药性使遗传工具的开发进一步复杂化。此外，对于生物恐怖主义制剂类鼻疽伯克霍尔德氏菌和鼻疽伯克霍尔德氏菌，禁止使用临床重要抗生素的抗性基因，因此需要开发额外的非抗生素遗传工具。开发了三个基于两个非抗生素选择标记、亚碲酸盐抗性 (Tel(r)) 和大肠杆菌天冬氨酸半醛脱氢酶 (asd(Ec)) 的缺乏抗生素选择的单拷贝系统，以促进伯克霍尔德杆菌物种的遗传操作。这些系统包括一个 mariner 转座子、一个 mini-Tn7 衍生的位点特异性转座子和六个基于 lacZ、gfp 和 luxCDABE 报告基因的 FRT 报告融合载体。最初，我们证明随机水手转座子 pBT20-Delta bla-Tel(r)-FRT 在新洋葱伯克霍尔德杆菌、泰国伯克霍尔德杆菌、伪鼻疽伯克霍尔德杆菌和鼻疽伯克霍尔德杆菌中有效转座。然后，我们利用基于 mini-Tn7-Tel(r) 的转座子载体 (mini-Tn7-Tel(r)-betBA) 和含有转座酶的辅助质粒 (pTNS3-asd(Ec)) 来补充泰国芽孢杆菌 Delta betBA 突变。接下来，FRT-lacZ 融合载体之一 (pFRT1-lacZ-Tel(r)) 通过 Flp（在辅助质粒 pCD13SK-Flp-oriT-asd(Ec) 上编码）进行整合，构建 B. thailandensis Delta betBA::FRT-lacZ-Tel(r) 报告融合菌株。通过对 B. thailandensis Delta betBA::FRT-lacZ-Tel(r) 融合菌株进行 β-半乳糖苷酶测定，显示 betBA 操纵子在胆碱存在和渗透胁迫条件下被诱导。最后，我们分别利用融合载体 pFRT1-gfp-Tel(r) 和 pFRT1-lux-Tel(r) 工程化了 Delta betBA::FRT-gfp-Tel(r) 和 Delta betBA::FRT-lux-Tel(r) 融合菌株。通过荧光显微镜和生物发光成像分析证实了胆碱和渗透压对 betBA 操纵子的诱导。
There are few appropriate single-copy genetic tools for most Burkholderia species, and the high level of antibiotic resistance in this genus further complicates the development of genetic tools. In addition, the utilization of resistance genes for clinically important antibiotics is prohibited for the bioterrorism agents Burkholderia pseudomallei and Burkholderia mallei, necessitating the development of additional nonantibiotic-based genetic tools. Three single-copy systems devoid of antibiotic selection based on two nonantibiotic selectable markers, tellurite resistance (Tel(r)) and Escherichia coli aspartate-semialdehyde dehydrogenase (asd(Ec)), were developed to facilitate genetic manipulation in Burkholderia species. These systems include one mariner transposon, a mini-Tn7-derived site-specific transposon, and six FRT reporter fusion vectors based on the lacZ, gfp, and luxCDABE reporter genes. Initially, we showed that the random mariner transposon pBT20-Delta bla-Tel(r)-FRT efficiently transposed within Burkholderia cenocepacia, Burkholderia thailandensis, B. pseudomallei, and B. mallei. We then utilized the mini-Tn7-Tel(r)-based transposon vector (mini-Tn7-Tel(r)-betBA) and a transposase-containing helper plasmid (pTNS3-asd(Ec)) to complement the B. thailandensis Delta betBA mutation. Next, one of the FRT-lacZ fusion vectors (pFRT1-lacZ-Tel(r)) was integrated by Flp (encoded on a helper plasmid, pCD13SK-Flp-oriT-asd(Ec)) to construct the B. thailandensis Delta betBA::FRT-lacZ-Tel(r) reporter fusion strain. The betBA operon was shown to be induced in the presence of choline and under osmotic stress conditions by performing beta-galactosidase assays on the B. thailandensis Delta betBA::FRT-lacZ-Tel(r) fusion strain. Finally, we engineered B. thailandensis Delta betBA::FRT-gfp-Tel(r) and Delta betBA::FRT-lux-Tel(r) fusion strains by utilizing fusion vectors pFRT1-gfp-Tel(r) and pFRT1-lux-Tel(r), respectively. The induction of the betBA operon by choline and osmotic stress was confirmed by performing fluorescent microscopy and bioluminescent imaging analyses.