The Mechanism Of Chromate And Selenite Resistance And Reduction In Alishewanella Sp.WH16-1

Chromium（Cr）is a transition metal and has very important application in industry.The long time mining and extend applications lead to the Cr pollution.Cr exists as Cr（VI）（chromate and dichromate）and Cr（III）two valences in the nature environment.Cr（VI）is high teratogenic,carcinogenic,soluble and easy to migrate with water,while Cr（III）is low toxic,low soluble and easy to be immobilized.Selenium（Se）is one of the essential trace elements in human body,but excessive selenium is harmful for human health.There are four valences:Se（IV）,Se（IV）（selenite）,Se（0）and Se（-II）in the nature environment.Among them,Se（IV）is the most toxic and high soluble,while Se（0）is nontoxic and low soluble.Therefore,it is of great environmental significance to reduce Cr（VI）and Se（IV）to Cr（III）and Se（0）,respectively.Microorganisms play an important role in the reduction of Cr（VI）and Se（IV）and even in the completely geochemical cycle of Cr and Se.The application of microorganism in the remediation of Cr and Se pollution takes the advantages of low price and no secondary pollution.However,the microbial Cr（VI）and Se（IV）resistance and reduction mechanism are still need to be further study.Alishewanella sp.WH16-1 is a facultative anaerobic bacterium isolated from soil of a copper and iron mine.This strain is Gram-negative,rod-shaped and motile.Its genome comprises 3,488,867 bp,50.4%G+C content,3,132 protein-coding genes.Under aerobic conditions,it was resistance to multiple heavy metal（loid）s Cr（VI）,Se（IV）,Cd（II）,Pb（II）,Cu（II）and As（III）,as well as efficiently Cr（VI）and Se（IV）reduction abilities.The reduction products of Cr（VI）and Se（IV）were of Cr（III）nanoparticles（CrNPs）and Se（0）nanoparticles（SeNPs）,respectively.In addition,it could also efficiently remove Cd（II）and Pb（II）.Various genes related to heavy metal resistance were found in the genome,which was consistent with the phenotype of heavy metal resistance,such as:a variety of heavy metal（loid）s transporters and the potential Cr（VI）reductive protein CsrF.Strain WH16-1 was a sulfate reducing bacterium,and complete sulfate reduction path was predicted by genome analysis.The sulfate reduction product S（-II）could form CdS and PbS precipitates with Cd（II）and Pb（II）,respectively.Through Tn5 transposon mutation,our research group found a putative Cr（VI）resistance gene cytbd（containing CydA and CydB coding genes）.This gene encodes Cytchrome bd（Cytbd）,and the Tn5 was located in the cydB gene.The analysis of this thesis showed that:（1）gene cydE（coding for a GbsR-type regulator）was co-transcribed with cydA and cydB,based on RT-PCR analysis and similar gene arrangements were also found in other Alteromonadaceae family strains;（2）the Cr（VI）resistance level was dramatically decreased and Cr（VI）reduction efficiency also decreased in strain△cytbd compared to the wild-type and a complemented strain（△cytbd-C）;（3）Cytbd could catalyze the decomposition of H₂O₂ according to the analyses of H₂O₂decomposition ability,cellular H₂O₂ contents,H₂O₂ inhibition zone,and H₂O₂sensitivity tests;（4）surprisingly,chromate was not an inducer of the expression of Cytbd,but sulfate induced expression of Cytbd,and sulfate/sulfide resistance levels were also decreased in the△cytbd strain;（5）the addition of sulfate enhanced the Cr（VI）resistance level and reduction efficiency;（6）Cytbd expression was repressed by CydE and derepressed by sulfate based on an in vivo bacterial one hybrid system and in vitro EMSA tests;and（7）DNA footprinting and short-fragment EMSA tests revealed two binding sites of CydE in its promoter region.All these results showed that Cytbd is negatively regulated by CydE and derepressed by sulfate.In addition,Cytbd contributes to the resistance of sulfate and sulfide,and sulfide could be used as a reductant to reduce chromate.Hence,Cytbd has an indirect effect on Cr（VI）reduction.Moreover,Cytbd is essential to decompose H₂O₂ to decrease cellular oxidative stress.Thus,the regulation and function of Cytbd may explain why sulfate could enhance chromate reduction.In order to further clarify the Cr（VI）and Se（IV）reduction mechanism in strain WH16-1,gene csrF encoding a flavoprotein was studied.Bioinformatics analysis showed that the amino acid similarity of CsrF and the reported Cr（VI）reductase was37%.Experimental results of gene knockout,complementation and heterogeneous overexpression showed that CsrF could reduce both Cr（VI）and Se（IV）in vivo.The purified CsrF was yellow and the absorption peak spectrum was consistent with FMN.The molecular weight of the monomer was 23,906,and the molecular weight of dimer was 47,960.CsrF could use NAD（P）H as electron donor to reduce Cr（VI）and Se（IV）in vitro,and the optimum pH was 7.0,the optimum temperature was 30 to 37℃.The enzyme could also reduce SO₄^2-and Fe³⁺,but not As（V）and NO^3-.The Km of Cr（VI）and Se（IV）were 250.6±23.46μmol/L and 204.1±27.91μmol/L,respectively.Site-direted mutagenesis results indicated that Arg13 and Gly113 are essential amino acids for CsrF.The csrF gene was cloned into Escherichia coli to improve the production of bio-SeNPs.Transmission electron microscope,element mapping,scanning electron microscope,energy spectrum analysis,Zeta potential,dynamic light scattering,infrared spectrum and X-ray photoelectron spectrum analyses results showed that the diameter of bio-SeNPs are 60-105 mm and composed of Se（0）,proteins,lipids and other substances.Further experiments showed that the bio-SeNPs could efficiently adsorption removal of anionic dyes（congo red）under acidic conditions and cationic dyes（safranine T and methylene blue）under alkaline conditions.Adsorption kinetics,isothermal adsorption lines and adsorption thermodynamics analyses indicated that the maximum adsorption capacity of bio-SeNPs on congo red,safranine T and methylene blue were 1,577.7,1,911.0 and 1,792.2 mg/g,respectively,which was 6.8%,25.2%and49%higher than that of traditional biomass materials.The adsorption process was spontaneous and primarily physical reaction.The adsorbed dye can be desorbed by 200mmol/L NaCl with good reusability.In summary,Alishewanella sp.WH16-1 was resistant to multiple heavy metal（loid）s and could efficiently reduce Cr（VI）and Se（IV）,and the reduction products were CrNPs and SeNPs,respectively.Moreover,a novel Cr（VI）resistant gene,cytbd was identified.Its transcription was repressed by CydE and derepressed by sulfate.Furthermore,a potential Cr（VI）reductase CsrF was analyzed.In vivo and in vitro experiments showed that this enzyme could reduce both Cr（VI）and Se（IV）,indicating this is a novel bacterial Cr（VI）and Se（IV）reductase.In addition,overexpression of CsrF in E.coli can promote the formation of bio-SeNPs.The extracted bio-SeNPs could efficiently adsorption removal of organic dyes.This is the first report of adsorption removal dyes by bio-SeNPs.