?phoB Phenotype Analysis Of Aeromonas Dhakensis Isolated From Siamese Crocodile

Aeromonas dhakensis is a new identified and classified species with high virulence for harboring a variety of virulence genes isolated from farmed fish in freshwater and clinical patients.Many evidences show that it is toxic to human blood cell lines and intestinal cells.In this study,three Aeromonas species were isolated from two diseased Siamese crocodile,and their virulence were tested by zebrafish infection.The results showed that the most toxic strain was A.dhakensis.PhoBR?Pho PR?two-component signal transduction system plays a key role in the survival and toxicity of bacteria in the host via regulating phosphorus metabolism balance and pathogenic virulence.A number of studies have found that PhoBR mutations attenuated the bacteria,and the pho P gene mutant of Mycobacterium tuberculosis has been conducted for the clinical evaluation stage as an attenuated vaccine.To investigate the function of PhoBR in Aeromonas,the isolate high virulent A.dhakensis strain was used a material for phoB gene deletion using the integrated plasmid p K18mobsac B,the morphological and virulence changes of the wild type strain and the knockout strain were compared to further understand the pathogen control and the function of PhoBR.Firstly,two about 1 kb homologous arms were amplified from the both sides of the upstream and the downstream of the phoB gene,and then the two fragments were combined together and cloned in the p K18mobsac B plasmid.The recombinant plasmid was introduced into?asd-deficient Escherichia coli X6097 by heat shock method.X6097and A.dhakensis were mixed and cultured in a certain ratio for importing the recombinant plasmid into A.dhakensis by conjugative transfer.The colonies which contained recombinant plasmid were selected by kanamycin and diaminopimelic acid?50?g/m L?.Finally,the phoB deleted mutant were constructed by homologous recombination and selected on high-concentration sucrose plates.The shape of A.dhakensis was not affected for the deletion of phoB gene,but the growth of the mutant was much slower than the wild in limited phosphorus medium concentration.Similarly,in term of the biofilm production,the expression levels of alkaline phosphatase,H₂O₂resistance,bacterial motility and antimicrobial peptide sensitivity,the capacity of?phoB strain was obviously reduced compared to the wild type strain.The half-lethal experiment result showed that the LD₅₀value of the?phoB strain was much higher than the LD₅₀value of the wild-type strain,which indicated that the phoBgene deletion was weakened the virulence of A.dhakensis.It was the primary report that A.dhakensis was isolated from diseased Siamese crocodile,the virulence of A.dhakensis was declined by phoB deletion.This study would be useful for A.dhakensis attenuated vaccine development.