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

Abstract

<i>Riemerella anatipestifer</i> is a member of the family <i>Flavobacteriaceae</i> 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 <i>R. anatipestifer</i> is also competent for natural transformation has not been investigated. Here, we showed that <i>R. anatipestifer</i> strain ATCC 11845 can uptake the chromosomal DNA of <i>R. anatipestifer</i> strain RA-CH-1 in all growth phases. Subsequently, a natural transformation-based knockout method was established for <i>R. anatipestifer</i> ATCC 11845. Targeted mutagenesis gave transformation frequencies of ∼10^-5 transformants. Competition assay experiments showed that <i>R. anatipestifer</i> ATCC 11845 preferentially took up its own DNA rather than heterogeneous DNA, such as <i>Escherichia coli</i> DNA. Transformation was less efficient with the shuttle plasmid pLMF03 (transformation frequencies of ∼10^-9 transformants). However, the efficiency of transformation was increased approximately 100-fold using pLMF03 derivatives containing <i>R. anatipestifer</i> DNA fragments (transformation frequencies of ∼10^-7 transformants). Finally, we found that the <i>R. anatipestifer</i> 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 <i>R. anatipestifer</i><b>IMPORTANCE</b><i>Riemerella anatipestifer</i> is an important duck pathogen that belongs to the family <i>Flavobacteriaceae</i> At least 21 different serotypes have been identified. Genetic diversity has been demonstrated among these serotypes. The genetic and pathogenic mechanisms of <i>R. anatipestifer</i> 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 <i>R. anatipestifer</i> For the first time, we showed that natural transformation occurred in <i>R. anatipestifer</i> ATCC 11845 and <i>R. anatipestifer</i> RA-CH-1. Then, we established an easy gene knockout method in <i>R. anatipestifer</i> based on natural transformation. This information is important for further studies of the genetic diversity and pathogenesis in <i>R. anatipestifer</i>.