Construction And Characterization Of Ahyi Deleted Mutant Srain In Aeromonas Hydrophila J-1

Aeromonas hydrophila is an important opportunistic pathogen which can cause infections in mammals and aquatic animals. A. hydrophila produces several virulence factors including hemolysins, enterotoxins, cytotoxic enterotoxin, GCAT and serine protease. These virulence factors have relationship with high concentration of bacteria in late exponential and early stationary growth phases. It can be supposed that quorum sensing may control these phenotypes.Quorum sensing (QS) means bacteria utilize signaling molecules to communicate between cells, which can monitor cell population density and to regulate gene expression in response to fluctuations in cell numbers. QS1can be found in a wide variety of gram-negative bacteria, and related with the pathogenicity of bacteria closely. The luxI gene encodes the sequences that mediate the formation of the signaling molecule. The ahyl gene is luxIs homologues in A. hydrophila. The primers of the ahyl gene were designed based on A. hydrophila ATCC7966genome sequence, and synthesized to amplify ahyl gene in A. hydrophila J-1. The desired size fragment (952bp) was obtained. By sequencing, the PCR products was confirmed to have high sequence homology with A. hydrophila ATCC7966and A. hydrophila SSU, and the percentage sequence similarity were99%and96%, respectively. The prevalence of ahyl gent in115Aeromonas different isolates was detected by PCR amplification. The detection result showed that:distribution rate of ahyl gene was60%in115Aeromonas strains, especially in100%(22/22) of A. hydrophila and50%(34/68) of A. veronii. There were no obvious characteristics in distribution of ahyl, irrespective of the nature of the isolates from water and fish, diseased or healthy.Two pairs of primers were designed based on ATCC7966genome for amplification of870bp of ahyl upstream-flanking and920bp of downstream-flanking sequences. Cmr gene cassette was linked between the two sequences to get a recombinant suicide plasmid pHM5(ahyI). Then the recombinant suicide plasmid was transformed into the donor E. coli SM10for further conjugal transfer into A. hydrophila J-1. A mutant strain was selected by antibiotics and sucrose. For genetic complementation, plasmid pUC18with the ORF and putative promoter of ahyl was transformed into the mutant strain. The biological characteristics of wild type strain, mutant strain and complementation strain were compared and analyzed. The result showed that, the mutant strain lost the ability to produce AI-1, but still could produce AI-2. Compared with wild strain, the activity of extracellular protease in mutant strain showed a great reduction. Meanwhile, SDS-PAGE analysis of total extracellular protein showed that there was a significant difference between wild and mutant strains. The abilities of invasion and adherence into cells in mutant strain were reduced, and restored in the complementation strain. The analysis of real-time PCR showed a decreased expression of serine protease gene. The biological characters, including hemolysis, biofilm formation, total protein, outer membrane protein and the50%lethal dose for zebrafish had no significant difference between wild and mutant strains.The disruption of the ahyl gene in A. hydrophila had an effect on AI-1production, extracellular protease activity, expression of serine protease, ability of invasion and adherence. However, the pathogenicity of mutant strain to zebrafish wasn’t influenced by these factors.