title: Use of Natural Transformation To Establish an Easy Knockout Method in Riemerella anatipestifer.
abstract: Riemerella anatipestiferis a member of the familyFlavobacteriaceaeand a major causative agent of duck serositis. Little is known about its genetics and pathogenesis. Several bacteria are competent for natural transformation; however, whetherR. anatipestiferis also competent for natural transformation has not been investigated. Here, we showed thatR. anatipestiferstrain ATCC 11845 can uptake the chromosomal DNA ofR. anatipestiferstrain RA-CH-1 in all growth phases. Subsequently, a natural transformation-based knockout method was established forR. anatipestiferATCC 11845. Targeted mutagenesis gave transformation frequencies of ∼10-5transformants. Competition assay experiments showed thatR. anatipestiferATCC 11845 preferentially took up its own DNA rather than heterogeneous DNA, such asEscherichia coliDNA. Transformation was less efficient with the shuttle plasmid pLMF03 (transformation frequencies of ∼10-9transformants). However, the efficiency of transformation was increased approximately 100-fold using pLMF03 derivatives containingR. anatipestiferDNA fragments (transformation frequencies of ∼10-7transformants). Finally, we found that theR. anatipestiferRA-CH-1 strain was also naturally transformable, suggesting that natural competence is widely applicable for this species. The findings described here provide important tools for the genetic manipulation ofR. anatipestiferIMPORTANCERiemerella anatipestiferis an important duck pathogen that belongs to the familyFlavobacteriaceaeAt least 21 different serotypes have been identified. Genetic diversity has been demonstrated among these serotypes. The genetic and pathogenic mechanisms ofR. anatipestiferremain largely unknown because no genetic tools are available for this bacterium. At present, natural transformation has been found in some bacteria but not inR. anatipestiferFor the first time, we showed that natural transformation occurred inR. anatipestiferATCC 11845 andR. anatipestiferRA-CH-1. Then, we established an easy gene knockout method inR. anatipestiferbased on natural transformation. This information is important for further studies of the genetic diversity and pathogenesis inR. anatipestifer.
authors.affiliation: Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu, Sichuan, People's Republic of China liumafengra@163.com chenganchun@vip.163.com.|Avian Disease Research Center, College of Veterinary Medicine of Sichuan Agricultural University, Chengdu, Sichuan, People's Republic of China.|Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu, Sichuan, People's Republic of China.
authors.forename: MaFeng
authors.initials: M
authors.lastname: Liu
authors.affiliation: Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu, Sichuan, People's Republic of China.|Avian Disease Research Center, College of Veterinary Medicine of Sichuan Agricultural University, Chengdu, Sichuan, People's Republic of China.|Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu, Sichuan, People's Republic of China.
authors.forename: Li
authors.initials: L
authors.lastname: Zhang
authors.affiliation: Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu, Sichuan, People's Republic of China.|Avian Disease Research Center, College of Veterinary Medicine of Sichuan Agricultural University, Chengdu, Sichuan, People's Republic of China.|Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu, Sichuan, People's Republic of China.
authors.forename: Li
authors.initials: L
authors.lastname: Huang
authors.affiliation: Unité des Infections Bactériennes Invasives, Département Infection et Epidémiologie, Institut Pasteur, Paris, France.
authors.forename: Francis
authors.initials: F
authors.lastname: Biville
authors.affiliation: Avian Disease Research Center, College of Veterinary Medicine of Sichuan Agricultural University, Chengdu, Sichuan, People's Republic of China.|Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu, Sichuan, People's Republic of China.
authors.forename: DeKang
authors.initials: D
authors.lastname: Zhu
authors.affiliation: Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu, Sichuan, People's Republic of China.|Avian Disease Research Center, College of Veterinary Medicine of Sichuan Agricultural University, Chengdu, Sichuan, People's Republic of China.|Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu, Sichuan, People's Republic of China.
authors.forename: MingShu
authors.initials: M
authors.lastname: Wang
authors.affiliation: Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu, Sichuan, People's Republic of China.|Avian Disease Research Center, College of Veterinary Medicine of Sichuan Agricultural University, Chengdu, Sichuan, People's Republic of China.|Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu, Sichuan, People's Republic of China.
authors.forename: RenYong
authors.initials: R
authors.lastname: Jia
authors.affiliation: Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu, Sichuan, People's Republic of China.|Avian Disease Research Center, College of Veterinary Medicine of Sichuan Agricultural University, Chengdu, Sichuan, People's Republic of China.|Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu, Sichuan, People's Republic of China.
authors.forename: Shun
authors.initials: S
authors.lastname: Chen
authors.affiliation: Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu, Sichuan, People's Republic of China.|Avian Disease Research Center, College of Veterinary Medicine of Sichuan Agricultural University, Chengdu, Sichuan, People's Republic of China.|Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu, Sichuan, People's Republic of China.
authors.forename: KunFeng
authors.initials: K
authors.lastname: Sun
authors.affiliation: Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu, Sichuan, People's Republic of China.|Avian Disease Research Center, College of Veterinary Medicine of Sichuan Agricultural University, Chengdu, Sichuan, People's Republic of China.|Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu, Sichuan, People's Republic of China.
authors.forename: Qiao
authors.initials: Q
authors.lastname: Yang
authors.affiliation: Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu, Sichuan, People's Republic of China.|Avian Disease Research Center, College of Veterinary Medicine of Sichuan Agricultural University, Chengdu, Sichuan, People's Republic of China.|Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu, Sichuan, People's Republic of China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Wu
authors.affiliation: Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu, Sichuan, People's Republic of China.|Avian Disease Research Center, College of Veterinary Medicine of Sichuan Agricultural University, Chengdu, Sichuan, People's Republic of China.|Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu, Sichuan, People's Republic of China.
authors.forename: XiaoYue
authors.initials: X
authors.lastname: Chen
authors.affiliation: Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu, Sichuan, People's Republic of China liumafengra@163.com chenganchun@vip.163.com.|Avian Disease Research Center, College of Veterinary Medicine of Sichuan Agricultural University, Chengdu, Sichuan, People's Republic of China.|Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu, Sichuan, People's Republic of China.
authors.forename: AnChun
authors.initials: A
authors.lastname: Cheng
mesh_terms: D055786:Gene Knockout Techniques / Q000379:methods*; D005827:Genetics, Microbial / Q000379:methods*; D060494:Riemerella / Q000235:genetics*; D014169:Transformation, Bacterial
keywords: Riemerella anatipestifer; natural transformation; targeted mutagenesis