Identification Of AO-1 Signal Molecules In Antimony Oxidizing Bacteria And Its Regulation Of Resistance And Oxidation Function

In recent years,the world’s demand for antimony is increasing.The long-term continuous high-intensity mining of antimony ore area breaks the natural balance of the mining area,speeds up the release of antimony to the environment,and makes the pollution form of antimony increasingly serious.Studies show that the dissolution and oxidation of antimony waste ore by antimony oxidizing bacteria under the effect of quorum sensing is more than ten thousand times that under the natural state.Therefore,it is a promising technology to use antimony oxidizing bacteria from the direction of quorum sensing to prevent and control antimony pollution at the source.In this paper,Pseudomonas sp.AO-1,a typical antimony oxidizing bacterium,was used as the research object to study the kinds and threshold of signal molecules in QS system,and the regulation of signal molecules on antimony resistance and oxidation of AO-1.The main work and achievements of this project are as follows:1.The species and spatial structure of signal molecules of typical antimony oxidizing bacterium Pseudomonas sp.AO-1 were explored,and its action threshold was defined.By studying the signal molecules of AO-1,it is found that:（1）AO-1 can produce long-chain signal molecules with quorum sensing phenomenon.（2）The signal molecules produced by AO-1 are C₈-HSL and C₁₂-HSL,of which C₈-HSL accounts for 32.5%and C₁₂-HSL accounts for 67.5%.The structures of these two signal molecules were determined.（3）When the concentrations of signal molecules are 0.05,0.10,0.15,0.20,0.30 and 0.40μg/L,the growth curve of AO-1 with C₈-HSL decreased by 5.43%,9.68%,13.52%,15.98%,15.53%and16.89%respectively compared with that without signal molecule,and the growth curve of AO-1 with C₁₂-HSL decreased by 6.21%,11.64%,13.84%,15.53%,16.44%and 18.26%respectively.When the concentration of signal molecules reaches 0.20μg/L,the influence on the growth of AO-1 does not increase with the increase of the concentration,which indicates that the concentration of signal molecule is 0.20μg/L can reach the threshold concentration of signal molecules of AO-1 QS system.2.The difference of AO-1 character expression under signal molecular stress was explored.By studying the changes of antimony resistance,Sb（Ⅲ）oxidation and EPS production of AO-1 before and after adding exogenous signal molecules,it was found that:（1）In the absence of Sb（Ⅲ）,the growth of C₈-HSL and C₁₂-HSL AO-1 was inhibited,but with the increase of Sb（Ⅲ）concentration in the medium,the two signaling molecules gradually promoted the growth of AO-1.（2）in 48h,adding C₈-HSL and c12-hsl can increase the oxidation rate by 9.8%and 5.97%,19.3%and 11.75%,respectively.（3）In the antimony free group,the content of EPS increased by 26.29%and 16.11%when C₈-HSL and C₁₂-HSL were added,respectively.In the antimony group,the EPS content of C₈-HSL and C₁₂-HSL increased 40.44%and 14.76%,respectively.When Sb（Ⅲ）and signal molecules were coupled,EPS content increased by 51.27%and 23.57%,respectively.These results indicate that the antimony resistance,Sb（Ⅲ）oxidation and EPS production of AO-1 can be improved under the stress of signal molecules.3.The gene expression differences of AO-1 under signal molecular stress,Sb（Ⅲ）stress and their coupling conditions were investigated.Transcriptome sequencing of AO-1 under no stress（A）,signal molecular stress（B）,Sb（Ⅲ）stress（C）and Sb（Ⅲ）and signal molecular coupling stress（D）showed that:（1）In B VS A,C VS A,DVS A and DVS C,316,998,2140,924 genes were up-regulated,107,305,188,157 genes were down-regulated,respectively.These results indicated that the AO-1 gene was significantly up-regulated under signal molecular stress,Sb（Ⅲ）stress and Sb（Ⅲ）and signal molecular coupling stress.Cluster analysis of genes showed that the direction of gene change was correlated,suggesting the possibility of domestication of AO-1 from this direction.（2）Differential genes mainly focus on metabolic process,catalytic activity,cell population,membrane transport,signal transduction,energy metabolism,cofactor transport,vitamin metabolism and amino acid metabolism,etc.These functional genes are closely related to the Sb（Ⅲ）tolerance and oxidation mechanism of AO-1,which should be paid more attention.（3）The signal molecules can positively regulate the expression of genes related to Sb（III）efflux,Sb（III）oxidation and EPS formation of AO-1.By reducing the concentration of Sb（Ⅲ）in AO-1,reducing the toxicity of Sb（Ⅲ）and increasing the extracellular adsorption of Sb（Ⅲ）oxidation,antimony resistance of strain AO-1 was improved.