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Complete genome sequence and comparative genomic analyses of the vancomycin-producing Amycolatopsis orientalis.

Xu L, Huang H, Wei W, Zhong Y, Tang B, Yuan H, Zhu L, Huang W, Ge M, Yang S, Zheng H, Jiang W, Chen D, Zhao GP, Zhao W - BMC Genomics (2014)

Bottom Line: Employing a customized PCR-targeting-based mutagenesis system along with the biochemical identification of vancomycin variants produced by the mutants, we were able to experimentally characterize a halogenase, a methyltransferase and two glycosyltransferases encoded in the vcm cluster.The broad substrate spectra characteristics of these modification enzymes were inferred.This study not only extended the genetic knowledge of the genus Amycolatopsis and the biochemical knowledge of vcm-related post-assembly tailoring enzymes, but also developed methodology useful for in vivo studies in A. orientalis, which has been widely considered as a barrier in this field.

View Article: PubMed Central - PubMed

Affiliation: Shanghai Laiyi Center for Biopharmaceutical R&D, Shanghai 200240, China. whjiang@sibs.ac.cn.

ABSTRACT

Background: Amycolatopsis orientalis is the type species of the genus and its industrial strain HCCB10007, derived from ATCC 43491, has been used for large-scale production of the vital antibiotic vancomycin. However, to date, neither the complete genomic sequence of this species nor a systemic characterization of the vancomycin biosynthesis cluster (vcm) has been reported. With only the whole genome sequence of Amycolatopsis mediterranei available, additional complete genomes of other species may facilitate intra-generic comparative analysis of the genus.

Results: The complete genome of A. orientalis HCCB10007 comprises an 8,948,591-bp circular chromosome and a 33,499-bp dissociated plasmid. In total, 8,121 protein-coding sequences were predicted, and the species-specific genomic features of A. orientalis were analyzed in comparison with that of A. mediterranei. The common characteristics of Amycolatopsis genomes were revealed via intra- and inter-generic comparative genomic analyses within the domain of actinomycetes, and led directly to the development of sequence-based Amycolatopsis molecular chemotaxonomic characteristics (MCCs). The chromosomal core/quasi-core and non-core configurations of the A. orientalis and the A. mediterranei genome were analyzed reciprocally, with respect to further understanding both the discriminable criteria and the evolutionary implementation. In addition, 26 gene clusters related to secondary metabolism, including the 64-kb vcm cluster, were identified in the genome. Employing a customized PCR-targeting-based mutagenesis system along with the biochemical identification of vancomycin variants produced by the mutants, we were able to experimentally characterize a halogenase, a methyltransferase and two glycosyltransferases encoded in the vcm cluster. The broad substrate spectra characteristics of these modification enzymes were inferred.

Conclusions: This study not only extended the genetic knowledge of the genus Amycolatopsis and the biochemical knowledge of vcm-related post-assembly tailoring enzymes, but also developed methodology useful for in vivo studies in A. orientalis, which has been widely considered as a barrier in this field.

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Genome atlas of theA. orientalisand gene clusters for secondary metabolism. The large circle represents the chromosome: the outer scale is numbered in megabases and indicates the core (red), quasi-core (orange), and non-core (sky blue) regions. The circles are numbered from the outside in. The genes in circles 1 and 2 (forward and reverse strands, respectively) are color-coded according to COG functional categories. Circle 3 shows selected essential genes (cell division, replication, transcription, translation, and amino-acid metabolism; the paralogs of essential genes in the non-core regions are not included). Circle 4 shows the secondary metabolic clusters, which are further enlarged outside the circle for detailed illustration. The vcm cluster is further illustrated in Figure 6. Circle 5 depicts the RNAs (blue, tRNA; red, rRNA). Circle 6 shows the mobile genetic elements (transposase, phage). Circle 7 depicts the GC content. Circle 8 shows the GC bias (pink, values > 0; green, values < 0). The small circle on the right side represents the plasmid DNA sequence. The outer scale is numbered in kilobases. All of the genes, regardless of the forward or reverse strands, are illustrated in the same circle. Circles 2 and 3 are the same as circles 7 and 8 of the large chromosome, respectively.
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Fig2: Genome atlas of theA. orientalisand gene clusters for secondary metabolism. The large circle represents the chromosome: the outer scale is numbered in megabases and indicates the core (red), quasi-core (orange), and non-core (sky blue) regions. The circles are numbered from the outside in. The genes in circles 1 and 2 (forward and reverse strands, respectively) are color-coded according to COG functional categories. Circle 3 shows selected essential genes (cell division, replication, transcription, translation, and amino-acid metabolism; the paralogs of essential genes in the non-core regions are not included). Circle 4 shows the secondary metabolic clusters, which are further enlarged outside the circle for detailed illustration. The vcm cluster is further illustrated in Figure 6. Circle 5 depicts the RNAs (blue, tRNA; red, rRNA). Circle 6 shows the mobile genetic elements (transposase, phage). Circle 7 depicts the GC content. Circle 8 shows the GC bias (pink, values > 0; green, values < 0). The small circle on the right side represents the plasmid DNA sequence. The outer scale is numbered in kilobases. All of the genes, regardless of the forward or reverse strands, are illustrated in the same circle. Circles 2 and 3 are the same as circles 7 and 8 of the large chromosome, respectively.

