Intracellular Compartmentalization Of Fungi To Arsenic And Gene Diversity Of Arsenic Transformation

Microorganisms participate in the biological earth cycle of arsenic in the environment,and also show good application potential in environmental restoration and detoxification.The efflux,transformation and compartmentalization of arsenic in the cells of environmental microorganisms are important ways of arsenic detoxification.However,it still lacks attention to the arsenic division and the key gene diversity of arsenic transformation of microgranisms at the subcellular level,especially for eukaryotic microorganisms.This paper employed three isolated fungi in the previous work,namely Fusarium oxysporum CZ-8F1,Penicillin jantihelium SM-12F4,and Trichoderma asperellum SM-12F1,as the target strains.The optimized enzymatic-differential centrifugation method was used to obtain each subcellular phase of eukaryotic microorganisms,and the ability of each subcellular phase to separate arsenic was quantitatively analyzed.Synchrotron radiation-based X-ray scanning transmission microscopy(STXM))was applied to in situ analyze the accumulation and distribution of arsenic at the subcellular level of fungi.On the basis of whole genome sequencing results,annotated genes related to arsenic accumulation and transformation of the target fungi were obtained,and the genetic diversity and mechanism of arsenic transformation related genes were systematically analyzed.The relevant results have important scientific significance for understanding the interaction mechanism between environmental microorganisms and arsenic and promoting its application in environmental remediation.(1)Based on the enzymatic-differential centrifugation method,the fungal subcellular phase were separated and the quantitative analysis of arsenic accumulation were carried out.This work optimized the condition of sepreating subcellular phase of the target fungi and successfully sepreated the cell walls,protoplast and vacuole.After exposure to 10 mg?m L^-1 of arsenate(As(V))2-5 days,the concentrations of As in different fungal organelles were generally increased followed the order of extracellular(65%)>cell wall(15%)>vacuole(10%)>other organelles(8%).The amount of As in vacuole accounted for 55%of the total As in protoplast and other organelles As accounted for 45%of total Asin protoplast.Inmobilization by cell wall and compartmentalization by vacuole were the main mechanisms of As resistance in the fungal strains tested.A further correlation analysis found the content of glutathione(GSH)presented a significantly positive correlation with accumulation of As(p<0.01)which can be used as a reasonable indicator to assess the sequestration ability of fungi.(2)The accumulation characteristics of arsenic in fungal subcellular phases were studied by insitu STXM technology.After culturing for 15 days with a concentration of 500 mg As(V)L^-1,transmission electron microscope(TEM)and scanning electron microscopy(SEM)results found that no significant changes was observed on the fungal intracellular and mycelium with the treatment of high concentration of arsenic stress,and the energy dispersive spectrometer(EDS)analysis obviously found the signals of arsenic accumulation on the mycelium of fungal cells.Fourier transform infrared spectroscopy(FT-IR)was used to scan the fungal cell wall revealing that the characteristic peaks of As-O are enhanced,while the C=O stretch of carboxylic acids,N-H stretch of amides and Si-O stretch are weakened.This results indicated that the bonding of carboxyl and amine groups on the fungal cell wall can bind As or replace Si in Si-O is the main mechanism for its adsorption and fixation of arsenic.Further,STXM analysis found that As was mainly accumulated in the septum and vacuole of fungal cells.The fixation of intracellular arsenic by fungal septum was the first time found which was supposed to help reduce the toxicity of arsenic to neighboring cells.In summary,eukaryotic microbial cell wall adsorption,vacuole compartmentation and membrane fixation are important mechanisms for its resistance and detoxification of arsenic.(3)Based on whole-genome sequencing to analyze the gene diversity related to arsenic accumulation and transformation by Trichoderma aculeatus.Assembly work was conducted by the Pac Bio and Illumina Hiseq platforms and compared with COG database and HMMER software,annotated proteins related to As accumulation and transformation of Trichoderma asperellum SM-12F1were obtained.It was found that the whole genome of the strain contained As(III)oxidation(1 aoxA,1AoxC)and As(III)efflux(1 ArsA,1 ArsB,3 Acr3),As(V)reduction(1 ArrA,1 ArsC2),2 Acr2),As(III)methylation(19 ArsM),organic arsenic oxidation(7 ArsH)and demethylation(2 ArsI),arsenic partition(2 Ycf1p),etc.41,12 types of arsenic functional annotation proteins.Among them,the most methylation genes accounted for 46%of the total annotated proteins;the number of As(V)reduction was greater than As(III)oxidizing proteins.Combined with the previous knowledge of the ability of this strain to transform and accumulate arsenic,the potential pathways and molecular mechanisms of eukaryotic microorganisms to arsenic metabolism are proposed.