| Bacillus thuringiensis(Bt)is a kind of Gram-positive endospore forming insecticidal bacterium,which can produce various insecticidal crystal proteins(ICPs).Therefore,Bt preparations are the most widely used microbial pesticides all over the world.The acrystalliferous mutant B.thuringiensis BMB171 possesses very high efficiency of electroporation transformation and favorable plasmid stability,and also is the ideal model strain.Our study devoted to the RNA chaperone Hfq and the mechanism of phenotype variation by c-di-GMP in B.thuringiensis BMB171.1)Functional analysis of the RNA chaperone Hfq in B.thuringiensisThe RNA chaperone Hfq is widely distributed in many bacteria and the homolog of Sm protein,which can participate in mRNA degradation in eukaryotic.Hfq is involved in virous pleiotropic phenotypes including the growth and metabolism and adaptiong to the environment stress for the Gram-negative bacteria,and Hfq plays an important role in several bacterial pathogens as well.The whole bacteria of Bacillus cereus group species can code two or more copies of hfq genes,and the number of copy for hfq genes is different from the majority of Gram-negative bacteria.This phenomenon suggests Hfq may reglate more physiological activities in B.thuringienisis.There are three hfq genes in our research bacterium B.thuringensis BMB171.They are RS08230(hfq1),RS18400(hfq2),and RS27540(hfq3).Firstly,we purified Hfq protein and studied its polymeric form and constructed different hfq mutants and investigated these mutants’ phenotype using kinds of phenotype examination.Based on our results,we found three hfq genes all are functional copies in BMB171 and the deletion of hfq can repress cell motility,enhance biolim formation,accelerate cell-cell aggregation,inhibit sporulation and increase virulence.At last,through RNA-seq analyses,we found there are 1730 differentially expressed genes in response to the deletion of hfq,and transcript level of 1414 genes were down-regulated,and that of 316 genes were up-regulated.Meanwhile,we predicted 55 potential sRNAs related to Hfq,and transcript of 14 potential sRNAs were repressed,and that of 18 potential sRNAs were increased.2)Mechanism research of phenotype alteration by high level c-di-GMP regulation in B.thuringiensisCyclic di-GMP is a universal second messenger in bacteria,which plays a key role in a wide range of bacterial physiological processes such as bacterial motility,biofilm formation,exopolysaccharides secretion,virulence and cell differentiation.The turnover of c-di-GMP is controlled by diguanylate cyclase(DGC)and c-di-GMP-specific phosphodiesterase(PDE).Our research group have improved that c-di-GMP can suppress motility,facilite biofilm formation,accelerate cell sedimentation and enhance vilurence.Therefore,we decided to study the mechanism of phenotype alteration betwenn BMB171 and Δ3pde.We detected respectively the concentration of c-di-GMP in BMB171 and Δ3pde and found the concentration of c-di-GMP higher than that of BMB171.We found c-di-GMP can affect cell motility by impeding transcription of flagellum assembly genes.In addition,we found the cells of Δ3pde were connected together by an extensive fibrous network to enhance biolim formation.This extensive fibrous network can facilitate cell-cell aggregation and affect the speed of sedimentation.c-di-GMP can enhance virulence for Helicoverpa armigerau through regulating the transcript of a pleiotropie regulatr PlcR.Finally,through whole-genome trancriptome profile analyses,we found some novel metabolism can be regulated by c-di-GMP directly or indirectly,and richment the regulatory network by the c-di-GMP signal transduction. |
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