Sorption Of DNA On Montmorillonite Influences The Horizontal Transfer Of Antibiotic Resistance Genes

Recently,microorganisms,plant or animal acquire antibiotic resistance genes(ARGs),due to antibiotics abuse and advances in transgenic technology.Large amounts of extracellular DNA are either secreted or released into environment from their living or lysed dead cells.After adsorption by soil clay minerals,DNA which carries ARGs can persist in the soil environment and maintain its transformation activity.Under appropriate conditions,ARGs may be transferred to other microorganisms even human pathogens through horizontal gene transfer(HGT),endangering public health and safety.Therefore,studies entailing DNA sorption and HGT of ARGs in soil are essential for people to understand the fate of ARGs,and avoid its potential risks.Montmorillonite is the principal clay mineral in the soil,with its surface properties affected by particle size and metal cations.However,the influences of sorption of DNA by montmorillonite on HGT of ARGs are still unclear in china and abroad.In this paper,three metal cations(Na+,Ca2+and Fe3+)were used to modify montmorillonite(MMT)to prepare the metal cation-modified MMT.The sorption of extracellular DNA on modified MMT and its mechanisms were studied.Montmorillonite was graded into three particle sizes of 1～5 μm,100 nm～1μm and<100 nm.The effect of DNA sorption by different particle sizes of MMT on HGT of ARGs and its mechanisms were studied.The main results are as follows:(1)The sorption of extracellular DNA on metal cation-modified MMT was illuminated.Modified MMT have high specific surface area(SSA)and cation exchange capacity(CEC),the highest were up to 77.42 m2·g-1 and 98.79 cmol·kg-1,respectively.Metal cation modification can promote the sorption of DNA on MMT.Compared with MMT(3.5 μg·mg-1),the amount of DNA sorption on modified MMT were increased by 0.74～5.38 times,and followed the descending order of Fe-MMT>Na-MMT>Ca-MMT>MMT.The sorption of DNA on modified MMT was affected by environmental factors,such as temperature,pH,electrolyte.Compared with 15 and 35℃,the sorption capacities of DNA on modified MMT were the largest at 25℃,and decreased with the increase of pH value(3.0～9.0).The addition of electrolytes(50 mM NaCl or CaCl2)can promote the sorption of DNA on MMT.The amounts of DNA sorption were 12.84、30.47μg·mg-1,respectively,reaching the promoting effect of DNA sorption on modified MMT.and the promoting effect of Ca2+ was stronger than that of Na+.(2)With the aid of XRD,FTIR,XPS,ICP-OES analysis and molecular dynamics simulation,the mechanisms of DNA sorption on metal cation-modified MMT were revealed.The results of XRD analysis showed that DNA whose diameter was 2 nm could not be intercalated into the modified MMT layers(～1.5 nm).After DNA sorption,the interlayer spaces of MMT,Na-MMT,Ca-MMT and Fe-MMT decreased by 0.06、0.15、0.08 and 0.28 nm,respectively,which was consistent with the rule of DNA sorption on modified MMT.The reduction indicated DNA sorption led to the loss of interlayer water of modified MMT,thus,causing the interlayer spaces of modified MMT decreased.Compared with the FTIR spectra of DNA,the changes after DNA sorption indicated that modified MMT interacted with the phosphate groups and bases of DNA,and the conformation of DNA changed from the B-form to the Z-form.Besides,the binding energy of O atom changed by 0.1 eV,and the binding energies of N atom and P atom did not change after DNA sorption,indicating that the sorption site of modified MMT and DNA were on O atom.Based on the results of FTIR assessment,the sorption sites were further determined to be O atom of DNA phosphate group(PO4-).Binding energies of Na,Ca and Fe atoms all changed after sorption,indicating that Na,Ca and Fe atoms were involved in DNA sorption.Moreover,metal cations bound on the surface of the modified MMT did not enter into the sorption solution.Combined with the results of XPS and ICP-OES analysis,we concluded that the mechanisms of metal cation modification promoting the sorption of DNA on MMT were the cation bridging of metal cations on the surface of modified MMT.GROMACS software was used to simulate the sorption of DNA on modified MMT,and the interaction energy and root mean square deviation(RMSD)were calculated.The interaction energy between DNA and MMT is mainly manifested as van der Waals energy,which is less than 60 kJ·mol-1;while that of Na-MMT,Ca-MMT and Fe-MMT are mainly exhibited as an electrostatic energy,which is greater than 1000,500 and 3000 kJ·mol-1,respectively.The results indicated that the interactions between DNA and modified MMT were stronger than those of MMT.RMSD showed that the systems of modified MMT and DNA were more stable than those of MMT.Finally,the sorption sites of O atoms in DNA phosphate group(PO4-)and mechanism of cation bridging can be directly determined by visualized analysis.The theoretical simulations and experimental results verified the sorption mechanism between DNA and modified MMT.(3)The effects of DNA sorption by different particle sizes of MMT on HGT of ARGs and its mechanisms were analyzed.Through wet sieving,sedimentation,centrifugation,and cross-flow ultrafiltration,MMT was effectively classified into three particle sizes:1～5 μm,100 nm～1 μm and<100 nm.The three particle sizes of MMT had no significant effect on the survival of E.coli.As the concentration of MMT increased,it first promoted and then inhibited HGT of ARGs,as the smaller particle size of MMT have a stronger inhibitory effect.When 1～5 μm,100 nm～1 μm and<100 nm fraction of MMT was at the concentrations of 0.05,0.005,and 0.005 mg·mL-1,the number of ARGs transformants reached the maximum at 28733,15300,and 23000 cfu·mL-1,respectively;when the concentrations of them were up to 0.5,0.25,0.05 mg·mL-1,no transformants occurred.Through fluorescence in situ hybridization,it was further verified that the resistance plasmid can be entered into E.coli more easily under the low concentrations of MMT.The results of sorption experiments showed that DNA could be completely absorbed by 1～5 μm,100 nm-1 μm and<100 nm fraction of MMT at the concentration of between 0.5 and 2 mg·mL-1,and then inhibiting the HGT of ARGs.The results of SEM images and flow cytometry showed that high concentrations of MMT caused holes on the membrane of E.coli,which increased the cell permeability.However,it could not promote HGT of ARGs.The results of ICP-OES showed that various metal ions were released by MMT.As the concentration of released metal ions increased with the increase of MMT concentration,this led to an initial promotion,then inhibition of HGT of ARGs,which is consistent with the effect of MMT on HGT of ARGs.Therefore,metal ions released by MMT can affect the physiological and biochemical activities of E.coli,thus promoting HGT of ARGs.