[Construction and characterization of some virulence determinants-deficient strain in Aeromonas hydrophila]

Cell conjugation was carried out between the donor E. coli CG120 with pAM120 (Tc(r)/Tn916) and the recipient Aeromonas hydrophila J-1 by filter mating. 3800 positive clones were gained according to the ability of growth on LB medium with 10 g/mL tetracycline (Tc) and 100 g/mL cephazolin (cfz). Conjugation efficiency was 3 x 10(-5) per donor. When 16S rDNA PCR amplification was performed with special primers, positive results were gained in all the 38 conjugants. To demonstrate the insertion of Tn916 into the genomes of conjugants, tetracycline gene (tet) PCR amplification was performed. A special band could be gained in the conjugants with Tc(r). Compared with the parent J-1 strain, some genes of main virulence determinants, such as proteases, hemolysins, DNase and amylases, could not be expressed in the conjugants. The pathogenicity capability of conjugants was greatly decreased and the 50% lethal dose for mice was more than 10(9) CFU. By serially passaged for 10 times, the above virulent characters had not been regained despite the disappearance of Tc(r). The results showed that by the insertion of Tn916, stable and avirulent A. hydrophila mutants could be gained. The mechanisms, by which Tn916 induced the changes of virulence characters in A. hydrophila, are not definite. It is suspected that there might exist hot spots of Tn916 or pathogenicity island on the chronosome of A. hydrophila.