Global Regulatory Mechanisms Of Chemotactic Two-component System In Novosphigobium Pentaromativorans US6-1 Under BaP Stress

Polycyclic aromatic hydrocarbons(PAHs)are composed of fused aromatic rings in linear,angular,or cluster arrangement,which are persistent organic pollutants widespread across in environments and foods with carcinogenic,teratogenetic and mutagenic properties.They have natural potential for bioaccumulation in various food chains to cause great harm to human health and ecosystem.Microbial degradation of PAHs is considered an efficient way to remove the PAH pollutants from environments.However,as the number of benzene rings increases,hydrophobicity,thermal stability,and biotoxicity of PAHs increase.Therefore,high molecular weight PAH degrading microorganisms are not only few,but also slow in the initiate of degradation,and the degradation rate is low.Bacteria can chemotactically respond to hydrophobic aromatic hydrocarbons,but the mechanism of sensing hydrophobic aromatic hydrocarbons and facilitating their degradation is unclear.At present,the researchers have more focused on the analysis of metabolic pathway for microbial degradation mechanism of refractory high molecular weight PAHs.However,the interaction between bacteria and PAHs,the physiological mechanism of how microorganisms mediate the PAHs into cells and how the PAH bacteria response the biotoxicity of PAHs to the promote the PAH degradation are less known,especially the molecular basis of this research.In this study,a marine bacterium Novosphigobium pentaromativorans US6-1 with the ability of chemotaxis and degradation of 2-5 ring PAHs was used as type strain,and 5-ring benzo[a]pyrene(BaP)was used as the object.Applying comparative transcriptomics,key gene knockout and complement,metabolites analysis,protein-small molecule interaction and protein-protein interaction to analyze the physiological response process of the initial degradation of BaP by US6-1,reveal the regulatory roles of chemotactic two-component system in sensing,transporting and degrading PAHs,as well as reveal global regulatory mechanism for adapting and degrading BaP in US6-1.This study provides the molecular foundation for the targeted optimization of microbial functions,the improvement of transform and degradation of PAHs(especially highly biotoxic BaP).The following shows the main results and conclusions of this study:1.The present study parsed the physiological response process of the initial degradation of BaP by US6-1.The degradation of 3-ring phenanthrene(PHE),4-ring pyrene(PYR)and 5-ring BaP were detected by using GC-MS.The results showed that the degradation rate of 10 ppm PHE and 10 ppm PYR were 100%and 52.88%after 2 days incubation,respectively,but the initiation of BaP degradation was slow,and the degradation rate of 10 ppm BaP was only 38.5%after 9 days of incubation,and then BaP degradation was stagnation.In order to understand the physiological response of the initial degradation of BaP by US6-1,using the precursor metabolite pyruvate(PA)as the control group,the easily degradable PHE and the bio-refractory and biotoxic BaP as the experimental group,the comparative transcriptomes of US6-1 in response to different carbon sources were analyzed.The results demonstrated that the expression of 20 genes encoding outer membrane protein and 15 genes encoding inner membrane protein were significantly upregulated under PHE and BaP stress.The properties of these membrane proteins showed that 32 membrane proteins were thermostable,except the outer membrane TBDT and OmpA family protein,other membrane proteins were hydrophobic.The expression of genes in central metabolic and energy metabolism pathways,such as pentose phosphate pathway,tricarboxylic acid cycle,oxidative phosphorylation and fatty acids biosynthesis were significantly downregulated under PHE and BaP stress.However,the genes involved in glyoxylic acid cycle were significantly upregulated only under BaP stress,and fatty acids catabolic gene HADH was significantly upregulated under PHE stress.In addition,the genes encoding catalase and the peroxidase were also downregulated in US6-1 under BaP stress,while the gene encoding glutathione peroxidase was no significant difference compared with PA condition,but the gene encoding glutathione reductase was downregulated in US6-1 under BaP stress.Furthermore,eight two-component system histidine kinase genes were significantly upregulated in US6-1 under PAH stress,and there was no significance difference between PHE and BaP stress.However,the chemotactic twocomponent system histidine kinase gene cheA was significantly up regulated under BaP stress at 12 h,24 h and 48 h compared with PHE stress.The results indicate that US61 can rapidly initiates global physiological response process including regulation of membrane stability,substance and energy supply,antioxidase system,and the chemotactic two-component