Multiple genetic tools for editing the genome of Riemerella anatipestifer using a counterselectable marker

Abstract

Riemerella anatipestifer (R. anatipestifer, RA) is an important bacterial pathogen of ducks and other birds; infection with RA causes high poultry mortality and heavy economic losses in the poultry industry. However, the pathogenesis of this bacterium is poorly understood, in part due to the lack of a suitable array of methods for genetic manipulation. In this study, we first examined the efficacy of the mutated pheS gene (pheS*) as a counterselectable marker in R. anatipestifer. A suicide vector carrying pheS*, pOES, was constructed and used for markerless deletion of the gene RA0C_2053 which encode a putative TonB-dependent receptor in RA ATCC11845. The suicide plasmid pOES was also used to introduce a "knock-in" Myc-tag into the C-terminus of RA0C_1912 which encode a putative Fur protein. Using pheS* as a counterselectable marker, markerless mutagenesis and "knock-in" genetic manipulation techniques were also developed based on natural transformation. Furthermore, this marker was used to generate a point mutation in the RA0C_1912 gene of the RA ATCC11845 genome. The genetic methods developed in this study provide new and useful tools required to investigate the physiology and pathogenic mechanisms of this bacterium. These techniques may also have wider application in many other members of the Flavobacteria.

DprA Is Essential for Natural Competence in Riemerella anatipestifer and Has a Conserved Evolutionary Mechanism

“…The competence machinery actively processes exogenous dsDNA and takes up internalized ssDNA to replace homologous (or partially homologous) chromosomal sequences via a mechanism catalyzed by RecA and facilitated by accessory factors, such as DprA (Kidane et al, 2012). Although it has been found that R. anatipestifer was natural competent and established multiple genome editing tools using it (Liu et al, 2018b), the mechanism of natural transformation in R. anatipestifer kept largely unknown. This study focused on the characterization of DprA in R. anatipestifer ATCC11845.…”

Prevalence of fluoroquinolone resistance and mutations in the gyrA, parC and parE genes of Riemerella anatipestifer isolated from ducks in China

“…Transconjugants were shaken in GC broth (GCB) at 37 °C overnight to lose the suicide plasmid. Then, 200 μL of the appropriate dilution was plated on GCB agar supplemented with 13 mM p-Cl-Phe to select clones without the plasmid [27]. Clones were screened by PCR using primers cfx P1/P2 to choose Cfx-sensitive colonies.…”

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

“…The E . coli - Capnocytophaga canimorsus shuttle plasmid pMM47.A [29] was digested with PstI to remove the replication region and generate the plasmid pMM47.B, which has been constructed in previous study [24]. To increase the conjugation efficiency, oriT was amplified from the plasmid pEX18GM [30] using the primers oriTP1 (introducing a HindIII site) and oriTP2 (introducing a SphI site) (Table 2) and was cloned into pMM47.B to generate the plasmid pMM47.C.…”

“…The process of knockout based on pORS was done as described in previous study with a little modification [24]. Briefly, the 780-bp upstream sequence of RA0C_1534 was amplified using the primers RA0C_1534 upP1 (containing a NcoI site) and RA0C_1534 upP2.…”

“…anatipestifer ATCC11845, the toxic effect of p- Cl-Phe on R . anatipestifer ATCC11845 grown on a blood agar plate was weak when the pheS mutant was expressed in trans [24]. In this study, we established a method for markerless gene deletion in the genome of R .…”