|Other Abstract||Columnris disease occurs in freshwater fish throughout the world. Its causative agent has been identified as the bacterium Flavobacterium columnare. Recent investigations on genetic diversity of this bacterium have revealed the occurrence of three genomovars of F. columnare. In order to understand the possible difference in virulence among different strains of the bacterium, two strains of F. columnare, i.e. G4 and G18, which were shown to belong to different genomovars in a previous study in our laboratory, were employed in the present study. F. columnare G4 strain, which has been recognized as a strong virulence strain, was examined for its specific genes when compared with a low virulence strain, G18, by Suppression Subtractive Hybridization (SSH). A total of 50 clones which were specifically detected in F. columnare G4 were sequenced, and 46 sequences of different genes obtained. 35 of the 46 genes showed some degree of homology with known proteins and can be classified into following categories, related with DNA replication, or recombination and repairing, inorganic ion transport or metabolism, outer-membrane proteins, enterotoxin, transposase, and metabolism etc.
The Type I restriction modification system (R-M system) in F. columnare G4 strain was described in this study. The complete sequence of Type I R-M system FclI was cloned by genomic DNA walking. Through analyzing the open reading frames (ORFs) of FclI, sequences encoding three subunits, i.e.m HsdM, HsdS and HsdR of Type I R-M system are identified, which were then named as fclIM, fclIS and fclIR in F. columnare G4, respectively. The ribose-binding site (RBS) and the putative promoter of fclIM and fclIS were analyzed, which are similar to those in F. hibernum. The transcription of fclIM, fclIS and fclIR in F. columnare G4 was determined by RT-PCR. fclIM and fclIS responsible for DNA methylation were transcribed, but fclIR associated with restriction was not. In addition, the size FclIM subunit was identified as about 90 kD by Western blotting. To identify the function of fclIM and fclIS in F. columnare G4, these genes were cloned into plasmid pACYC184 and transformed into E. coli TOP10. The expression of fclIM and fclIS in E. coli was identified by RT-PCR. The integrative plasmid pGL006 was transformed into E. coli containing fclIM and fclIS and was methylated. Using the methylated pGL006, the electroporation efficiency of F. columnare G4 increased 3.6 times under the voltage of 2.0 kV/cm.
Several genes encoding putative virulence factors in F. columanre strain G4, such as TonB-dependent receptor, transposases, as well as ABC transporter permease protein were cloned by genome walking. he flanking sequence of a putative transposase was cloned. A putative Rhs element was located at the downstream of the putative transposase.|