A study by researchers in Institute of Microbiology, Chinese Academy of Sciences revealed a novel strategy to achieve targeted gene deletion in Clostridium.
The Gram-positive bacteria genus Clostridium is closely related to human life. This genus includespathogenic clostridia producing exotoxinsuch as C. botulinum and C. tetani,as well as Clostridium species of industrial importance, such as C. thermocellum, C. acetobutylicum.
Targeted gene inactivation or deletion is one of the basic requirementsfor understanding the gene functions. Targeted gene inactivation in Clostridium was very difficult until a gene knockout system employing the Lactococcus lactis Ll.LtrB group II intron has been developed, which significantly increase the insertional mutagenesis efficiency. However, targeted gene deletion in Clostridium via homologous recombination is of very low efficiency and low reproducibility.
Inspired by the high-efficiency of group II intron mediated retrotransposition, Dr. JIA Kaizhi and his co-tutor Prof. LI Yin designed a novel strategy to achieve targeted gene deletion in Clostridium. This strategy combines the advantage of intron retrotransposition and homologous recombination. First, an allele homologous to the upstream or downstream of the intron target site was constructed together with the intron. Second, the intron carrying the allele was integrated into clostridial genome.Finally, the target gene, operon, or gene cluster will be deleted by homologous recombination.
A functional unknown operon CAC1493-1494 located in the chromosome, and an operon ctfAB located in the megaplasmid of C. acetobutylicum DSM1731, were successfully deleted by using this approach, without leaving antibiotic marker in the genome. This strategy was named as “Group II intron-anchored gene deletion in Clostridium”-the possibility of homologous recombination was enhanced after anchoring the intron into the genome. The authors claim that this strategy is also applicable for gene deletion in other bacterial species, given that the group II intron retrotransposition system is established.
This work has been published on PLoS ONE 6(1): e16693. doi:10.1371/journal.pone.0016693.
LI Yin, Ph.D., Professor
Institute of Microbiology, Chinese Academy of Sciences, Beijing, China.