A new simple method for introducing an unmarked mutation into a large gene of non-competent Gram-negative bacteria by FLP/FRT recombination.
A new simple method for introducing an unmarked mutation into a large gene of non-competent Gram-negative bacteria by FLP/FRT recombination.
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DOI：
10.1186/1471-2180-13-86
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发表时间：
2013-04-17
期刊：
影响因子：
4.2
通讯作者：
Hori K
中科院分区：
文献类型：
--
作者：
Ishikawa M;Hori K
对于分子微生物学中靶基因的破坏，无标记诱变优于标记诱变，因为前一种方法不考虑极性效应并且不留下选择标记。与天然感受态细菌相比，没有有用的方法将未标记的突变引入非感受态细菌的大基因中。然而，编码巨大蛋白质的大基因存在于多种细菌中，对于生理学和潜在应用来说是有趣且重要的。在这里，我们提出了一种新方法，将未标记的突变引入非感受态革兰氏阴性细菌的大基因中。构建了两个基因替换质粒pJQFRT和pKFRT/FLP，应用FLP/FRT重组系统将未标记的突变引入非感受态革兰氏阴性菌的大基因中。在我们的方法中，pJQFRT和pKFRT/FLP通过同源重组分别整合到靶基因的上游和下游区域。所得突变体具有抗生素抗性标记、sacB 反选择标记、tetR 调节器控制下的 flp 重组酶以及将靶基因及其染色体上的标记夹在中间的相同 FRT 位点。通过诱导flp重组酶的表达，目标基因与来自整合质粒的其他基因一起被完全删除，导致产生未标记的突变。通过这种方法，我们在非感受态革兰氏阴性细菌不动杆菌中构建了 atA 的未标记突变体，该突变体编码巨大的三聚体自转运蛋白粘附素（3,630 个氨基酸）。 Tol 5。未标记的ataA突变体表现出与野生型Tol 5相同的生长速率，但失去了Tol 5的粘附特性，类似于我们之前生成的ataA的转座子插入突变体。在 Tol 5 菌株中构建未标记的 atA 突变体证明了我们方法的可行性。由于 FLP/FRT 重组可以切除超过 100kb 的长 DNA 区域，因此我们的方法有可能选择性地破坏比 ataA 更大的基因或更长的基因簇区域。我们的方法允许直接有效地将未标记的突变引入非肠杆菌革兰氏阴性菌的大基因或基因簇中。
For the disruption of a target gene in molecular microbiology, unmarked mutagenesis is preferable to marked mutagenesis because the former method raises no concern about the polar effect and leaves no selection marker. In contrast to naturally competent bacteria, there is no useful method for introducing an unmarked mutation into a large gene of non-competent bacteria. Nevertheless, large genes encoding huge proteins exist in diverse bacteria and are interesting and important for physiology and potential applications. Here we present a new method for introducing an unmarked mutation into such large genes of non-competent Gram-negative bacteria. Two gene replacement plasmids, pJQFRT and pKFRT/FLP, were constructed to apply the FLP/FRT recombination system to introduce an unmarked mutation into a large gene of non-competent Gram-negative bacteria. In our methodology, pJQFRT and pKFRT/FLP are integrated into the upstream and the downstream regions of a target gene, respectively, through homologous recombination. The resultant mutant has antibiotic resistance markers, the sacB counter-selection marker, flp recombinase under the control of the tetR regulator, and identical FRT sites sandwiching the target gene and the markers on its chromosome. By inducing the expression of flp recombinase, the target gene is completely deleted together with the other genes derived from the integrated plasmids, resulting in the generation of an unmarked mutation. By this method, we constructed an unmarked mutant of ataA, which encodes the huge trimeric autotransporter adhesin (3,630 aa), in a non-competent Gram-negative bacterium, Acinetobacter sp. Tol 5. The unmarked ataA mutant showed the same growth rate as wild type Tol 5, but lost the adhesive properties of Tol 5, similar to the transposon-inserted mutant of ataA that we generated previously. The feasibility of our methodology was evidenced by the construction of an unmarked ataA mutant in the Tol 5 strain. Since FLP/FRT recombination can excise a long region of DNA exceeding 100 kb, our method has the potential to selectively disrupt much larger genes or longer regions of gene clusters than ataA. Our methodology allows the straightforward and efficient introduction of an unmarked mutation into a large gene or gene cluster of non-enterobacterial Gram-negative bacteria.
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DOI：
10.1038/nbt1183-784
发表时间：
1983-01-01
期刊：
BIO-TECHNOLOGY
影响因子：
--
作者：
SIMON, R;PRIEFER, U;PUHLER, A
通讯作者：
PUHLER, A
影响因子：
3.2
作者：
Loehfelm, Thomas W.;Luke, Nicole R.;Campagnari, Anthony A.
通讯作者：
Campagnari, Anthony A.
影响因子：
14.9
作者：
Metzgar, D;Bacher, JM;de Crécy-Lagard, V
通讯作者：
de Crécy-Lagard, V
影响因子：
4.4
作者：
Ishii, S;Koki, J;Hori, K
通讯作者：
Hori, K
DOI：
10.1073/pnas.120163297
发表时间：
2000-06-06
影响因子：
11.1
作者：
Datsenko, KA;Wanner, BL
通讯作者：
Wanner, BL