Regulation Of Aeromonas Hydrophila Quorum Sensing System On Its Biological Characteristics

Aeromonas hydrophila ATCC7966 is a Gram negative bacteria, which exists widely in nature, and it is also a typical human-animal-fish zoonosis pathogen.There are two kinds of quorum sensing systems in this bacteria, QS1 in which AHL as the signal molecule and QS2 in which AI-2 as the signal molecule. The gene ahy I encodes the synthsae of signal molecule AHL and the gene lux S encodes the synthsae of signal molecule AI-2. In this research, the regulation of A. hydrophila quorum sensing system on its biological characteristics was studied.Based on the principle of homologous recombination, recombinant gene knock-out vector was constructed consisting of Kanr cassette with the flanking homologous regions of gene lux S. The constructed recombinant plasmid was transformed into competent E. coli SM 10(λpir) cells and was further transferred into A. hydrophila ATCC 7966 by conjugation. The luxS gene deletion mutant strain was confirmed by resistance screening, PCR and DNA sequencing. Growth rates, ability of AI-2 synthesis, extracellular protease synthesis and biofilm formation were compared between the wild strain and lux S gene deletion mutant strain. Results suggested that lux S gene deletion mutant strain of A. hydrophila ATCC 7966 was successfully constructed. Compared with the wild strain, the growth cycle of the mutant strain was extended, the ability to synthesize AI-2 and extracellular protease was lost. Biofilm forming ability of the mutant strain was decreased, which was 14.5% and 60.0% of that of the wild strain at 24 h and 36 h respectively. Compared with the wild strain, the biofilm formation rate of the mutant strain was slower and the biofilm structure was looser.Based on the principle of homologous recombination, recombinant gene knock-out vector was constructed consisting of Cmr cassette with the flanking homologous regions of gene ahyI. The constructed recombinant plasmid was transformed into competent E. coli SM 10(λpir) cells and was further transferred into A. hydrophila ATCC 7966 by conjugation. The ahy I gene deletion mutant strain was confirmed by resistance screening, PCR and DNA sequencing. Growth rates, extracellular protease synthesis and biofilm formation were compared between the wild strain and ahyI gene deletion mutant strain. Results suggested that ahy I gene deletion mutant strain of A. hydrophila ATCC 7966 was successfully constructed. Compared with the wild strain, the growth cycle of the mutant strain was extended and the extracellular protease was more. Biofilm forming ability of the mutant strain was decreased, which was 12.7% and 31.6% of that of the wild strain at 12 h and 24 h respectively.It was indicated that QS1 and QS2 were both involved in the regulation of different biological characteristics of A. hydrophila ATCC 7966, such as growth rates, extracellular protease and biofilm. This study laid the foundation for further investigation of the role of quorum sensing system in the pathogenesis of A. hydrophila.