Expression And Characterization Of YeaZ Interaction Related Protein Genes Of Vibrio Harveyi

There are rich microorganism resources in natural environments,most of which are in an unculturable or difficult-to-cultivate state,and only 0.01%-10%could be obtained by the pure culture method.Many bacteria form a dormant state in the adverse environment,which is called as viable but nonculturable state?VBNC?.Under appropriate conditions,bacteria in VBNC state can recover as a normal cultureable form.Vibrio harveyi is a Gram-negative bacterium widely existing in natural environments,and is also one of the most important aquaculture animal pathogens in China.The bacterium enters into viable but nonculturable state in adverse environments.The V.harveyi cells in VBNC state remain their pathogenicity after resuscitation.The mechanisms of recovering from the dormant state to a cultureable state have not yet been clarified.In this paper,the environmental adaptation related proteins YeaZ,YjeE and YgjD of V.harveyi were studied.The yjeE,ygjD genes were cloned and expressed in Escherichia coli.Their structures and enzyme activities were also analyzed.The activity of YeaZ and site-directed mutated protein was determined and their enzyme activities and biological acivities were also studied.The main purpose is to explore the mechanisms of environmental adaptation and survival of V.harveyi,the main research content is as follow:The yjeE gene of V.harveyi SF-1 was amplified,which contains 465bp,and encoded a polypeptide of 153 amino acids.Sequence analysis showed that the similarities of yjeE gene with homologous gene sequences of E.coli,V.parahaemolyticus,V.alginolyticus and V.mimicus were78%-94%.The amino acid sequence similarities with homologous proteins of E.coli,Haemophilusinfluenzae and Bucillus subtilis were 55%-68%.The yjeE gene was cloned into the pCzn1 expression vector and transformed into E.coli cells,which were induced by IPTG.The separation and purification of YjeE was completed by Ni²⁺-affinity chromatography.SDS-PAGE showed that the purified protein had a molecular weight of 18.997 kDa.The expressed and purified YjeE protein had ATPase activity,with a specific activity of 60.72U/mg.It was found that addition of the purified YjeE promoted growth of the normal V.harveyi cells.The ygjD gene was amplified from the genome of V.harveyi SF-1,which consisted of 1020bp and encoded a polypeptide of 340 amino acids.Sequence analysis showed that the sequence similarities of ygjD gene with those of E.coli,V.parahaemolyticus,V.alginolyticus,V.cholerae,V.campbellii,V.mmicus,V.fluvialis were 77%-94%.The ygjD gene was cloned into the pET-28a?+?expression vector.The expression was carried out in E.coli by inducing with IPTG at 37°C.The expressed protein was purified by Ni²⁺-affinity chromatography.SDS-PAGE analysis showed that the molecular weight of the expressed protein was 39.183 kDa.Western-blotting confirmed the expression of YgjD protein.The YeaZ and the site-directed proteins with Asp⁸⁸,Ser¹⁸⁵and Trp¹⁶⁹and Asp⁸⁸-Ser¹⁸⁵were expressed and purified,respectively.The activities of purified proteins were determined by azocasein method.It was found that the activities of the mutated proteins were decreased.The protease activity of the YeaZ mutant with Asp⁸⁸-Ser¹⁸⁵was completely lost.The mutant without protease activity lost their promoting effect on the VBNC cells of V.harveyi.