High-Frequency Targeted Mutagenesis inPseudomonas stutzeriUsing a Vector-Free Allele-Exchange Protocol.
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 genusPseudomonas. 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 self-ligation (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 bacteriumPseudomonas stutzeriA1501. The results showed that theP. stutzeriA1501 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 inP. stutzeri, which may also be applicable for other gram-negative bacteria.
Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, P.R. China.
Ahmed E
AE
Gomaa
Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, P.R. China.
Zhiping
Z
Deng
Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, P.R. China.
Zhimin
Z
Yang
Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, P.R. China.
Liguo
L
Shang
Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, P.R. China.
Yuhua
Y
Zhan
Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, P.R. China.
Wei
W
Lu
Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, P.R. China.
Min
M
Lin
Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, P.R. China.
Yongliang
Y
Yan
D000483:Alleles; D024881:Drug Resistance, Bacterial / Q000235:genetics; D000072669:Gene Editing; D055786:Gene Knockout Techniques; D005798:Genes, Bacterial; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors; D016680:Genome, Bacterial; D016296:Mutagenesis; D010957:Plasmids; D016133:Polymerase Chain Reaction; D044223:Pseudomonas stutzeri / Q000235:genetics*; D011995:Recombination, Genetic
Pseudomonas stutzeri; Vector-free allele exchange (VFAE); genome editing; homologous recombination; mutagensis