High-Frequency Targeted Mutagenesis in Pseudomonas stutzeri Using a Vector-Free Allele-Exchange Protocol

文献类型： 外文期刊

第一作者： Gomaa, Ahmed E.

作者： Gomaa, Ahmed E.;Deng, Zhiping;Yang, Zhimin;Shang, Liguo;Zhan, Yuhua;Lu, Wei;Lin, Min;Yan, Yongliang

作者机构：

关键词： Vector-free allele-exchange (VFAE);homologous recombination;Pseudomonas stutzeri;mutagensis;genome editing

期刊名称：JOURNAL OF MICROBIOLOGY AND BIOTECHNOLOGY （ 影响因子：2.351； 五年影响因子：2.65 ）

ISSN：

年卷期：

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收录情况： SCI

摘要： The complexity of the bacterial recombination system is a barrier for the construction of bacterial mutants for the further functional investigation of specific genes. Several protocols have been developed to inactivate genes from the genus Pseudomonas. Those protocols are complicated and time-consuming and mostly do not enable easy construction of multiple knock-ins/outs. The current study describes a single and double crossover-recombination system using an optimized vector-free allele-exchange protocol for gene disruption and gene replacement in a single species of the family Pseudomonadaceae. The protocol is based on selfligation (circularization) for the DNA cassette which has been obtained by overlapping polymerase chain reaction (Fusion-PCR), and carries an antibiotic resistance cassette flanked by homologous internal regions of the target locus. To establish the reproducibility of the approach, three different chromosomal genes (ncRNA31, rpoN, rpoS) were knocked-out from the root-associative bacterium Pseudomonas stutzeri A1501. The results showed that the P. stutzeri A1501 mutants, which are free of any plasmid backbone, could be obtained via a single or double crossover recombination. In order to optimize this protocol, three key factors that were found to have great effect on the efficiency of the homologous recombination were further investigated. Moreover, the modified protocol does not require further cloning steps, and it enables the construction of multiple gene knock-in/out mutants sequentially. This work provides a simple and rapid mutagenesis strategy for genome editing in P. stutzeri, which may also be applicable for other gram-negative bacteria.

分类号： Q93

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