Characterization Of The Three Typical Transmembrane Chemoreceptors In Azorhizobium Caulinodans Symbiosed With Coastal Salt Tolerant Plant Sesbania Rostrata

Tidal flat is the intertidal shoal in the coastal zone,which is rich in land resources.Coastal salinization is becoming more and more serious,and it is the oretical and practical significance to study salt-tolerant plant-microorganism interaction and restoration.Azorhizobium caulinodans ORS571 belongs to alpha-subdivision of proteobacteria that symbiotic nodulated on the roots and stems of its host coastal salt tolerant plant Sesbania rostrata,and can also fix nitrogen during free-living growth or as an endophyte in other plants.S.rostrata has strong resistance to salt and waterlogging.It is used as a pioneer plant in the phytoremediation of saline and alkaline soil in tidal flat and has great potential in the improvement and utilization of tidal flat resources.S.rostrata also has high economic value and could be used in making feed and food stabilizer.Chemotaxis is a response process in which bacteria sense the chemical gradients and move toward an environment conducive to their growth.In the chemotactic signaling pathway of bacteria,chemoreceptors are located in the initial part,which contain transmembrane chemoreceptors and soluble chemoreceptors.Transmembrane chemoreceptors are mainly responsible for sensing extracellular signals.Three transmembrane chemoreceptors TlpA1,AZC₀719 and AZC₁721 of A.caulinodans ORS571 are selected in this study to explore the sensing mechanism,which is helpful to explain the symbiosis mechanism of A.caulinodans ORS571 and its host S.rostrata from the molecular level.The main results were as follows:Domain analysis showed that there were 43 chemoreceptors in A.caulinodans ORS571,37 of which were transmembrane chemoreceptors.TlpA1 was located at the front of the chemotaxis gene cluster and had obvious transmembrane region,but its extracellular sensing domain was unknown.AZC₀719 had a 4HB_MCP domain.It is generally found in prokaryotic signal transduction proteins.Both ends of the domain are accompanied by transmembrane domains,and only extracellular signals are sensed.The chemoattractants are mainly organic acids and amino acids.AZC₁721 had a CHASE3 domain,which was an extracellular sensing domain that existed in histone kinase,MCPs,etc.It was involved in bacterial chemotaxis and metabolic pathways.Protein binding site prediction showed that TlpA1,AZC₀719 and AZC₁721 Nterminal extracellular parts all had complete protein binding sites,and the three binding sites predicted had the highest homology with the extracellular part of E.coli K12 histidine kinase NarQ,which is sensing nitrate or nitrite,lipid and carbon.The N-terminal expression strains of TlpA1,AZC₀719 and AZC₁721 were constructed.PCR and gene sequencing results showed that the expression strain was successfully constructed.SDS-PAGE electrophoresis results showed that the target protein was soluble.Isothermal titration calorimetry（ITC）and protein thermal shift assay results showed that there was interaction between TlpA1 and succinic acid,and between AZC₁721 and 3-hydroxy-2-butanone.Mutant strain ΔTlpA1 was provided by our laboratory.The mutant strains ΔAZC₀719 and ΔAZC₁721 were constructed by this study.For mutants ΔTlpA1,ΔAZC₀719 and ΔAZC₁721,the growth rate,chemotaxis behavior,extracellular polysaccharide,biofilm formation,flocculation rate were detected.The results showed that there was no significant difference in the growth rate between the mutant and the wild type.Chemotaxis behavior test results showed the mutant strains ΔAZC₁721 fomed the chemotaxis ring significantly greater than the wild type,when 3-hydroxy-2-butanone used as carbon source.But at semisolid plates with different carbon source,the mutant strain ΔAZC₀719 formed the same chemotaxis ring with the wild type.The exopolysaccharide test results showed that mutant strains ΔAZC₀719 and ΔAZC₁721 had no significant difference in Congo red colony morphology and the secretion of extracellular polysaccharide compared with wild type.Biofilm formation test results showed that the biofilm formation ability of three mutant strains ΔTlpA1,ΔAZC₀719 and ΔAZC₁721 is significantly lower than the wild type in the presence or absence nitrogen source.Flocculation test results showed there was no significant difference between three mutant strains ΔTlpA1,ΔAZC₀719,ΔAZC₁721 with wild type.