Physiological Characterizations Of Response Regulator GacA And Small RNA CsrB In Rahnella Aquatilis ML12and Their Regulatory Role In Biocontrol Phenotype

Rahnella aquatilis ML12is a potential biocontrol bacteria with antagonistic effect on pathogenicbacteria and fungi isolated. Based on the whole genome of ML12, gene encoding gacA, the regulatorof two component regulatory system GacS/GacA, and a noncoding small RNA csrB have beenlocalized and focused for studying the regulatory mechanism referring to biocontrol function in thisdissertation. Thereafter, the biocontrol-related physiological characteristics, including bacterialgrowth, biofilm formation, swimming and swarming behavior, phosphate solubilization, plantgrowth-promoting and biocontrol efficiency, have been analysed.In the first step, gacA and csrB have been deleted in R. aquatilis ML12, producing R. aquatilisML12ΔgacA and ML12ΔcsrB on the strategy of markerless exchange mutagenesis, respectively.Firstly, the gene encoding gacA and csrB have been identified in the genome. The DNA frank regionsof targeted genes have been inserted into the vector pSR47s, producing pSR47-gacA and pSR47-csrB.Mutants ML12ΔgacA and ML12ΔcsrB have been constructed and identified through two-step ofhomologous recombination and DNA sequencing. Secondly, the constructed mutants have been usedfor regulatory roles of gene gacA and small RNA csrB referring to biocontrol exhibition bacteriaML12. The related assay, including colony morphology, swimming, swarming, biofilm formation,antibiotic material producing, biocontrol efficienty, etc, have been performed. The wild type strainML12were set as control.The results have showed that the swimming speed of ML12ΔcsrB and ML12ΔgacA were bothslower than the wide type ML12. The swimming diameter reduced25%in32hours. MutantML12ΔgacA lost the swarming behavior. The capacity of biofilm formation of mutant ML12ΔcsrB wasabout twice as much of the wide type. However, the mutant ML12ΔgacA decreased13.2%. The role ofphosphate solubilization of the two deletion mutants decreased20%. The growth promoting ofML12ΔgacA and ML12ΔcsrB was decreased30%and18%compared to wide type. According to thedouble plate bacteriostatic experiment we have found that the antibacterial circle diameter ofML12ΔgacA to C58bacteria reduced81.8%. Anothor the bacteriostatic circle diameter of ML12ΔcsrBand ML12ΔgacA to tomato grey mould decreased37%and16%, respectively.The biocontrol bacteria have some limitations during to unstable efficiency after application inthe field currently. In this dissertation, it is demonstrated that the regulatory gene gacA and smallRNA csrB regulate biocontrol ability of R. aquatilis ML12positively. The results here clarify thebiocontrol regulatory mechanism of R. aquatilis ML12.