Characterization Of The Soluble Chemoreceptors In Azorhizobium Caulinodans ORS571

Azorhizobium caulinodans belongs to the alpha-subdivision of proteobacteria and has the capacity of fixing nitrogen both during free-living growth and in a symbiotic interaction with Sesbania rostrata. Chemotaxis is a stimulated process enabling motile bacterial species to detect chemical gradients and to move in a benefical direction. In bacterial chemotaxis, chemoreceptors sense environmental changes and transmit this information to the chemotactic machinery to guide motile bacteria to preferred niches. In this paper, the chemotaxis system of A. caulinodans was systematically analyzed and the functions of soluble chemoreceptors were preliminarily discussed. Both under free- living and symbiotic states, the role of Icp B was lucubrated, the research contents and results are as follows:(1) Comparative genome analysis of chemotaxis system of A. caulinodans.Che clusters and chemoreceptor genes were analyzed via bioinformatic and comparative genomic methods. We found that there is a unique che cluster, a che Y and a cheZ are outside the major che cluster. We identified 43 methyl-accepting chemotaxis protein(MCP) homologs containing conserved methylated domain and core domain in which cytoplasmic domains are composed of 38 heptad repeats. The methylase CheR is pentapeptide- independent. The che cluster is predicted to regulate by σ54 promoter.(2) Preliminary studies of the soluble chemoreceptors.We have constructed mutants of six soluble chemoreceptors. The chemotactic rings of all six mutants were smaller than that of wild type strain and this difference was more obvious when bacteria were cultured under nitrogen fixation condition. To visualize the subcellular localization of chemoreceptor in vivo, we expressed the green fluorescent protein(GFP) fused to the C-terminal region of six chemoreceptors. The result showed that All chemoreceptors localized to the cell poles. The fluorescence of polar foci was more stronger under nitrogen fixation condition. In nodulation competition assay, all five PAS containing chemoreceptors could not compete with the wild type strain except the PiLZ containing chemoreceptor(AZC-3349).(3) The study on function of soluble chemoreceptor IcpB during free- living and symbiotic stage.This spectrophotometric analysis confirmed that the Icp B N-terminal PAS domain possessed an absorption spectrum typical of oxygen-bound heme proteins. The histidine residue at position 154 is the key site for bind heme. Site-directed mutagenesis confirmed that H154 plays a key role in bacterial chemotaxis and aerotaxis. Mutation of the icp B gene affected the production of biofilm and exopolysaccharides, especially under nitrogen fixation condition. The nodules induced by the icp B mutant lacked sufficient leghemoglobin and were smaller than those induced by the wild type strain.