The Silence Mechanism Of Nematicidal Crystal Protein Cry5Ba And Identification Of The Nematicidal Virulence Factors In Bacillus Thuringiensis YBT-1518

Bacillus thuringiensis forms proteinaceous crystals during sporulation,which account for 20-30%of the sporulated culture.The B.thuringiensis cell produces large amounts of insecticidal crystal proteins due to it has a lot of positive adjustment factor to adjust the cry gene expression regulation.There are many reports focused on the positive regulate mechanism of the crystal proteins.But there is few reports on negative control.B.thuringiensis YBT-1518 produces rice-shaped protein crystals with 2 components of molecular mass 45 kDa(cry55Aa)and 54 kDa(cry6Aa)which are toxic to root-knot nematode.Based on a novel gene isolation strategy,we isolated from this strain the third crystal gene cry5Ba which can direct the expression of 140 kDa Cry5Ba protein in a heterologous B.thuringiensis host BMB171.But in YBT-1518 we cannot observe the expression of Cry5Ba under the detection with an optical microscope and SDS-PAGE.After eliminating the plasmid pBMB0228,which harbors cry55Aa and cry6Aa,there is not effect on the expression of Cry5Ba.RT-PCR detection showed a transit transcription at the onset of the stationary phase.More than 20kb flank sequences(each side)of cry5Ba can not be found to affect the expression of Cry5Ba.After finishing the genome sequence and bioinformatics analysis,a gene locus BMBsr1 which reversely complements the ribosome binding site of cry5Ba was predicted as a sRNA coding sequence.By heterologous expression,gene BMBsrl was found to inhibit the expression of Cry5Ba in strain YBT-1518.When applying gfp and lacZ as report marker,BMBsr1 was found gene silencing effect either.Base scanning mutagenesis demonstrated the key bases in BMBsr1 determining the gene silencing effect.Gene BMBsr1 will be the first identified negative control factor in crystal protein gene expression in B.thuringiensis.And this finding will help to discover the potential highly toxic crystal protein genes that are silencing in B.thuringiensis sources.Some 5.thuringiensis strains have high toxicity to nematodes.Nematicidal activity has been found in several families of crystal proteins,such as Cry5,Cry6,and Cry55.The B.thuringiensis strain YBT-1518,has three cry genes that have high nematicidal activity.The whole genome sequence of this strain contains multiple potential virulence factors.To evaluate the pathogenic potential of virulence factors,we focused on a metalloproteinase called Bmp1.It encompasses a consecutive N-terminal signal peptide,a FTP superfamily domain,a M4 neutral protease GluZincin superfamily,two Big 3 superfamily motifs and Gram positive anchor superfamily motif as C-terminal domain.Here,we showed that purified Bmp1 protein showed metalloproteinase activity and toxicity against Caenorhabditis elegans(LC50 is 610±9.37 ?g/ml).In addition,mixing CrySBa with Bmpl protein enhanced the toxicity 7.9 times(the expected toxicity of the two proteins calculated from their separate toxicities)against C.elegans.Confocal microscopic observation revealed that Bmpl protein was detected around mouth and esophagus to intestine.Striking microscopic images revealed that Bmpl degrades intestine tissues,and the Cry5Ba causes intestinal shrinkage from the body wall.Thus,the B.thuringiensis Bmpl metalloproteinase is a nematicidal virulence factor.These findings give a new insight into the relationship between B.thuringiensis and its host nematodes.