Characterizations Of Lysis-related Proteins From Deep-sea Thermophilic Bacteriophage GVE2

As the important components of geobiological system,deep-sea microbes play important roles in ecology,resources and environments,and also may light the way to the development of new drugs,industrial processes,and other products useful to us all.In the past,microbes are thought to be the key factors affecting the hydrothermal vent biological community.However the existence of bacteriophages in the relatively original and extreme deep-sea ecosystems proves that bacteriophages may be its controller.Studies on deep-sea thermophilic bacteriophages will be helpful for understanding the control of hydrothermal vent biological communities,but also of great potential for exploring the origin of life. The lysis of bacteria by bacteriophages leads to the death of bacteria,and further influences the multiplication of microbes and the food chain.Therefore,it is meaningful to study on bacteriophage lysis.In this investigation,the lysin and holin of thermophilic bacteriophage GVE2 isolated from a deep-sea thermophilic bacterium were characterized to understand the lysis mechanism of bacteriophage at high temperature.In some cases,the lytic range of a lysin may considerably exceed that of the phage from which it is derived,but also display a narrow spectrum of the same genus. However,amidases have been suggested to display a broader spectrum of antibacterial activity than other classes of endolysins because it can cleave the amide bond between N-acetylmuramic acid and L-alanine in peptidoglycan.The specificity of lytic range makes it have no harmful to healthy.Lysin as a new antibacterial has great potential. The use of the lysin attracts people's interest.Most lysins are obtained from mesophilic bacteriophages and only very few lysin is from thermophilic bacteriophage.In this study,the GVE2 lysin belonging to N-acetylmuramoyl-L -alanine amidases groups was characterized.The recombinant lysin was active over a range of temperature from 40℃to 80℃,with an optimum at 60℃.Its optimal pH was 6.0,and it was stable over a wide range of pH from 4.0 to 10.0.when the Lysin was pre-incubated in a series of pH buffers pH5.0-10.0 for 1 hour,it still show 50% relative activity.The recombinant lysine exhibited good tolerances at acid pH values.The lysin was highly active when some enzyme inhibitors ordetergents (phenylmethylsulfonyl fluoride,Tween-20,TritonX-100,and chaps) were used. However,it was strongly inhibited by sodium dodecyl sulfate and ethylene diamine tetraacetic acid.Its enzymatic activity could be slightlystimulated in the presence of K⁺,Li⁺ and Na⁺.But the metal ions Mn²⁺,Ca²⁺,Zn²⁺,Ba²⁺ and Mg²⁺ showed inhibitions to the lysin activity.In the double-strand DNA bacteriophages,the lysin lacks a secretory signal sequence to mediate its release across the cytoplasmic membrane.In this case,the holin forms pores in the cytoplasmic membrane of the host to allow the lysin to access the peptidoglycan layer of host cell.Our study indicated that there was interaction between GVE2 lysin and holin by Far-Western blot analysis.The followed investigation using bacterial two-hybrid system revealed that the holin could bind the 81-233-aa domain of lysin.Based on GST pull down and co-immunoprecipitation,it was found that the host ABC transporter protein,alcohol dehydrogenase zinc-binding domain protein and EF-Tu were interacted with GVE2 lysin.The results showed that the GVE2 lysin might interact with the host alcohol dehydrogenase zinc-binding domain protein and ABC transporter protein in the lysis protein complex.