The Molecular Mechanism Of Reduced Glutathione Involved In Monomethylarsonate Tolerance And Reduction In Enterobacter Sp.CZ-1

Arsenic(As)is a toxic metalloid that is widely distributed in nature.There are some common arsenic species in the environment like arsenite As(?),arsenate As(?),and various methylated arsenicals.In order to cope with the toxic stress of arsenicals,microorganisms in the habitat have evolved various detoxification strategies,including reduction,oxidation,efflux and methylation mechanism.Arsenic methylation refers to the process of continuously adding methyl groups to the arsenic atoms of inorganic arsenicals by using methyl donors.The products of this process include monomethylarsonate MAs(?),dimethylarsinic acid DMAs(?),pentavalent trimethylarsine oxide TMAs(?)O and trimethylarsenite TMAs(?),etc.MAs(?)is a common product of arsenic methylation in the environment.It had been widely used in industry,agriculture and animal husbandry,because MAs(?)has weeding effect and its toxicity is low to human body.Although MAs(?)is less toxic than inorganic arsenic,it still has potential environmental hazards.Excessive application of MAs(?)in the soil will inhibit rice growth,affect rice reproduction and grain development.MAs(?)also will accumulate in rice roots,stems,leaves and grains.So that As from soil can move to plants and food chain,threaten ecosystems and human health.In addition,MAs(?)will also be transformed by microorganisms and be reduced to highly toxic methylarsonous acid MAs(?).MAs(?)can be demethylated to highly toxic As(?),and also can be further methylated to DMAs(?)(can induce rice straighthead)and TMAs(?)(a highly volatile arsenical with highly toxic)in the environment.Enterobacter sp.CZ-1 which isolated from an As-contaminated paddy soil collected in Chenzhou city,Hunan province was selected as this study material.In contrast to other strains,Enterobacter sp.CZ-1 had higher MAs(?)tolerance under the situation that MAs(?)is less toxic to many microorganisms.So this study started with the screening of the CZ-1's MAs(?)tolerance gene,and used a variety of molecular biology methods such as shotgun method.heterologous expression and gene knockout technology to obtain a better understanding on how the microorganisms cope with MAs(?)in the environment.The main results of this article are as follows:(1)The difference between the E.coli Trans5? and Enterobacter sp.CZ-1 about MAs(?)tolerance was significant.E.coli Trans5? could tolerate only 1.75 mM MAs(?)in ST10"1 solid culture medium,while Enterobacter sp.CZ-1,which is closely related to it,could tolerate 750 mM MAs(?).So that the gshA gene involved in the MAs(?)tolerance was identified in the genome of strain CZ-1 by constructing the gene library in strain Trans5a,and its encoding product was responsible for catalyzing the first step reaction of microbial intracellular GSH biosynthesis.Heterologous expression of gshA increased the intracellular GSH content in E.coli Trans 5? and conferred E.coli Trans 5? with much higher tolerance to MAs(?).In order to further explore the roles of gshA gene and endogenous GSH in strain CZ1's MAs(?)tolerance,this study performed a knockout experiment and got ?gshA mutant and ?gshB mutant(the encoded product of gshB is responsible for the second step of GSH biosynthesis).Compared with wild-type strain,the intracellular GSH synthesis ability of the two mutant strains was basically lost and the MAs(?)tolerance both decreased significantly.Exogenous addition of 50 ?M GSH could restore the MAs(?)tolerance.This result indicated that endogenous GSH was involved in the MAs(?)detoxification of strain CZ-1.(2)Further experiments showed that strain CZ-1 have the ability to reduce MAs(?)and 5 ?M MAs(?)can be almost completely reduced to MAs(?)within 24 h in ST10-1 fluid medium by strain CZ-1.However the MAs(?)reducing capacity of ?AgshA mutant and ?gshB mutant decreased by about 50%and 65%respectively.Exogenous addition of GSH could restore the MAs(?)reducing capacities of ?gshA mutant and ?gshB mutant to 80%and 73%of strain CZ-1 respectively.Turning to the experimental evidence on MAs(?)tolerance,the knockout of gshA and gshB did not affect the MAs(?)tolerance of strain CZ-1.These results indicated that endogenous GSH was involved in the MAs(?)reduction,but did not participate in the MAs(?)detoxification process in strain CZ-1.Surprisingly,E.coli Trans 5? also had extremely weak ability to reduce MAs(?),heterologous expression of gshA gene could improve the MAs(?)reducing capacity of E.coli Trans 5? significantly.For further explain how the GSH involved in MAs(?)detoxification,intracellular As content and reactive oxygen species(ROS)in wild-type strain and two mutant strains under MAs(?)stress were measured.The results showed that the intracellular As content of strain CZ-1 remained stably under the treatment of MAs(?).but the intracellular As content of the ?gshA mutant and ?gshB mutant both increased and were higher than the wild-type strain,the?gshA mutant is more susceptible to MAs(?)than the ?gshB mutant.About ROS,strain CZ1's ROS hardly changed with the 2.5 mM MAs(?),while the ROS of the ?gshA mutant increased significantly.Together these results provided important insights into two detoxification mechanisms of MAs(?)in Enterobacter sp.CZ-1,a)GSH participates in the reduction of MAs(?)and the reduction product eliminate through the efflux channel of MAs(?)thereby decreasing the accumulation of As in Enterobacter sp.CZ-1;b)GSH can stabilize the intracellular ROS level and avoid the cellular damage caused by As stress.MAs(?)is ubiquitous in the environment,the microbial transformation and detoxification mechanism about it are important components of the biogeochemical cycle of arsenic.However,there are no reports about the detoxification mechanism of MAs(?)in microorganisms.This study focused on the MAs(?)tolerance and reduction mechanism of microorganisms,An important gene gshA involved in MAs(?)tolerance was screened,and two reasonable conjectures about the detoxification mechanism of MAs(?)were obtained.These findings have important implications for developing the transformation mechanism between different As in the environment by microorganisms,and help people to understand the mechanism of microorganisms dealing with As stress in ecological environment.