Genome Sequencing And Comparative Genomic Analysis Of Amycolatopsis BJA-103

The genus Amycolatopsis is a kind of Actinomycete that produces important antibiotics,which belongs to the family pseudonocardiaceae.Among this genus,varieties of strains are widely used in the industrial production of antibiotics,such as A.orientalis HCCB10007 and A.mediterranei U32,which produce vancomycin and rifamycin respectively.In a screening program for the isolation of Valeriana associated actinomycetes,strain BJA-103 was isolated in the rhizomes of Valeriana jatamansi Jones from the Qinling Mountains.In this study,we successfully sequenced a new member of the genus Amycolatopsis strain BJA-103 and performed comparative genomic analysis to provide comprehensive understandings of the genus Amycolatopsis at the pan-genomic,phylogenetic,gene content and secondary metabolic levels.The research progress is summarized as follow:Complete genome sequence of Amycolatopsis BJA-103 was obtained by whole-genome sequencing(GenBank Accession Number: CP017780).The complete genome of BJA-103 contains a circular chromosome of 9488490 bp with GC content of 68.83 %,and 8644 CDSs,4 rRNA operons and 55 tRNA genes were detected in it.Thirty secondary metabolic gene clusters were predicted using antiSMASH 4,suggesting that strain BJA-103 may harbor abundant secondary metabolic resources.The general genome features of BJA-103 and 4 other completely sequenced strains were compared in this section.The genome features of strain BJA-103 are similar to that of A.orientalis HCCB10007 and A.japonica MG417-CF17 in the respects of genome size,GC content,CDSs number and coding density.Results of collinearity analysis revealed that the genome of BJA-103 displayed complete synteny with A.orientalis HCCB10007,the intensively studied strains would provide references for mining the potential properties of BJA-103.Via Pan-genomic analysis,all genes of the 23 Amycolatopsis genomes were clustered into 37,019 orthologous gene clusters,and core genome consisted of 1629 orthologous gene clusters present in all strains.Total 20421 gene clusters were identified as strain-specific genes that provide species diversity.As shown in the pan-genome curve,the size of pan-genome was expanding as the increasing of sequenced strains,indicating that the genus Amycolatopsis has an “open” pan-genome.We can firmly predict that new gene clusters will continue to be identified with the addition of strains isolated and sequenced,then following the growth of pan-genome size,however,the number of core genome will be moderately adjusted to reach a plateau in the future.Similar to the most strains,BJA-103 had a medium quantity of strain-specific genes,indicating that BJA-103 is definitely not the same species as other 22 strains.Strain BJA-103 would be the first genome sequenced strain in its species,or even hold the possibility of being a new species.We constructed the phylogenetic tree based on all concatenated SNPs in the 1629 core orthologous gene clusters,the result elucidated the phylogenetic relationship and status of each strains.As shown in dendrogram,BJA-103 possessed the closest phylogenetic relationship with A.coloradensis DSM 44225.Furthermore,our results certainly reflect the high credibility of analyses based on SNPs of core genome and MLSA of 7 housekeeping genes in phylogenetic analysis.Meanwhile,intergenomic ANI and dDDH values can be used to provide definite evidences for classifying the members of this genus and support the results of future phylogenetic analyses.To clarify the gene content of Amycolatopsis,the UPGMA phylogenetic tree was constructed based on the genetic distance matrix of the pan-genome using binary data.BJA-103 clustered in the same clade with A.alba DSM 44262 and A.azurea DSM 43854,which did not strictly follow their phylogenetic relationships as shown in the SNP dendrogram,suggesting the phylogeny of gene repertoire in Amycolatopsis was performed not only through vertical inheritance in evolution process,but also suffered lots of horizontal gene transfer events.Furthermore,we noticed the dendrogram based on gene content of pan-genome also represented the capability of antibiotic production.We found the orthologs encoding StrR-like pathway-specific regulator are present only in glycopeptide antibiotic-producing strains and play a positive regulatory effect on the production of antibiotics,which can be used for activation of the ristocetin biosynthetic gene cluster in BJA-103.The biosynthetic genes of these glycopeptide gene clusters had an identical arrangement of domains in spite of synthesizing distinct glycopeptide antibiotics,and the deciding factors of antibiotic differentiation are the positions and substrate types of A domains in gene clusters.We found 3 residues differences in the middle of binding pocket of A3 domains between the vancomycin and balhimycin biosynthetic gene clusters.It's probably on account of some SNPs that lead to this antibiotics differentiation.Nevertheless,the biosynthetic mechanism of these antibiotics remains further in-depth investigations.