Phosphodiesterase In Bacillus Cereus Involved In Biofilm Formantion And The Virulence Of Helicoverpa Armi Gera

Phosphodiesterases(PDEs)are a type of cellular ubiquitous enzyme that modulate various signals within cells by hydrolyzing phosphodiester bonds such as cAMP and GMP conduction and physical activity.At present,11 PDEs with different structures have been found in biological cells.Different types of PDEs have different distributions,expressions,and modes of regulation and tolerance to inhibitors in cells,and they exert different physiological functions.YmdB was a PDE that is present in Bacillus subtilis and was involved in the regulation of bacterial biofilm formation,motility,extracellular polysaccharide production,bistimulus,and other physiological properties.0-9 was a biocontrol bacterium of soil-borne wheat diseases obtained in the previous study.It could colonize the roots of wheat and had the characteristics of easy cultivation,biofilm formation,and movement capacity.Though analyzing the amino acid sequence of the coding gene in the genome of the 0-9 strain,we have found that there was a gene whose amino acid sequence is 34%homologous to YmdB in B.subtilis,and that both had a similar structure with YmdB.The function of this gene and its encoded product in Bacillus cereus have not yet been reported.Here,we studied the function of this gene(tentatively named ymdB)by gene knockout and replenishment,and analyzed the effect of this gene on biofilm formation and on the pathogenicity of Helicoverpa armigera.In order to analyze the function of the ymdB gene,the ymdB gene deletion strain,?ymdB,was constructed by homologous recombination.The colony morphology,extracellular protease production capacity,motility,and biofilm formation of the ?ymdB strain and the wild-type 0-9 were determined.Differences in biological traits such as hemolytic activity showed that the mutant ?ymdB significantly decreased the population growth,motility,salt sensitivity,and hemolytic activity compared to the wild-type 0-9 strain,while the extracellular protease production ability were increased.At the same time,it was found that the surface of the colony formed by ?ymdB was smooth.The above results indicated that ymdB gene had a variety of physiological regulatory effects in Bacillus cereus.By constructing ymdB two heterologous complementary strains and determining the above biological phenotype,it was found that the complementation of ymdB gene could restore the biological characteristics of 0-9.After comparing its amino acid sequence,its functional amino acid was found to be glutamic acid.Site-directed mutagenesis was performed on the amino acid encoding gene sequence to construct a ymdB gene site-directed mutant complementary strain,and the above biological traits were determined.It was found that none of these traits could be recovered.This showed that the ymdB gene had multiple effects on the physiological functions of Bacillus cereus.In order to verify that the ymdB gene encoded product is a phosphodiesterase,a protein expression strain of ymdB gene was constructed in this study.Protein expression,purification,and enzyme activity were measured.The results showed that the ymdB gene encoding product was a phosphodiesterase.The study found that in the 0-9,a regulatory system centered on SinR/SinI regulates biofilms,and SinR regulated the formation of biofilms by sipW,hag,calY,tasA,and motA.The negative repression factor of the operon.In order to study whether YmdB was affected by SinR/SinI-mediated regulation of biofilm formation,we used the method of homologous recombination to construct the gene deletion strains?sinR,?sinI,?ymdBAsinl,?ymdBAsinR,and determine the biofilm formation.The results showed that AsinR biofilm formation was significantly greater than that of 0-9;?sinl biofilm formation was significantly reduced compared to 0-9;and ?ymdB?sinl,?ymdB?sinR were two genomic deletion strains of biology.To further analyzed the effect of YmdB on the formation of biofilms in 0-9,the sipW,hag,calY,motA,tasA regulated by SinR in wild-type B.cereus 0-9 and AymdB were measured by fluorescence PCR in different culture periods.The expression levels of this genes were found to be significantly lower in the AymdB than in the wild type 0-9.At the same time,?sinR,?sinl,?ymdB?sinl,?ymdB?sinR and their complementary strains were tested for biological characteristics associated with biofilm formation(creation of surface colonies and production of extracellular proteases).The results showed that ?sinR was responsible for colony morphology and extracellular protease production capacity.There was no significant difference between ?sinl and 0-9,but there was no significant difference between ?ymdB?sinI and?ymdB?sinR and ?ymdB;and the complementary strains could restore their traits.The results showed that?sinR was responsible for colony morphology and extracellular protease production capacity.There was no significant difference between Asinl and 0-9,but there was no significant difference between AymdBAsinI and ?ymdB?sinR and ?ymdB;and the complementary strains could restore their traits.In this study,the expression of YmdB protein and the expression of ymdB gene in different culture periods 0-9 were determined by Western blot and real-time fluorescence quantitative PCR.The results showed that the expression level of YmdB in 0-9 would decrease with the increase of culture time;at the same time,the expression of ymdB gene would decrease with the increase of culture time.0-9 was highly similar to Bacillus thuringiensis,and Bacillus thuringiensis have been reported to have insecticidal effects,and numerous studies have shown that YmdB was involved in toxic effects in microorganisms.Therefore,0-9 GFP-tagged strains and ?ymdB GFP-tagged strains were constructed in this study and injected into the cotton bollworm to observe the lethal effect on the ccotton bollworm.Meanwhile,the lethal effect of ymdB gene-complementing strains on cotton bollworm was also determined.The results showed that the final mortality rate of Helicoverpa armigera was significantly higher than that of ?AymdB in the 0-9 GFP-labeled strain group;the ymdB gene-complemented strain restored the lethal effect of 0-9 to Helicoverpa armigera;The 0-9 effect on the death of the bollworm was restored YmdB was found to have a promoting effect on its hemolytic ability in 0-9 in the determination of biological traits.From the above results,it was concluded that YmdB had a toxic effect on Helicoverpa armigera in 0-9.