Study On The Second Messenger C-di-GMP Of Biocontrol Agents Bacillus PG12 And 905

The second messenger cyclic di-GMP(c-di-GMP)is widely distributed among bacteria and coordinates diverse aspects of bacterial growth and behaviour,including motility,biofilm formation and antibiotic biosynthesis.The bacillus spp.exist widely in the environment and offer a number of advantages for their application in agricultural.Antagonism to pathogens and colonization in plants to compete space and nutrition are the main mechanisms of biocontrol.Bacillus amyloliquefaciens PG12 and Bacillus cereus 905 are two effective biocontrol agents.To clarify the function of c-di-GMP pathway in Bacillus,we studied the c-di-GMP pathway in B.amyloliquefaciens and B.cereus for the first time.Specifically,we identified diguanylate cyclases(which catalyze c-di-GMP biosynthesis)and c-di-GMP phosphodiesterase(which degrades c-di-GMP)and confirmed that several phenotypes related with biological control were influenced by c-di-GMP turnover.Our study contributed to not only exploring the molecular mechanisms of c-di-GMP in Bacillus against plant disease,but also searching for new ways to increase the stability of the biocontrol efficacy.The main experimental results are as follows:(1)In this part of experiment,we determined that there were four GGDEF domain proteins which maybe relate to c-di-GMP biosynthesis and two EAL domain proteins which relate to c-di-GMP degradation in silico.And we preliminarily inferred YhcK and YtrP were two diguanylate cyclases and YuxH was the only c-di-GMP phosphodiesterase in PG12 by active sites analysis.We further confirmed that YhcK and YtrP were two diguanylate cyclases by heterologous expressing all the GGDEF proteins in E.coli BL21.We also constructed knockout mutants of all the predicted genes,overexpression strains of YtrP and YuxH with or without active domain.We quantified intracellular c-di-GMP levels of all the mutants using LC-MS/MS and finally determined in PG12 YhcK and YtrP were two diguanylate cyclases and YuxH was the only c-di-GMP phosphodiesterase.(2)We assessed the swimming and swarming motility of knockout strains and overexpression mutants.The motility of ?yuxH was reduced,while ?yhcK and ?ytrP showed a dramatic increase in motility compared with WT and motility was reduced in ytrP overexpression strains and increased in yuxH overexpression strains.Thus,the c-di-GMP level had an inverse effect on motility.Analysis of the biofilm formation indicated none of the knockout mutants showed altered biofilm compared to WT while biofilm formation in ytrP overexpression was enhanced and yuxH overexpression strains showed diminished biofilm formation.This demonstrated that c-di-GMP pathway also influenced biofilm formation.The crude extract of ?ytrP and ?yuxH had the same antagonistic capability to Botryosphaeria dothidea with WT and cells of ?yhcK,?ytrP,and ?yuxH had the same antagonistic capability to Acidovorax citrulli with WT.The biocontrol assay showed that the control efficacy to bacterial fruit blotch of overexpression strains was almost the same with PG12 control.However,strains overexpressing yuxH showed a significant lower control efficacy to rot ring of apple.We could infer that c-di-GMP pathway possibly participate in this process.(3)We determined that there were five GGDEF domain proteins,five GGDEF+EAL domain proteins and one EAL domain protein in B.cereus 905 using bioinformatics method.And we preliminarily inferred diguanylate cyclases were CdgB,CdgA,and CdgE while c-di-GMP phosphodiesterase were CdgH and CdgE by active sites analysis.We constructed knockout and complementary mutants of CdgB,CdgA,and CdgH to study the influences of c-di-GMP pathway to submerged biofilm formation.The result demonstrated that GGDEF domain proteins CdgB and CdgA enhanced biofilm formation and EAL domain protein CdgH reduced biofilm formation.The phenotypes were partly restored in complementary strains.We inferred that c-di-GMP pathway took part in submerged biofilm formation of B.cereus 905.