Characterization Of The Chemoreceptor MCP18870 In Novosphingobium Pentaromativorans US6-1

Polycyclic aromatic hydrocarbons(PAHs)are a large group of organic compounds comprised of two or more fused benzene rings bonded in linear,cluster,or angular arrangements,which originate from natural and anthropogenic sources.They have toxic effects on foods and environment.Biodegradation is the most efficient way to remove PAHs from the environment.Chemotaxis is the mechanism by which bacteria temporally detect chemical gradients and swim toward higher concentrations of attractants or lower concentrations of reprllents.Bacteria chemotaxis contributes to the metabolism of substrates.Many bacterial strains have been found they exhibit chemotaxis toward aromatic compounds.Novosphingobium pentaromativorans US6-1 is an excellent bioremediator,which can metabolize different kinds of PAHs for its growth.In this paper,the chemotactic genes,chemotactic proteins and the chemotaxis toward different chemicals in strain Novosphingobium pentaromativorans US6-1 were studied using genomics,transcriptomes,site-specific deletion and other techniques.The main results are as follows:We analyse the chemotactic genes and chemotactic proteins based on the complete genome of strain US6-1,29 genes were assigned to bacterial chemotaxis pathway of KEGG,including 6 MCP,1 sensor kinase CheA,14 coupling protein CheW,1 response regulator CheY,5 MCP related protein and 3 flagellar related protein.Most of them were encoded on the chromosome,and a few chemotactic proteins were encoded on the plasmid.In addition,we compared the transcriptomes with or without BaP.In contrast to the control,the gene expressions of MCP were up-regulation in different degree.The significant differential expressed proteins were MCP2035(JI59_RS02035)and MCP3030(JI59_RS03030).The analyses showed the strain US6-1 possessed many chemotaxis genes to form a complete chemotaxis pathway.Domain architecture of MCPs in strain US6-1 were visualized using SMART and Phyre 2.From the results,we got that only MCP3030(JI59_RS03030)and MCP18870(JI59_RS18870)possessed ligand binding domains(LBDs).Both of the two LBDs were predicted to adopt a four-helix bundle fold(4-HB).The prediction of LBDs structures is helpful to understand the molecular mechanism of chemotaxis.Drop assays and swimming plate assays were used to detect the chemotaxis towards simple compounds and PAHs of the strain US6-1.The results showed the strain exhibited chemotaxis towards several simple compounds after 3h incubation,however,the chemotactic responses to PAHs were observed after 4d incubation.The difficult degradation of PAHs leads to a slow chemotactic responses.This suggests the chemotaxis to PAHs may be metabolism dependent.In order to research the link of chemotaxis and degradation of BaP,we constructed a non-chemotactic mutant,US6-lAcheA,by disruption of sensor kinase CheA in strain US6-1.Compared to the wild type,the degradation rate of BaP by US6-1?cheA was makely decreased,suggesting that chemotaxis strongly affect the degradation.Genetic site-specific deletion and cloning,expression techniques were used to explore the function of MCP18870.At first,we constructed gene mcp18870 deleted mutant.According to chemotactic assays,we found the mutant lost the chemotactic response to catechol.At the same time,MCP18870-LBD gene was cloned,expressed and purified.Fluorescence spectroscopy analysis indicated that the LBD of MCP18870 specifically binds to gentisic acid.Based on the results,MCP18870 is involved in triggering chemotaxis towards catechol and gentisic acid in US6-1.