Genomic Analysis Of Bacillus Thuringiensis Phage And Characterization Of A New Phage Holin Protein

Bacillus thuringiensis(Bt)is a gram-positive,spore-forming soil bacterium.It can produce parasporal crystals(Cry)which exhibit specific toxicity against variety insects and nematodes.Bt has been developed as the leading biopesticide for use as an alternative or supplement to synthetic chemical pesticides.However,83%of Bt strains contain lysogenic phage which resulted in serious losses during Bt fermentation.The environmental adaptation of Bt strains in soil were also associated with the lytic phage.In order to better understand Bt phage and provide effective strategy for the control of Bt phage,this study focused on several aspects of Bt phage,including the isolation and genome analysis of Bt phage and studies of a new phage holin protein.(1)The isolation and genome analysis of Bacillus thuringiensis phage.Up to now,some Bt phages have been isolated and studied,but still too few to analys Bt phages systematically.In this study,11 lysogenic phages and 4 lytic phages of Bt were isolated,Which contain Tectiviridae,Siphoviridae and Myoviridae phage.Spectrum detection indicate all the 15 phages expressed strong host specificity.All the phages only have lytic activity to B.cereus group strains and lytic phages have wider lytic spectrum than lysogenic phages.The genome size lysogenic phages is between 30 kb and 60 kb except BMBtpll,but that of lytic phages is much larger,about 30?60 kb.Genome annotation indicate that the majority genes encode unknown function proteins and genes with related functions always clustered together and form into a functional module.The results of whole genome comparision analysis suggest that 9 of the 15 phages are new type Bacillus phages.BMBtp5,BMBtp6 and BMBtp9 are very similar to each other and form a new Bacillus phage cluster.BMBtp1,BMBtp3,BMBtp4,BMBtp7,BMBtp8 and BMBtp10 represent 6 new Bacillus phage type respectively.This study enlarged the database of Bt phage greatly and will help for the production and application of Bt(2)The discovery of low abundance phage and its wide prevalence.Phages are the most abundant biological entities in diverse environments and bacterial genomes that play important roles ranging from nutrient cycling to gene transfer.However,a large number of lysogenic phages in bacterial genomes are unknown.Especially,the low-level induction prophages have been ignored for a long time.In this study,we reported 5 extremely low abundance phages revealed by high-throughput sequencing from Bt.Only 0.02%to 0.22%of the total reads matched these 5 phage genomes.The extremely low abundance feature made them easy to ignore by traditional approaches.These 5 phages have 27 to 51-kb dsDNA genome,which harbor genes encoding proteins with great amount of unknown functions and novel ORFs that are rarely found in public databases,and many unusual phage-encoded ORFs,including bacterial virulence factor,chromosome or plasmid segregation proteins,toxin-antitoxin system component,transcriptional regulator,Cytosine DNA methyltransferase,and CRISPR/Cas system component,etc.The whole genome nucleotide comparison and terminase phylogeny analysis of BMBtpLAs with reported 118 fully sequenced Bacillus phages revealed these 5 low abundance phages represent 3 new Bacillus phage clusters.Furthermore,similar sequences aligned to 3 of them were widespread(range from 8.58%to 27.49%of 862)in bacterial genomes isolated from soil samples.Our work suggested the novelty and the wide prevalence of these phage types in bacterial genomes,which may common present in other bacterial species.(3)Characterization of a new phage holin protein Bthl.Holin is the key component when phage lysis host cell.But genome analysis indicate that lysogenic phage BMBtp1 has no typical holin protein.Bioinformatic analysis of the product of gene Bthl suggest it has some basic features of phage holin protein and the following qPCR also demonstrate that gene Bthl is associated with phage lytic growth.Then heterologous expression of Bthl in E.coli indicate that Bthl can form a hole on membrane and the growth of E.coli was also depressed.The oligomeric states was detected and the dimers and trimers were observed.Bth1-GFP fusion expression also indicate Bthl locates on membrane.When gene Bth1 was deleted from YBT-1765,phage BMBtpl cannot lysis host cell,this told us Bthl is a key component of phage lytic system.Bthl-like protein also prevalence in bacteria and phage.Gene Bth2 is also a Bthl-like gene in YBT-1765,but biofilm formation and environment tolerance experiment demonstrate that gene Bth2 is more likely a bacterial gene which is associated with host function.Our result indicate Bthl protein is a new type of phage holin.This novel holin protein may be useful for understanding the interaction between Bt and its phage.It also enriches the substantial library of holin and may be useful for the comparative studies to elucidate the evolution and mode of action of holin.