Use of Natural Transformation To Establish an Easy Knockout Method in Riemerella anatipestifer

is a member of the family and a major causative agent of duck serositis. Little is known about its genetics and pathogenesis. Several bacteria are competent for natural transformation; however, whether is also competent for natural transformation has not been investigated. Here, we showed that strain ATCC 11845 can uptake the chromosomal DNA of strain RA-CH-1 in all growth phases. Subsequently, a natural transformation-based knockout method was established for ATCC 11845. Targeted mutagenesis gave transformation frequencies of ∼10 transformants. Competition assay experiments showed that ATCC 11845 preferentially took up its own DNA rather than heterogeneous DNA, such as DNA. Transformation was less efficient with the shuttle plasmid pLMF03 (transformation frequencies of ∼10 transformants). However, the efficiency of transformation was increased approximately 100-fold using pLMF03 derivatives containing DNA fragments (transformation frequencies of ∼10 transformants). Finally, we found that the RA-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 of is an important duck pathogen that belongs to the family At least 21 different serotypes have been identified. Genetic diversity has been demonstrated among these serotypes. The genetic and pathogenic mechanisms of remain largely unknown because no genetic tools are available for this bacterium. At present, natural transformation has been found in some bacteria but not in For the first time, we showed that natural transformation occurred in ATCC 11845 and RA-CH-1. Then, we established an easy gene knockout method in based on natural transformation. This information is important for further studies of the genetic diversity and pathogenesis in .