system of US6-1 is particularly active in response to the highly biotoxic BaP.2.The present study found and verified the function of methyl-accepting chemotaxis proteins MCPs of US6-1 in chemotaxis and degradation of BaP.The chemotactic response of US6-1 to PAHs showed that the US6-1 not only chemotactically responded to PHE、PYR and BaP,but also chemotactically responded to salicylate,catechol,gentisate,protocatechuate,benzoate,phthalate.In order to investigate the molecular mechanism of recognition of PAHs and PAH metabolites,and the specific mechanism of the chemotactic two-component system in regulating the degradation of PAHs,the MCP was used as the object of study.Firstly,phylogenetic profile and domain architectures of MCPs showed that four MCPs with Ligand binding domain(LBD)have the potential ability to chemotactically attracted PAHs and their metabolites.Secondly,analysis of chemotactic response of four MCPs deletion mutants showed only in the Δmcp03030 lost the chemotactic responses to benzoate,phthalate,PHE and BaP.Thirdly,the specifically bound of MCP03030LBD with PHE,PYR,BaP and phthalate was confirmed using surface plasmon resonance(SPR)assays.However,MCP18870LBD bound only PYR.The mutant Δmcp03030-Δmcp18870 was then constructed and was shown to have lost the chemotactic response to benzoate,phthalate,PHE,PYR and BaP.The degradation of PHE,PYR and BaP by Δmcp03030Δmcp18870 were also decreased.Finally,compared with US6-1,the chemotactic response of Δtbdt-11 without the outer membrane transporter TBDT-11 to PHE,PYR and BaP was lower.Our study demonstrated that MCP03030 and MCP18870 could recognize PAHs and their metabolites,especially BaP,and trigger metabolismdependent and metabolism-independent chemotaxis,as well as paly an important role in US6-1 transport,adaptation and degradation of PAHs through the downstream signaling pathway of the chemotactic two-component system.3.The present study primarily revealed that the global regulatory pattern of chemotactic two-component system of US6-1 in adaptation and degradation of BaP.The phenotypic characteristic of mutant Δmcp03030-Δmcp18870 were analyzed,and the results showed that MCP03030 and MCP18870 co-deleting mutant not only lost the ability to chemotactic response to PAHs and their metabolites,but also lost the ability of using the branched-chain amino acids Val and Ile as sole carbon source.In order to explore the global regulatory pattern of the chemotactic two-component system,the potential functions of downstream signaling pathway of chemotactic two-component system in US6-1 under BaP pressure were investigated.Firstly,the results of the bacterial two-hybrid system demonstrated that that MCP03030 and MCP18870 could not only interact directly with the histidine kinase CheA,but also interacts indirectly with CheA through the coupling protein CheW.Therefore,the phenotypic characteristic of mutant ΔcheA were analyzed,and the results displayed that the mutantΔcheA lost the ability to chemotactic response to PHE,PYR,BaP,benzoate and phthalate,and the degradation rate of BaP was also significantly decreased.Meanwhile,the proportion of odd chain fatty acids was increased while the proportion of even chain fatty acids was decreased in mutant ΔcheA,then cell surface hydrophobicity(CSH)and biofilm formation capacity of mutant ΔcheA under BaP stress at early stage were also significantly decreased.Furthermore,the mutant ΔcheA lost the ability to metabolize branched-chain amino acids Val and Ile.And then,the pull-down assay was performed to test the interaction of heterologously expressed and purified CheA with the total proteins from US6-1 under BaP stress at 1 d and 6 d.The results showed that CheA can interact with CheY2 under BaP stress at 1 d,and the Lrp/AsnC family transcriptional regulator with the potential to regulate the metabolism of branched-amino acids can interact with CheA under BaP stress at 6 d.Meanwhile,we found that Ic1R family transcriptional regulators with potential ability of regulating the metabolism of branched-chain amino acids,TetR family transcriptional regulators near PAHs degradation genes,and Crp/Fnr family transcriptional regulators with potential ability of regulating glyoxylic acid circulation pathway might interact with CheA under BaP stress at 1 d and 6 d.The physiological state of US6-1 and mutant ΔcheA were tested,and the results demonstrated that the concentrations of free branched-chain amino acids were significantly increased in mutant ΔcheA compared with US6-1.In summary,the chemotactic two-component system can not only link with transport system to regulate the chemotaxis and degradation of PAHs by sensing PAHs and their metabolites,but also interact with the regulators from the process of BaP degradation to regulate the physiological process of branched-chain amino acid metabolism,and enable US6-1 to adapt and resist the biotoxicity of BaP and degrade BaP.