Study On The Function Of MCP03030 And CheA In Chemotaxis Pathway Of Novosphingobium Pentaromativorans US6-1

Bacterial chemotaxis is the movement towards environments that contain higher concentrations of beneficial substrates,or lower concentrations of toxic compounds.Chemotaxis facilitates degrading bacteria to sense and migrate toward polycyclic aromatic hydrocarbons(PAHs),thereby increasing its biodegradation rate.Novosphingobium pentaromativorans US6-1 is a g ram-negative bacterium which could efficiently degrade PAHs and possessed observable chemotaxis toward it.Despite the limited number of methyl-accepting chemotaxis proteins(MCPs)in US6-1,the furnction and substrate range of them remain unk:nown.Moreover,regulatory mechanisms the histidine kinases involved in US6-1 are still unclear.In order to explore the function of key components in the chemotactic system,we focused on the MCP03030 and key histidine kinases CheA to reveal the potential link between chemotactic system and cellular metabolic,biofilm formation and cell surface hydrophobicity in US6-1.The main results are as follows:1.TMHMM Server v.2.0 was used to predict the transmembrane helix of MCPs in US6-1 and analyzed the conserved domains of them by using the SMART database.The results showed that MCP03030 belongs to the type la chemoreceptor containing a periplasmic 4-helix bundle(4HB)ligand binding domain(LBD).In addition,we analyzed the conserved domains of 27 histidine kinases in US6-1 and found that the histidine kinase CheA is one of the soluble histidine kinases with special structure.By analyzing the chemotaxis pathway genes and the genes adjacent to cheA,we found that US6-1 has a complete chemotaxis pathway including a chemotactic gene cluster consisted of cheX-cheA-cheR-cheB-cheD-cheY-che W.Chemotactic gene cluster locates in one operon and it is adjacent to multiple transporters and transcriptional regulators.2.The mutant A03030 was constructed by using homologous recombination.The results of the titration plate assay,swim plate assay and quantitative capillary assay showed that the mutant △03030 lost the ability of chemotaxis toward benzoic acid and phthalic acid.The deletion of the mcp03030 gene resulted in a drop in the chemotactic response to salicylic acid.Surprisingly,the chemotactic response of △03030 to Phenanthrene(Phe)was faster than US6-1.The deletion of mcp03030 gene did not affect the degradation of Phe verified by the sublimation experiment.3.The mutant △cheA was also obtained by using homologous recombination.The deletion of cheA gene did not affect chemotaxis of US6-1.The results of metabolism experiments with different amino acids as single carbon source showed that the deletion of cheA gene caused the strain to lose the ability to metabolize proline and isoleucine.By comparing the difference in biofilm formation ability and cell surface hydrophobicity between the wild type and mutant strains under different environmental stresses,the result showed that cheA significantly affected the formation of biofilms at early stage and the cell surface hydrophobicity(CSH).Those results concluded that cheA regulates the biofilm formation ability and CSH of strain in response to environmental stress.