The Role Of Extracellular DNA During Soil Bacterial Biofilm Development And Structure Construction

Soil is a highly structured ecosystem composed of heterogeneous microhabitats.More than 80% soil microorganisms adhere to soil minerals or mineral-organic complexes' surface,forming microcolonies or biofilm.Biofilm can gain competitive advantages through extracellular polymers secreted by microorganisms,such as enhancing quorum sensing between cells,assisting cells in colonizing on the surface,alleviating environmental stress.Extracellular DNA(eDNA),an essential biofilm matrix,plays a vital role in biofilm development and structure construction.However,it is still unknown whether eDNA plays the same role in the biofilm formation of soil bacteria.Moreover,the understanding eDNA's function on biofilm spatial distribution,especially at molecular and single-cell levels,is minimal.Therefore,our study explores eDNA's role in the initial biofilm formation and spatial structure construction of grampositive bacterium Bacillus subtilis and gram-negative bacterium Pseudomonas putida,which are the representative soil bacteria.Then the typical chemical analysis method,molecular biology technology and modern instrumental analysis methods such as laser confocal microscope(CLSM),super-resolution structure light microscope(SIM),atomic force microscope(AFM),quartz crystal microbalance with dissipation monitoring(QCM-D),isothermal titration microcalorimetric instrument(ITC)have been employed in the study.The main results are as follows:(1)The dynamic releasing process of eDNA from different soil bacteria and its influence on biofilm formation has been revealed.The results show that the amount of eDNA released by B.subtilis increased from 6.21 ng/m L to 75.56 ng/m L with time,and27.5% of eDNA was in the biofilm matrix.The amount of eDNA released by P.putida increased from 135.05 ng/m L to 328.99 ng/m L with time,and 81.2% of eDNA was located in the biofilm matrix.DNase I treatment significantly inhibited the B.subtilis biofilm formation,but had no significant effect on the biofilm formation of P.putida,suggesting that eDNA may play different roles in the different soil bacterial biofilm development.The eDNA sequencing analysis showed that the eDNA fragment of B.subtilis is about 500 bp in size,and its DNA is related to the genes related to biofilm formation.The size of eDNA fragment in P.putida biofilm is more than 1200 bp,and its DNA is related to the protein gene in the conserved region.Based on the bacterial live and dead staining technique,it was found that the eDNA was released by active secretion at the initial stage of B.subtilis biofilm,while that of P.putida was released by cell lysis.CLSM observation and co-localization analysis showed that the eDNA of B.subtilis overlaps and forms clusters with the cells,while the eDNA of P.putida does not form a specific structure with the cells.These results suggest that the spatial distribution of eDNA in biofilm may be the main factor affecting bacterial biofilms' formation.Further combined with SIM technology,it was found that the eDNA encapsulated cells and led to a longitudinal expansion of cells during the initial stage of B.subtilis biofilm,while the cells in P.putida aggregated irregularly,indicating that eDNA was actively released by B.subtilis to connect with cells,which affected the biofilm spatial structure construction,thus regulated the formation of biofilm.(2)The role of eDNA during the adhesion of B.subtilis on the goethite surface has been clarified.The results showed that in the condition of high ionic strength(100mmol/L Na Cl solution),the adhesion rate(?f/?t)of B.subtilis cell with eDNA on goethite surface is the fastest and the cells are easy to form micro-aggregates.Moreover,DNase I treatment can significantly inhibit the cell adhesion,while in low ionic strength conditions(1 mmol/L,10 mmol/L Na Cl solution),eDNA had no significant effect on bacterial adhesion.The QCM-D model fitting results showed that the contact viscoelasticity and contact area between B.subtilis cell and goethite were increased by6 times and 2 times under high ionic strength,respectively,while the effect of eDNA was not obvious under low ionic strength.Combined with ATR-FTIR and 2D-COS analysis,it was found that eDNA was beneficial to the formation of Fephosphate/phosphate ester complex between cell and goethite under the condition of high ionic strength,which promoted the stable adhesion of B.subtilis to goethite surface.With the decrease of Na Cl concentration from 100 mmol/L to 1 mmol/L,the characteristic peak of phosphodiester bond on cell with eDNA gradually weakened.In the process of cell adhesion,polysaccharide related groups such as glycosidic bond on cell surface preferentially adsorbed on goethite surface.Combined with extended DLVO(Derjaguin-Landau-Verwey-Overbeek)theory,electrostatic force and van der Waals force play an important role in the interaction between the interaction of goethite and cell with eDNA,while acid-base reaction mainly function in the interaction of goethite and cell without eDNA.The results of this study provide a useful basement for better understanding the function of eDNA in bacterial colonization.(3)The role of extracellular matrix in the dynamic process of spatial structure construction of B.subtilis has been clarified.DNase I treatment significantly inhibited B.subtilis biofilm formation in the early phases of biofilm development.Confocal laser scanning microscopy(CLSM)and image analysis revealed that eDNA was cooperative with exopolysaccharide in the early stages of B.subtilis biofilm development,while exopolysaccharide played a major structural role in the later stages.In addition,deletion of the exopolysaccharide production gene epsG in B.subtilis resulted in loss of the interaction between exopolysaccharide and eDNA and reduced the biofilm biomass in pellicles at the air-liquid interface.The physical interaction between these two essential biofilm matrix components was confirmed by ITC.Biofilm 3D structures become interconnected through surrounding eDNA and exopolysaccharide.eDNA interacts with exopolysaccharide in the early phases of biofilm development,while exopolysaccharide mainly participates in the maturation of biofilms.The findings of this study provide a better understanding of the role of the interaction between eDNA and exopolysaccharide in shaping the biofilm 3D matrix structure and biofilm formation.