The Function Of Flagella In The Extracellular Electron Transfer Of Geobacter Species And Its Underlying Mechanisms

Geobacter species are a kind of anaerobic bacteria with extracellular respiration ability,are abundant in soil and groundwater sediments.They may be completely oxidize organic compounds intracellular,and generated electrons are transmitted to the extracellular electron acceptors(such as Fe(?)oxides,humus,electrodes,syntrophic partners,etc.)through the intracellular respiratory chain,reduce them and generate energy to maintain their own growth.The unique extracellular electron transfer capability of Geobacter species has important practical application in elemental biogeochemical cycles,soil bioremediation,pollutant degradation,and anaerobic digestion of methane production.Geobacter sulfurreducens,as a model strain in Geobacter species for which has complete genome sequence and complete genetic manipulation method,and is widely used to study the metabolism,gene regulation and extracellular electron transfer.G.sulfurreducens is rich in extracellular cytochrome c,extracellular polymers,pili,and flagella.Early studies have shown that extracellular cytochrome c,extracellular polymers,and pili of G.sulfurreducens can mediate extracellular electron transport.However,the role of flagella in extracellular electron transfer of Geobacter species has not been reported.There are two strains in G.sulfurreducens,the strain KN400 can produce flagella,is motility,while the strain PCA is not.Through the genetic manipulation by inhibiting flagellin expression of strain KN400 or restoring flagellin expression of strain PCA to investigate the function of flagella in the extracellular electron transfer of Geobacter species by using ferrihydrite,graphite electrodes,and Geobacter metallireducens as extracellular electron acceptors.The main results are as follows:(1)Based on analysis of the gene encoding flagellin,flagellin mutant was constructed.Through the genetic manipulation by inhibiting flagellin expression of strain KN400 and strain GS-15 or restoring flagellin expression of strain PCA to obtain mutant strains such as KN?FilC? GS-15?FilC and PCA-PRG5fgrM.(2)Flagella promote the reduction of extracellular insoluble electron acceptors(ferrihydrite)by G.sulfurreducens.The results showed that the rate of ferrihydrite reduction of the strain PCA-PRG5FgrM was higher than that of the wild-type strain PCA,while the ferrihydrite reduction rate of the strain KN?FilC was lower than the wild-type strain KN400.It shows that flagella promote the reduction of ferrihydrite by G.sulfurreducens.Considering flagella as a motility organ can help G.sulfurreducens locate much fresh ferrihydrite and promote the reduction of ferrihydrite.(3)Flagella can improve the current production of G.sulfurreducens.Through analysis of biofilm thickness and total protein content,it was found that flagella promotes the formation of biofilms;in terms of extracellular cytochrome c content and composition,the absence of flagella reduces the content of extracellular cytochrome c,but its composition has no effect;meanwhile,electrochemical methods are used to characterize the anode biofilm and it is found that the expression of the flagella can increase the ability of the strain to transfer electrons to the electrode.The results showed that flagella can be used as a scaffold structure to promote the formation of electrogenic biofilm,increase the area of the electrode production of electrocatalytic biomass and extracellular cytochrome c content,thereby increasing its current production.(4)Flagella play no role in direct electron transfer of Geobacter species.The co-culture system of Geobacter metallireducens GS-15 and Geobacter sulfurreducens KN400 was constructed to study the role of KN?FilC and GS-15 ?FilC in co-culture.As a result,it was found that the mutants lacking the flagella can still form co-cultures,but their growth cycle is prolonged.This shows that flagella are not directly involved in the direct electron transfer between species,but favor the formation of co-culture system.In this study,we constructed the mutants with flagellin expression and deletion in G.sulfurreducens,indicating that flagella promotes extracellular electron transfer processes such as reduction of ferrihydrite,electrode,and syntrophic partners,which not only changes the traditional understanding of flagella only as a motility organ of bacteria,but also provides a new perspective for understanding the extracellular electron transfer process of microorganisms.