Isolation And Identification Of Aeromonas Hydrophila And Functional Characterization Of Hfq On Its Bioprocesses

Aeromonas hydrophila is a major pathogen causing the bacterial disease and brings about huge damage and economic losses in aquaculture all over the world. Meanwhile, this bacterial, a zoonotic pathogen, can also infect human and is a threat to human health. Thus, to study the virulence regulation of this bacterial will do some favor to the control of its resulting disease. Hfq, the sRNA chaperon and the global post-transcriptional regulator, is widespreaded in bacteria and involved in various bioprocesses and virulence regulation in kinds of pathogensa by promoting the interaction of different sRNAs and their target mRNAs. In this study, a pathogenic strain, isolated from the diseased grouper in the fish farm in Yantai, Shandong Province, was characterized to be A. hydrophila by physiological, biochemical, and molecular methods. Then the hfq gene in this strain was cloned and the △hfq mutant strain was constructed by markerless gene deletion strategies. Furthermore, the regulation of Hfq on the physiological functions and virulence in A. hydrophila was figured out. This will broaden the knowledgment about the virulence regulation mechanism in A. hydrophila and also lay some theorial foundation for scientific prevention of this pathogen. The results were just as follows:(1) A dominant strain was isolated from the pancreatic tissue of the diseased grouper. The strain, which was Gram negative short-rod bacterial, can form β hemolysis circle, utilize glucose to generate acid and gas, reduce the nitrate, decomposite malose, glucose, mannitol, sucrose an so on, but not the citrate, xylose, inositol and so on through the study on its growth and physiological and biochemical characteristics. In further, the 16S rRNA sequence was cloned and blasted. Then the phylogenetic tree was constructed. The 16S rRNA sequence of this strain shared 99% identity with that of A. hydrophila and located to the same branch in the phylogenetic tree. Therefore, taken all the results together, this strain was characterized to be A. hydrophila. And the artifitial infection experiment demonstrated that this strain was pathogenic.(2) The hfq gene of A. hydrophila was cloned, and a specific fragment of 264 bp, encoded for 88 amino acids, was obtained. The predicted secondary structure of Hfq protein was made up of an a-helix consisting of 12 amino acids, five β-folds consisting of 32 amino acids and the coil. The three-dimensional structure of Hfq protein was predicted to be a hollow annular dimer. The recombinant expression plasmid pET28a (+)-hfq was successfully constructed and a specific band of 12 kDa size was detected after induction and the protein production was enbanced with the induction time.(3) The markerless inframe-deletion Ahfq mutant strain was successfully generated using the suicide plasmid, pDM4 by homologous recombination method. By comparison of the phenotypes of the wild-type and Ahfq strain, it was indicated that:during the logarithmic phase, the concentration of Ahfq strain was lower than that of wild type strain, but reached the same level withwild type when entering the stationary phase, and evev higher at the late stationary phase. Hfq positively regulated the swimming ability, but has no obvious influences on the swarming ability and hemolysis activity in A. hydrophila. As Hfq expression was impaired, the extracellular protease production was greatly increased, which means Hfq severely repressed the extracellular protease production in A. hydrophila. Moreover, the growth of the Ahfq strain was seriously inhibited under 30% sucrose and 3% NaCl conditions, and repressed to some extent under different concentration of SDS and ethonal. The isolated A. hydrophila strain was highly pathogenic to zebra fish, while the Ahfq mutant strain was apparently attenuated as the Hfq expression was impaired.