Studies On Mutantion Of The YjeE And YeaZ Genes Of Vibrio Harveyi And Their Biological Characteristics

Vibrio harveyi exists widely in the marine environments.The bacterium could enter into the viable but non-culturable state under extreme environments.VBNC cells are recovered to a cultureable form under appropriate conditions.This study investigated the VBNC status of V.harveyi induced by hydrogen peroxide and low temperature.The cell growth and environment stress related proteins YjeE and YeaZ of V.harveyi were expressed in E.coli.The biological properties of the recombinant proteins were investigated.And their growth-promoting and cell-resuscitation effects on V.harveyi cells were also studied.The main results are as follows:After induction of V.harveyi SF-1 at 4?for 70 days,the cells entered into a viable but non-culturable state.When induced with 50 mM H₂O₂,V.harveyi cells could enter into the VBNC state at 96h.The LIVE/DEAD BacLight bacterial viability kit was used to measure the changes in the number of the live and total cell numbers during the induction of hydrogen peroxide.It was found that the total cell numbers and the live cell numbers changesd slightly,while the culturable cell numbers decreased to zero,which indicated that the bacterial cells entered into the VBNC state.Catalase,sodium pyruvate and its analogues were added to the VBNC cells to study their resuscitation promoting effects on V.harveyi cells.It was found that addition of the catalase sodium pyruvate and?-Ketobutyric acid sodium salt could promote recovery of the VBNC cells,while other pyruvate analogs did not have promoting effect on the VBNC cells.There was no significant difference in the growth characteristics between the resuscitated V.harveyi cells and the normal cells.The prokaryotic expression vector pET-28a-yjeE containing yjeE gene of V.harveyi was transformed into E.coli BL21?DE3?,and induced by IPTG.The recombinant protein was purified by nickel affinity chromatography.SDS-PAGE electrophoresis analysis showed that the purified YjeE protein had a molecular weight of 19 kDa.The recombinant protein showed ATPase activity,with an enzyme specific activity of 0.953 U/mg.The mutants of enzyme activity related amino acids of YjeE were constructed with site-directed mutations and expressed in E.coli.When compared with the wild-type YjeE protein,the enzyme activities of mutant proteins with different substituted amino acid such as Lys⁴¹,Thr⁴²,Ser⁶⁰,Pro⁶¹,Thr⁶²,Asp⁸⁰,Tyr⁸²,Arg⁸³,Cys¹⁰⁵,Trp¹⁰⁹,and Glu¹⁰⁸-Trp¹⁰⁹ were decreased to 9.99%,21.96%,10.09%,27.03%,8.38%,37.29%,8.38%,1.65%,16.74%,6.77%,and 9.96%,while the enzyme activities of Cys²⁵,His⁷⁸,and Glu¹⁰⁸ mutants was completely lost,indicating that these three amino acids played the most important role in maintaining enzyme activity.The wild-type recombinant YjeE protein can promote the normal cell growth,while the growth-promoting effects on V.harveyi cells of the different mutants weakened or disappeared,indicating that their growth-promoting effects are related to the ATPase activity.However,recombinant YjeE did not significantly promote the recovery of V.harveyi cells in VBNC state.The yeaZ gene of V.harveyi and its different mutant genes in the active sites such as Asp⁸⁸,Ser¹⁸⁵,Asp⁸⁸-Ser¹⁸⁵,Thr⁷¹,and Asp¹¹² were expressed and purified in E.coli.The protease activity of recombinant YeaZ with different mutants was measured using azocasein as a substrate.It was found that the enzyme activity of the wild-type recombinant YeaZ was 39130 U/mg,and the protease activity of YeaZ mutants containing Asp⁸⁸ and Ser¹⁸⁵ decreased to 24.28%and 35.27%.The protease activities of YeaZ mutants containing the two sites of Asp⁸⁸-Ser¹⁸⁵ were completely lost.The muralytic activity of YeaZ was determined with the artificial substrate4-methylumbelliferyl-?-DN,N?,N?-triacetylchitotrioside.The muralytic activity of wild-type YeaZ was 8.32 U/mg,while the muralytic activities at the mutation points Thr⁷¹ and Asp¹¹² were decreased by 33.22%and 59.74%.The recombinant YeaZ did not promote the normal cell growth of V.harveyi.However,it could significantly promote the resuscitation of VBNC state cells,and its resuscitation promoting abilities was related to the protease activity,but not to the muralytic activities.