Rifampin-resistant Mutations In Bacillus Amyloliquefaciens CC09 And Its Biological And Genetic Effects

Bacillus amyloliquefaciens CC09 can produce a vast array of biologically active molecules potentially inhibitory for phytopathogen growth.Among these active compounds,the most widely studied chemical is cyclic lipopeptides(CLPs).However,due to the low capacity of lipopeptide production,CLPs produced by Bacillus has not been industrialized so far.The LB medium containing Rifampin(50 μg/mL)was used to select Rifampin resistance(Rif)mutants.The mutations conferring Rif are proved to be in rpoB encoding β subunit of RNA polymerase.The major results are as follows:1、14 Rif mutants were isolated and characterized six single-site Rifampin resistance(Rif)mutations H485Y,H485C,H485D,H485R,Q472R and S490L,as well as one double-site mutation S490L and S617F in RNA polymerase β subunit of B.amyloliquefaciens CC09.2、The effect of Rif mutation on antifungal activity against phytopathogen Geotrichum candidum in vitro were evaluated.Among them,H485R and H485D mutants enhanced the production of Iturin A by 1.7-2.0 fold as high as that of the wild strain.H485Y,S490L,Q472R and S490L/S617F mutants decreased the antifungal activity and Iturin A production,while the H485C mutant was a neutral mutant at aspect of antifungal activity and Iturin A production.3、The effect of Rifr mutation on biological traits was studied and the results were listed following:(1)Different mutations make differences in Rifampin resistance,cell colony,cell size and sporulation.All mutants showed defective effects on sporulation.Mutants Q472R and S490L severely influenced the morphology of cell colony.(2)Rif mutations make great differences in motility,biofilm formation and cell colonization on wheats.All mutants showed defective effects on motility,especially mutant S490L.The mutation on 485H made no difference in the biofilm formation time;S490L mutation postponed the biofilm formation significant;The appearance of biofilm among mutants carrying 485H mutation were severely wrinkle.In addition,it looks like the biofilm of negative mutants 0917(S490L)and 0954(S490L/617F)were made up of floating cell debris,while the biofilm of 0943(Q472R)was very smooth and bright compared to the other strains.Except the Rifr mutant 0954(S490L/617F)made no difference in colonization of interior leaves with the WT,other mutants showed defects in colonization of the wheat leaves.(3)Different mutations make difference in nutrition and stress resistance.The availability of carbon and nitrogen sources of different Rifr mutants was different,and the tolerance of Rifr mutants to temperature,pH and NaCl was different.(4)The defective effects of mutation S490L on cell size,motility and sporulation could be partially recovered by the intragenic second site mutation S617F.4、14 genes involving B.amyloliquefaciens CC09 biocontrol traits were selected for qPCR analysis.The results indicated that Rifr mutations affected the gene expression on transcription,and these changes make difference in biocontrol traits of the strain.