Development of a markerless gene deletion strategy using rpsL as a counterselectable marker and characterization of the function of RA0C_1534 in Riemerella anatipestifer ATCC11845 using this strategy

• DOI: 10.1371/journal.pone.0218241
• Publication Date: 2019-06-10T17:37:37Z
• Authors: MaFeng Liu; Xiu Tian; MengYi Wang; DeKang Zhu; MingShu Wang; RenYong Jia; Shun Chen; XinXin Zhao; Qiao Yang; Ying Wu; ShaQiu Zhang; Juan Huang; Bin Tian; XiaoYue Chen; YunYa Liu; Ling Zhang; YanLing Yu; Francis Biville; LeiChang Pan; Mujeeb Ur Rehman; AnChun Cheng
• Keywords: Genetic Markers; 0303 health sciences; Ribosomal Protein S9; Science; Escherichia coli Proteins; Q; R; Drug Resistance, Microbial; Mutagenesis, Insertional; Riemerella; 03 medical and health sciences; Bacterial Proteins; Mutation; Escherichia coli; Medicine; Selection, Genetic; Genetic Engineering; Gene Deletion; Research Article; Sequence Deletion

Riemerella anatipestifer is a gram-negative bacterium that mainly infects ducks, turkeys and other birds. In a previous study, we established a markerless mutation system based on the pheS mutant as a counterselectable marker. However, the toxic effect of p-Cl-Phe on the R. anatipestifer strain expressing the pheS mutant was weak on blood agar plates. In this study, we successfully obtained streptomycin-resistant derivative of R. anatipestifer ATCC11845 using 100 μg/mL streptomycin as a selection pressure. Then, we demonstrate that rpsL can be used as a counterselectable marker in the R. anatipestifer ATCC11845 rpsL mutant strain, namely, R. anatipestifer ATCCs. A suicide vector carrying wild-type rpsL, namely, pORS, was constructed and used for markerless deletion of the gene RA0C_1534, which encodes a putative sigma-70 family RNA polymerase sigma factor. Using rpsL as a counterselectable marker, markerless mutagenesis of RA0C_1534 was also performed based on natural transformation. R. anatipestifer ATCCsΔRA0C_1534 was more sensitive to H2O2-generated oxidative stress than R. anatipestifer ATCCs. Moreover, transcription of RA0C_1534 was upregulated under 10 mM H2O2 treatment and upon mutation of fur. These results suggest that RA0C_1534 is involved in oxidative stress response in R. anatipestifer. The markerless gene mutation method developed in this study provides new tools for investigation of the physiology and pathogenic mechanisms of this bacterium.