Isolation And Identification Of The Silver-Nano Particles Resistance Bacterium Enterobacter Sp.IS-1 And Investigation Of The Molecular Mechanism Of The Tolerance Of Silver-Nano Particles

The use of silver-based microbicides has a long history,and it could even date back to 400 BC.Common silver-based bactericides,such as silver nitrate and silver sulfadiazine,their bacteriocidal activities rely on silver ions completely and therefore they are commonly-used in a variety range of fields,such as medical facilities,chemistry,food packeting and other daily products.Silvernanoparticals(AgNPs)is a new type of silver-based antibacterial material under the background of nanotechnology,which has both the advantages of nanomaterials and silver atoms.First,it can achieve antibacterial effect by releasing sil-ver ions(Ag+);Besides,due to its large specific surface area,it has excellent photochemical and electrochemical properties compared with other antimicrobial and consequently it can have a better bactericidal effect.While the high-efficiency antibacterial effect of AgNPs is widely reported,the explosive growth of its use has led to its increasing release into the environment,which could be a potential threat to environmental safety and human health.Among this issue,people are more concerned about whether the massive use of AgNPs can induce bacterial resistance,and if there is any AgNPs resistant strain in the environment now.In this study,we aimed at the strain Enterobacter sp.IS-1,a AgNPs resistant strain isolated from industrial sludge,attempting to reveal its molecular mechanism to AgNPs toxicity.The research results are as follows:1)First,we treated sludges with AgNPs for 72 hours,and carried out a high-throughput sequencing of 16S rRNA of bacteria and archaea of the sludge.The results indicate that Enterobacter is the dominant microflora in the sludge environment.Subsequently,we found a strain that could tolerate about 75 mg/L AgNPs from the sludge sample and we named it IS-1.Strain IS-1 was identified as an Enterobacter through the 16S rRNA gene sequence analysis.In order to illuminate the molecular mechanism of this strain towards AgNPs,we sequenced the whole genome of strain IS-1.It was found that the strain IS-1 contains a typical silver ion resistance gene cluster-silPABCRSE,which contains the following 7 genes:silP,silA,silB,silC,silR,silS,silE.Among them,silP and silABC encode two sets of Ag+efflux systems respectively,which are responsible for removing intracellular Ag+;silR and silS encode a cellular kinase and a transcriptional regulatory element respectively,and their main function are to recognize Ag+;The encoded product of silE is a heavy metal bind-ing protein,which can complex the free silver ions in the cell to ease its toxi-city.We hypothesize that silPABCRSE is not only involved in the detoxification of silver ion,but also participates in the detoxification of silver nanoparticles.To verify this hypothesis,we knocked out the silPABCRSE.After clarifying the difference in silver resistance between the mutant strain IS-1(?silPABCRSE)and the original bacteria IS-1,we found that there is also a clear difference in the resistance towards AgNPs.Under the treatment of the same concentrations of AgNPs,the growth of mutant strain IS-1(?silPABCRSE)is greatly com-promised when compared with strain IS-1,which indicates that silPABCRSE is involved in the nanosilver resistance of strain IS-1 indeed.2)We found that IS-1(?silPABCRSE)mutant strain can still grow in TYE liquid medium containing 25 mg/L AgNPs and surprisingly during the growth of this strain,there would be some black sediments at the bottom of the flask.Similarly,there would be a black-brown precipitation circle when the mutant strain was cultured on TYE solid plate containing 25 mg/L AgNPs.In order to figure out whether the formation of black precipitate is an another way for the strain IS-1 to deal with the toxicity of AgNPs,we conducted a SEM-EDS analysis of the black precipitate and found that the Ag content accounted for 49.4%,and C?N accounted for 25.7%and 24.2%respectively.Based on this results and previous research,we speculate that the black precipitate may be caused by the aggragation and flagellin may be linked with it.In order to verify this hypothesis,we knocked out the flagellin gene fliC and obtained two mutant strains:IS-1(?fliC)and IS-1(?silPABCRSE&fliC),and simultaneously carried out the experiment of adding ellagic acid to suppress the function of flagellin.We found that the knockout of fliC and the addition of ellagic acid couldn't affect the formation of black precipitates,and neither to the tolerance of AgNPs.This article systematically studied the possible mechanism of AgNPs resistance in strain IS-1.First,it was confirmed that the Ag+resistance gene cluster silPABCRSE is an important mechanism for strain IS-1 towards AgNPs;Secondly,aiming at the cause of AgNPs aggregation,combined with relevant researches,the experiment of flagellin main gene fliC knockout and flagellin inhibitor PGRE were carried out.The results showed that flagellin has nothing to do with the AgNPs aggregation and the specific mechanism needs further investigation.