Function Analysis Of Flagellar Genes FliN,fliM And A Two-component Transduction System Gene AZC₀619 In Azorhizobium Caulinodans ORS571

Azorhizobium caulinodans ORS571 not only fix nitrogen in a symbiotic interaction with its host Sesbania rostrata,but also has the capacity of fixing nitrogen during free-living station or as an endophyte in other hosts.Flagella is the motile organ of bacteria.The bacteria are driven by the flagella to the environment that is beneficial to their survival.The rotation direction of the flagella is controlled by the flagellar motor.The rotor of the flagellar motor is located at the end of the bacterial chemotaxis pathway,and it is an important link between the external signal stimulation and bacterial movement.At present,there are few reports about the flagellar motor proteins in the A.caulinodans ORS571.We have previously discovered a two-component system transcriptional regulator AZC 0619 in ORS 571 genome,whose function has not been annotated.To elucidate the function and mechanism of the flagellar motor genes fliN,fliM and the transcriptional regulatory factor AZC₀619 in ORS571,we constructed the gene knock-out mutant strains ?fliN,?fliM,and ?0619 by homologous recombination and triparental conjugation,respectively.We systematically analyzed the effects of these genes on the cell growth,motility,chemotaxis,exopolysaccharide production,biofilm formation and cell flocculation.Combination of bioinformatics analysis methods and fluorescence quantitative PCR technology proved that AZC₀619 has a positive regulatory effect on multiple flagellins in ORS571.These findings laid the foundation for further exploration of the functions and mechanisms of the fliN,fliM and AZC₀619 genes.The main findings are described as the followings:1.The fliN and fliM genes were cloned from the ORS571 genome by PCR.The mutants ?fliN and ?fliM were constructed.The PCR and sequence analysis results showed that the fliN and fliM genes were successfully knocked out.The mutants ?fliN and ?fliM didn't shown any difference in cell growth compared to the wild-type strain.However,both mutants lost their flagella showed by observation under electron microscope.Their motility and chemotaxis on semi-solid medium were attenuated.The wild-type strain and the mutants had significant different in colony morphology and exopolysaccharide?EPS?secretion on 0.8%L3 nitrogen-free medium.The WT colony surface was more humid and smooth due to the higher content of EPS,while the EPS content of the mutants decreased significantly.The surface of mutant colonies were generally dry and rough.In addition,?fliN and AfliM were impaired in biofilm formation.To the contrary,the flocculation ratio of both mutants was higher than that of the wild-type strain within the same amount of time.Therefore,such data indicates that the flagellar genes fliN and fliM could regulate the flagellum formation,cell motility,exopolysaccharide production,biofilm formation and cell flocculation of Azorhizobium caulinodans ORS571.2.The AZC₀619 gene was cloned from the ORS571 genome and the mutant A0619 was successfully constructed.The phenotype of the mutan A0619 was similar to that of the mutants ?fliN and AfliM.Compared with the wild type,?0619 exhibited characteristics including no flagella,obvious defects of motile and chemotaxis,reduction of biofilm formation and extracellular polysaccharide content,but increased flocculation ratio.In addition,the ability of adhesion to plants root and competitive nodulation of the mutant ?0619 was significantly lower than that of wild type.Bioinformatics analysis showed that AZC₀619 in ORS571 has 53%of amino acid sequence homology with gene ftcR which is a major regulator involved in flagellin gene expression in Brucella melitensis 16M.Therefore,we selected 9 flagellar genes in the ORS571 flagellum cluster for qRT-PCR analyze on their expressions in in WT and?0619.These 9 gene expressions were dowm-regulated in the mutant ?0619 compared to the WT.Based on these phenotypes,we hypothesis that AZC 0619 is probably a positive transcriptional regulator involved in flagellin gene expression in ORS571.