Whole Genome Sequencing And Analysis Of Two Purine Nucleoside Industrial Producers

In this study, two purine nucleoside industrial producers B. subtilis ATCC 13952 and B. subtilis ATCC 19217 were chosen as research objects. Whole-genome sequence of ATCC 13952 and ATCC 19217 were obtained by Illumina Solexa sequencing and preliminary analysis were carried out by various bioinformatic software. The results from this study lay the foundation for further study of background information on genetics, mechanism of purine nucleoside accumulation, and molecular breeding. The main results were achieved and shown as follows:(1) Whole genome sequencing and function annotation of B. subtilis ATCC 13952 and B. subtilis ATCC 19217Whole-genome sequencing of ATCC 13952 and ATCC 19217 were carried out by Illumina sequencing to produce 689 Mb and 1,675 Mb filtered sequences, representing a 170-fold and 400-fold coverage of the genome. The genome was assembled using paired-end reads by Velvet software, producing 48 and 54 contigs, and N50 was 412,061 bp and 999,463 bp repectively. Then 26 and 33 scaffolds were got after scaffolding by SSPACE software, genome continuity increasing further.Then Mummer and OSLay tool were used for maping the scaffolds to the reference genomes, ordered and oriented all scaffolds, facilitate efficient gap closure.The inner and external gaps were filled by sequencing the PCR amplified products.At last,the complete genome sequence were obtained.And the sequence had already been submited to NCBI Gen Bank database,the accession number is CP009748 and CP009749. The size of B. subtilis ATCC 13952 genome is 3,876,276 bp with GC content of 45.8%, containing 3,852 CDSs,7 r RNA operons and 72 t RNAs. The size of B. subtilis ATCC 19217 genome is 3,959,897 bp with GC content of 46.4%, containing 3,677 CDSs,7 r RNA operons and 72 t RNAs.(2) Analysis of B.subtils ATCC 13952 and B.subtils ATCC 19217 genomeThe main analysis content includes: COG and GO functional classification, basic metabolism analysis, comparative genomics and analysis of genes relate to purine metabolism. All protein sequences BLAST with COG and Inter Pro database,then sorted them by function.The number of genes in metabolism,transcription and biosynthesis category is especially large,maybe means that metabolism of both strains was active.The metabolism pathways in both two strains were reconstructed by KEGG pathway database, adenine synthesis pathway was absent. Comparative genomic study between B.subtils ATCC 13952, B.subtils ATCC 19217 and other four close Bacillus strains B. amyloliquefaciens XH7,B. amyloliquefaciens TA208,B. amyloliquefaciens DSM7,B. subtilis 168,general features of six strains were very similar,and the genome structure remain highly conservative in addition to a small amount of insert and deletion. Synteny Block showed that the synteny relation of six strains was good, but there was a big DNA fragment inversion in B. amyloliquefaciens XH7 and B.amyloliquefaceins TA208.Maybe the inversion was due to horizontal gene transfer mediate by prophages owing to six other prophage and prophage remnants were found flanking the fragment region.Genes relate to purine metabolism in B.subtils ATCC 13952 and B.subtils ATCC 19217 BLAST with standard strains B. amyloliquefaciens DSM7 which didn’t accumulate purine nucleoside, pur A、drm、pbu X genes were absent,this would promoted the producing of purine nucleoside.Mutations occurred in pur F、gua A、pgi、pur M、pur L、pur C genes,some of them may good for purine nucleoside accumulation.However, most of the other genes remain unchanged, indicating the potential for further strain improvement.In this study,inosine industrial producer B. subtilis ATCC 13952 and guanosine industrial producer B. subtilis ATCC 19217 were sequenced,the complete sequences were obtianed after assembling and filling gaps.Then ORF prediction,function annotation and classification analysis,comparative genome analysis were carried by some useful tools.Proteins coded by purine metabolism-related genes had been resolved.These work would lay good foundation for molecular breeding study of inosine and guanosine producers.