Type I restriction-modification system and its resistance in electroporation efficiency in Flavobacterium columnare

Flavobacterium columnare, the causative agent of columnaris disease, infects freshwater fish worldwide. However, the pathogenicity of this bacterium is poorly understood due possibly to the lack of an efficient in-frame knockout technique. In order to improve electroporation efficiency, the type I restriction-modification system (R-M system) was cloned and its role in electroporation was examined in F. columnare G(4) strain. The complete sequence of type I R-M system in the bacterium, designated as Fcl, contains all three subunits of type I R-M system, named as fclM, fclS, fclR, respectively, with the identification of a hypothetical gene, fclX. Constitutive transcription of the three genes was observed in F. columnare G(4) by RT-PCR. The ORF of fclM and fclS was cloned into the plasmid pACYC184 and transformed into Escherichia coli TOP10. The resultant E. coli strain, designated as E. coli TOPmt, was transformed with the integrative plasmid pGL006 constructed for F. columnare G(4). The integrative plasmid was re-isolated from TOPmt and incubated with the lysate of F. columnare G(4). The re-isolated integrative plasmid, designated as pGL006', showed higher resistance than pGL006. With pGL006', the electroporation efficiency of the strain G(4) increased 2.6 times, while that of F. columnare G(18) was not obviously improved. Furthermore, a method to improve the electroporation efficiency of F. columnare G(4) was developed using the integrative plasmid methylated by E. coli TOPmt which contains the fclM and fclS gene of F. columnare G(4). Further analyses showed that the fcl gene cluster may be a unique type I R-M system in F. columnare G(4). It will be of significant interest to examine the composition and diversity of R-M systems in strains of F. columnare in order to set up a suitable genetic manipulation system for the bacterium.