Effect And Mechanism Of Zinc Ion And Transcription Factor RBMA?RS17245 On Bacillus Amyloliquefaciens FZB42 Biofilm Formation

Biofilm is a key factor which may influence the colonization of Bacillus spp.around plant roots.Recently,there are many studies on the mechanism related to the biofilm formation of Bacillus,but the influence of metal ions has not been reported.There are a lot of metal ions in the soil,and some metal ions are necessary for the organism,but the excess metal ions will influence their physiological activity and even lead to poisoning to the organism.The average content of zinc ions in the soil of southern China is high.This brings a test for the application of Bacillus spp.in soil:Does zinc ion affect the ability of Bacillus spp.biofilm formation,and what's the mechanism?This study focused on this issue and explained the role of zinc ions in the B.amyloliquefaciens biofilm formation.On the other hand,previous studies in our laboratory have found that spermidine is a very important biomass in the metabolites of Bacillus and can affect the formation of biofilms.B.amyloliquefaciens FZB42 possesses a complete spermidine synthesis gene cluster but cannot detect the production of spermidine.Through sequence analysis,we found a gene locating in the upstream of the FZB42 spermidine synthesis gene cluster,which contains a typical DNA binding site and could be a transcription factor.In this study,the RBAM?RS17245 deletion mutant and its complement mutant were constructed,the regulation mechanism of this transcription factor in FZB42 spermidine biosynthesis was studied by HPLC and qRT-PCR.1.Study of the inhibition of bacillus amyloliquefaciens biofilm formation ability by Zinc ion with non-growth-inhibiting doseBiofilms are architecturally complex communities of microbial cells held together by a self-produced extracellular matrix.Biofilm can help Bacillis spp.to colonize around plant roots successfully and keep their tolerance to biotic and abiotic stress.Considerable research has focused on the environmental signals that trigger or inhibit biofilm formation by affecting cellular signaling pathways.However,response to soil cues in plant-associated Bacillus has remained largely unaddressed.The concentration of zinc ions in the soil of southern China is relatively high,and B.amyloliquefaciens FZB42 has been commercialized for use in farming operations.Whether biofilm formation can be formed is the key to its biocontrol effect.Therefore,we aimed to investigate the effect of Zn(?)ions in biofilm formation of Bacillus amyloliquefaciens FZB42.We found that the biofilm formation ability of B.amyloliquefaciens FZB42 was inhibited by Zn(?)at 100-250 ?M zinc ion conditions(non-growth inhibitory concentration),which was characterized by reduced wrinkles and thinning,100 and 200?M Zn(?)The thickness after treatment is reduced by 25%and 75%,respectively.This influence results from the inhibition of the production of extracellular polysaccharides and TasA proteins by Zn(?),which are the main components of the extracellular matrix.The deletion of these two proteins prevents the formation of biofilms by Bacillus.Furthermore,the presence of Zn(?)suppressed activity of the response regulator SpoOF,but not of sensor histidine kinases KinA,KinB,KinC,and KinD.The phosphorelay downregulated Sinl and de-repressed SinR expression,leading to repression of the transcription level of EPS and TasA protein.2.Study of the inhibition of Mn(?)uptake in Bacillus amyloliquefaciens by zinc ion with non-growth-inhibiting dose.Many metals can participate in the life activities of Bacillus spp..However,the amount of various metal ions in the cells has their own thresholds,and excessive concentrations of metal ions tend to cause damage to the cells themselves.This study shows that when the concentration of Zn(?)in the environment is too high,Zn(?)can compete with Mn(?)for the binding site of solute-binding protein MntA,thereby reducing intracellular Mn(?)concentration.The concentration of Mn(?)was reduced by 2 and 8 times after treatment with 100 and 200 ?M Zn(?),respectively.The process of competing for binding sites is irreversible.In addition,Zn(?)can also inhibit the transcription levels of mntABCD and mntH by up-regulating the metal regulatory protein MntR,further reducing intracellular Mn(?)levels.In the Bacillus biofilm formation system,Mn(?)can promote the formation of biofilm by regulating the activity of spoOF.Biofilm phenotypic experiments showed that the biofilm formation ability of mntA mutants was weakened and the damage of Zn(?)to biofilm could not be completely relieved.Exogenous supplementation of Mn(?)also failed to relieve the inhibition of Zn(?)on biofilms.These results indicate that the metal ion Zn(II)has a passive negative effect on plant growth promotion and biofilm formation of the biocontrol bacteria Bacillus amyloliquefaciens FZB42,which is derived from the fact that Zn(II)inhibits FZB42 from extracellular uptake of Mn(?)and causes intracellular Mn(II)concentration to be too low,thereby inhibiting the phosphorylation level of SpoOA,blocking the major components of biofilm EPS and TasA protein synthesis.3.Study on the mechanism of transcription factor RBAM?RS17245 regulating the synthesis of spermidine in Bacillus amyloliquefaciens FZB42Spermidine is a very important biomass in the metabolites of Bacillus and can affect the formation of biofilms.Previous studies of our lab have showed that B.amyloliquefaciens F7B42 possesses a complete cluster of spermidine synthesis-related genes,but its spermidine production cannot be detected by HPLC.Through sequence alignment,it was found that there are seven genes with unknown function upstream of the spermidine synthesis-related spe gene cluster of FZB42 which were called:RBAM?RS17245?RBAM?RS17250?RBAM?RS17255?RBAM?RS17260?RBAM?RS17265?RBAM?RS17270 and RBAM?RS17275.Among the 7 genes,RBAM?RS17425 is located upstream of speB and has a typical DNA binding site,which may be a transcription factor.In this study,the RBAM?RS17425 deletion mutant and its functional revertant mutant were constructed.The qRT-PCR results showed that the gene expression level related to spermidine synthesis was significantly up-regulated,especially speE gene,whose expression level was upregulated by 5.5 times,indicating that the transcription factor has an negative regulation effect on the synthesis of FZB42 spermidine.Through EMSA experiments,we found that this transcription factor binds to the promoter region of the spermidine synthesis-related gene speE,thereby inhibiting its transcription.Finally,we found through a series of phenotypic experiments that the deletion of transcription factor RBAM?RS 17245 can promote the formation of FZB42 biofilm,while also improving its salt tolerance.The deletion mutant can grow normally in LB medium supplemented with 10%NaCl exogenously,while the wild type can only tolerate LB medium supplemented with exogenous 8%NaCl..In summary,the transcription factor RBAM?RS 17425 can inhibit the biosynthesis of spermidine by inhibiting the expression of the gene cluster of spermidine synthesis of B.amyloliquefaciens FZB42.Through the study on the effect of zinc ion and transcription factor RBAM?RS 17245 on the biocontrol function of Bacillus amyloliquefaciens FZB42,we defined the inhibition ability and specific mechanism of the zinc ion and transcription factor RBAM?RS1745 on FZB42 biofilm formation and spermidine biosynthesis,respectively.This will play a guiding role in the future commercial production and field application measures of FZB42 for its fully exerted application value and better growth promoting and disease prevention abilities.