Research Article: 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

Date Published: June 10, 2019

Publisher: Public Library of Science

Author(s): 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, Timothy J. Johnson.


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.

Partial Text

Riemerella anatipestifer (referred to herein as R. anatipestifer or RA) is a gram-negative bacterium belonging to the family Flavobacteriaceae, phylum Bacteroidetes, and genus Riemerella [1]. To date, more than 21 serotypes have been reported, and there is no cross-protection among them, and there may exist different epidemic serotypes in the same duck farm [2–4]. Thus, the effects of vaccination have been unsatisfactory. In addition, R. anatipestifer is naturally resistant to various antibiotics [5–9]. The application of antibiotics to prevent and treat the disease leads to severe contamination and poses a threat to human health. To prevent this disease completely, an understanding of the pathogenic mechanisms is required.

The genetic tools were critical for the study on the physiology and pathogenic mechanism of the pathogens, however, it was imperfect for the R. anatipestifer, which is an important bacterial pathogen for duck industry. In a previous study, we established a markerless mutation method based on a pheS mutant as a counterselection marker [24]. However, the toxic effect of p-Cl-Phe on R. anatipestifer cells expressing the pheS mutant on blood agar plates was weak [24]. Here, we described a dominant gene marker encoding the S12 protein from R. anatipestifer ATCC11845, indicating the feasibility of this system for negative selection. A comparison of the rpsL alleles of several bacteria revealed that most of these alleles encoded a conserved lysine at position 43, which is precisely the amino acid that is known to confer streptomycin resistance when mutated in various species [22, 23, 33]. Consistent with this finding, we screened 7 of 10 mutants that exhibited A-to-G transversions at position 128. Furthermore, we showed that streptomycin-sensitive rpsL alleles were dominant compared to streptomycin-resistant rpsL alleles, and rpsL is a suitable counterselection marker for R. anatipestifer ATCCs. Subsequently, we developed a markerless mutant based on the constructed suicide plasmid. In this case, Cfx-resistant colonies were obtained for the first step (plasmid integration) at a frequency of 10−5. Streptomycin-resistant colonies were obtained for the second step (plasmid loss) at a frequency of 10−3, and approximately 30% of the streptomycin-resistant colonies carried the deletion. It’s worth noting that the frequency for each step recombination should be various according the different target genes.




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