The Study On The Mechanism Of H-antigen Determination Of Bacillus Thuringiensis

Bacillus thuringiensis(Bt)is a kind of rod-shaped Gram-positive bacteria.The parasporal crystal protein produced by Bacillus thuringiensis is toxic to Lepidoptera,Coleoptera,Hymenoptera and Diptera.Bt is the most successful microbial pesticide.At the same time,the insecticidal gene carried by Bt has also been successfully used to construct insect resistant transgenic crops.Due to the long-term use of insect resistance and the greater harm caused by new pests,more Bt resources mining has been concerned by researchers.Efficient classification and identification system is the key to the new type of Bt resource mining.The traditional H-serotyping method based on H-antigen(flagellin antigen)immune response has been applied to the classification and identification of tens of thousands of Bt strains.However,this method has been stopped because of its complicated operation.This study intends to explore the genetic determinants of Bt H-antigen from multiple levels and provide theoretical basis for the development of efficient Bt classification and identification system.At the same time,the elucidation of the genetic determination mechanism of Bt's H-antigen can also provide the basis for the other bacteria classifyied by H-serotype.In this study,we systematically understand the genetic basis of Bt's H-antigen from the perspectives of diversity of Bt flagellum synthesis gene cluster,transcription of key genes involved in H-antigen determination,translation of key genes and post-translational modification.The main results are as follows:(1)There are abundant diversity of flagellum synthesis gene clusters existed among Bt and Bacillus cereus.In order to understand the genetic diversity of Bt strains at the H-serotype level,B.cereus and B.mycoides were also analyzed.By comparing the flagellin synthesis gene clusters,we analyzed the flagellin determinant genes of the three groups,and found that the flagellin synthesis gene clusters of the members of B.cereus group were rich in diversity.Among them,Bt's flagellin synthesis gene clusters are almost the same as that of B.cereus,but it was quite different from that of B.mycoides.However,there is little significant difference in the sequence of flagellin gene and copy number of flagellin gene.It is speculated that Bacillus cereus strains have similar genetic basis with Bt's H-serotype.For Bt,the gene sequence and copy number of flagellin can partly reflect the diversity of H-antigen,indicating that the formation of Bt's different H-antigens is affected by multiple levels.(2)Our results showed that flagellin genes of all Bt strains without flagella structure can be transcribed normally,indicating that the H-antigen determination of Bt occurred at other levels of gene expression.Because many strains have similar structure of flagellin synthesis gene cluster and sequence of flagellin gene,but have great differences in H-antigen composition.We first explored the influence of gene expression level on H-antigen composition.The results showed that all the flagellin genes of Bt strains with flagellum structure could be transcribed,and the flagellin genes of the strains without flagella could also be transcribed whose flagellin genes are the same as those of other strains.However,flagellin of the strains without flagella could not be detected outside the cell,indicating that the transcription product of the gene was not translated into protein or the flagellin could not be secreted to participate in the assembly of flagellum,indicating that the H-antigen composition of Bt was affected by the level of translation or post-translation.(3)Although there are multiple copies of flagellin synthesis genes in many Bt's genomes,only one of them is expressed in general strains,indicating that translation level is crucial for the determination of H-antigen composition.In order to explore the expression of flagellin genes of different serotypes at translation level,we sequenced the extracted flagellin proteins based on mass spectrometry.The results showed that only one flagellin was detected in all the flagellin samples extracted from Bt strains,including Bt strains with multiple copies of flagellin genes.The flagellin expressed by these strains is often the longest sequence in their genome which is always be the Hag protein.When the strain do not encode the hag gene,it may generally express another flagellin called Fla B.When hag or fla B were multiple copies,Bt tends to express Hag A or fla B1 which have longer sequence length than other copies of flagellins.(4)The post-translational modification sites of flagellin encoded by Bt strains whose flagellin gene are similar but serotype are quite different,indicating that post-translational modification plays a decisive role in the composition of H-antigen of these kind of Bt strains.In order to explore the effect of post-translational modification on the composition of H-antigen,we used protein mass spectrometry to analyze the flagellin of different serotype with the same or highly similar flagellin gene sequence.We found that there were four kinds of post-translational modifications in the flagellin of different serotype of BT strains,namely acetylation modification and O-linked glycosylation modification,ubiquitination modification and phosphorylation modification.In addition,the post-translational modification sites of flagellin gene in Bt strains with high sequence identity were also different.These results indicate that post-translational modification directly determines the diversity of H-antigen composition of many Bt strains.(5)The H-antigen composition diversity of different sub-serotype of Bt strains is mainly determined by the V-region of flagellin gene,and the regulation is mainly affected at translation and post-translation levels.Because the flagellin gene is conserved at both ends and changeable in the center,the sequences or sites determining the composition of H-antigen is mainly located in the V-region of flagellin.Considering that post-translational modification can affect the diversity of H-antigen composition by regulating the V-region of flagellin sequence,we show the sites,sequences and modification sites of Bt strains whose flagellin V-region sequence are highly identical,and analyzed and speculated the determinants of H-antigen of these kind of strains.It is found that in serotypes of H3 ac,H3ad,H3 ade,H4ac,H5 ab,H7,H9,H23,H25,H26,H31,H37,H41 and H42 of Bt strains,suggesting that the H-antigen composition of these serotypes might be determined by post-translational modification.Although flagellin gene were highly similar in the Bt strains of serotype H20 ab,H20ac,H27 and H28 ac,there were differences in flg E gene and post-translational modification site in flagellin synthesis gene cluster.It is suggested that the H-antigen composition of these serotypes of Bt strains may be determined by flg E or post-translational modification.The H-antigen composition of other serotypes of Bt strain may be determined by the difference of V-region sequence.In our study,the determinants of H-antigen in different serotype of Bt strains were analyzed and speculated from the gene level,transcription level,translation level and post translation level.The expression of flagellin genes in different serotypes of Bt strains and the sites,sequences and post-translational modification sites that may determine the composition of H-antigens were revealed.Other genes that may affect H-serotypes were also found,which provided new ideas and insights for the improvement of the typing system of Bt's H-serotype and the classification of other strains.