Molecular Response Mechanism Of Pannonibacter Phragmitetus BB To Cr(Ⅵ)

Due to the rapid development of chromate production,ferrochrome mining,and other industries,large amount of chromium slag had been generated.The long-term heaping of chromium slag has posed a great threat to the soil environment.The great concealment and migration of Cr(Ⅵ)compel the Cr(Ⅵ)pollution control to become a difficult issue in heavy metal contaminated soil remediation.Fortunately,certain bacteria in the Cr(Ⅵ)-contaminated soil possess the ability of resistance and reduction for Cr(Ⅵ),which provide a novel strategy for the remediation of Cr(Ⅵ)-contaminated soil.To date,a large number of Cr(Ⅵ)-reducing bacteria(CRB)have been isolated and applied to Cr(Ⅵ)treatment.However,the lack of studies on bacterial Cr(Ⅵ)response mechanism limits the development and popularization of Cr(Ⅵ)bioremediation.In this study,an efficient CRB Pannonibacter phragmitetus BB(here after BB),isolated from Cr(Ⅵ)-contaminated soil,was selected as the research object to comprehensively reveal the molecular response mechanism of CRB to Cr(Ⅵ)through the genomics,proteomics,and metabolomics analyses.This study provides theoretical foundation for bioremediation of Cr(Ⅵ)-contaminated soil.The main research contents and innovative achievements are as follow:(1)The unique phenotype and genotype of strain BB were revealed,and the reason why BB showed such a high Cr(Ⅵ)resistance and reduction was preliminarily explained.A comparative study of BB,P.phragmitetus D1(here after D1)and P.phragmitetus D4(here after D4)revealed that the Cr(Ⅵ)tolerance and reduction of BB was much higher than that of D1 and D4;The whole genome analysis showed that the genome size,GC content,and the number of coding genes of BB,D1,and D4 were similar,it indicated that there was no significant difference on the whole genome scale for three P.phragmitetus;A large number of unique Cr(Ⅵ)resistance genes located on the chromosomes and plasmids in the genome of BB through the comparative analysis of the number and spatial distribution of Cr(Ⅵ)resistance genes in genomes of three strains.(2)The multi-enzyme system involved in Cr(Ⅵ)resistance and reduction of BB was identified,and the Cr(Ⅵ)reduction pathway catalyzed by various reductases in BB was also clarified.Proteomics showed that the key enzyme system of bacterial Cr(Ⅵ)resistance,including sulfate transporters,antioxidant enzymes,DNA repair enzymes,and chromate transporters were overexpressed under Cr(Ⅵ)stress;Various reductases,such as intracellular soluble reductases and membrane-bound reductases,also significantly up-regulated.Intracellular reductases reduced Cr(Ⅵ)to Cr(Ⅲ)by one or more steps of electron transfer,while membrane bound reductases were part of extracellular electron transport system and used extracellular chromate as the terminal electron acceptor.(3)Numerous reducing metabolites and a potential endogenous extracellular electron shuttle were identified from the differential metabolites,revealing multiple non-enzymatic mechanisms for the Cr(Ⅵ)reduction by BB.The amount of differential metabolites significantly decreased along with the complete reduction of Cr(Ⅵ),which confirmed that the process of non-enzymatic Cr(Ⅵ)reduction were closely associated with metabolites;Metabolites with amine and hydroxyl groups,such as vitamin and glutathione,were found to be responsible for the absorption and reduction of Cr(Ⅵ),respectively;There was a potential endogenous electron shuttle pyrrole-2-carboxylic acid among these differential metabolites,which promoted extracellular non-enzymatic Cr(Ⅵ)reduction by long-distance electron transfer across the membrane.(4)Quorum sensing was found in BB during Cr(Ⅵ)reduction.The synthetic pathway of signal molecules in BB was reconstructed,confirming the regulatory role of LuxR/LuxI quorum sensing on Cr(Ⅵ)tolerance/reduction.The typical quorum sensing phenomenon appeared in Cr(Ⅵ)reduction until the bacterial biomass reached a certain level;The potential quorum sensing genes luxI/luxR were identified from the genome of BB.The synthetic pathway of signal molecules in BB was reconstructed according to the up-regulated expression of LuxI/LuxR under Cr(Ⅵ)stress;In addition,a mutant BB Gm/ΔluxI::Gm with the deletion of luxI gene was constructed.No signal molecules was produced in mutant,and its ability of Cr(Ⅵ)tolerance and reduction was significantly lower than that of the wild type,indicating quorum sensing participated in the regulation of bacterial Cr(Ⅵ)response.(5)Cr(Ⅵ)was found as a chemical repellent for BB.The potential chemotaxis genes were identified via their overexpressed under Cr(Ⅵ)stress,then we reconstructed the pathway of chemotaxis.The chemotaxis experiment and observation of bacterial morphology preliminarily concluded that strain BB with visible flagellum showed negative chemotaxis to Cr(Ⅵ);The potential chemotaxis genes included mcp,cheA,cheW,cheY,motA,fliG,fliM were identified;Proteomics clarified the overexpression of chemotaxis proteins were stimulated by Cr(Ⅵ),preliminarily confirming the chemotaxis of BB to Cr(Ⅵ);The chemotaxis pathway in BB stimulated by Cr(Ⅵ)was reconstructed according to KEGG metabolic pathway,indicating chemotaxis played an important role in bacterial Cr(Ⅵ)response.87 figures,29 tables and 206 references...