Mentions: The genome of A. orientalis HCCB10007 comprises two replicons (Figure 2), a large circular chromosome (8,948,591 bp) and a small, dissociated circular plasmid (33,499 bp). The same circular chromosomal topology with that of A. mediterranei U32 [24] and A. mediterranei S699 [25, 26], which are the other two complete genomes of the Amycolatopsis genus currently available, implies that this is a common topological feature that differs from the Streptomyces linear chromosomes [27]. The genome of A. orientalis HCCB10007 is much smaller (1.3 Mbp) than that of A. mediterranei, and only 8,121 protein-coding sequences (CDSs) were predicted, which is approximately 1,100 fewer CDSs than those identified in the genome of A. mediterranei (Table 1). The difference is mainly accounted for ~1.1 Mbp shorter in the length of the non-core regions of A. orientalis. Furthermore, this difference is also enhanced to a certain extent (about 0.2 Mbp) by the smaller average size of the intergenic region (IR) both in the core and the non-core regions of the A. orientalis genome (Table 1), resulting in a more compact arrangement of genes (coding density of 90.4%) compared with that of A. mediterranei (89.1-89.3%).Figure 2


Complete genome sequence and comparative genomic analyses of the vancomycin-producing Amycolatopsis orientalis.

Xu L, Huang H, Wei W, Zhong Y, Tang B, Yuan H, Zhu L, Huang W, Ge M, Yang S, Zheng H, Jiang W, Chen D, Zhao GP, Zhao W - BMC Genomics (2014)

Genome atlas of theA. orientalisand gene clusters for secondary metabolism. The large circle represents the chromosome: the outer scale is numbered in megabases and indicates the core (red), quasi-core (orange), and non-core (sky blue) regions. The circles are numbered from the outside in. The genes in circles 1 and 2 (forward and reverse strands, respectively) are color-coded according to COG functional categories. Circle 3 shows selected essential genes (cell division, replication, transcription, translation, and amino-acid metabolism; the paralogs of essential genes in the non-core regions are not included). Circle 4 shows the secondary metabolic clusters, which are further enlarged outside the circle for detailed illustration. The vcm cluster is further illustrated in Figure 6. Circle 5 depicts the RNAs (blue, tRNA; red, rRNA). Circle 6 shows the mobile genetic elements (transposase, phage). Circle 7 depicts the GC content. Circle 8 shows the GC bias (pink, values > 0; green, values < 0). The small circle on the right side represents the plasmid DNA sequence. The outer scale is numbered in kilobases. All of the genes, regardless of the forward or reverse strands, are illustrated in the same circle. Circles 2 and 3 are the same as circles 7 and 8 of the large chromosome, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4048454&req=5

Fig2: Genome atlas of theA. orientalisand gene clusters for secondary metabolism. The large circle represents the chromosome: the outer scale is numbered in megabases and indicates the core (red), quasi-core (orange), and non-core (sky blue) regions. The circles are numbered from the outside in. The genes in circles 1 and 2 (forward and reverse strands, respectively) are color-coded according to COG functional categories. Circle 3 shows selected essential genes (cell division, replication, transcription, translation, and amino-acid metabolism; the paralogs of essential genes in the non-core regions are not included). Circle 4 shows the secondary metabolic clusters, which are further enlarged outside the circle for detailed illustration. The vcm cluster is further illustrated in Figure 6. Circle 5 depicts the RNAs (blue, tRNA; red, rRNA). Circle 6 shows the mobile genetic elements (transposase, phage). Circle 7 depicts the GC content. Circle 8 shows the GC bias (pink, values > 0; green, values < 0). The small circle on the right side represents the plasmid DNA sequence. The outer scale is numbered in kilobases. All of the genes, regardless of the forward or reverse strands, are illustrated in the same circle. Circles 2 and 3 are the same as circles 7 and 8 of the large chromosome, respectively.
Mentions: The genome of A. orientalis HCCB10007 comprises two replicons (Figure 2), a large circular chromosome (8,948,591 bp) and a small, dissociated circular plasmid (33,499 bp). The same circular chromosomal topology with that of A. mediterranei U32 [24] and A. mediterranei S699 [25, 26], which are the other two complete genomes of the Amycolatopsis genus currently available, implies that this is a common topological feature that differs from the Streptomyces linear chromosomes [27]. The genome of A. orientalis HCCB10007 is much smaller (1.3 Mbp) than that of A. mediterranei, and only 8,121 protein-coding sequences (CDSs) were predicted, which is approximately 1,100 fewer CDSs than those identified in the genome of A. mediterranei (Table 1). The difference is mainly accounted for ~1.1 Mbp shorter in the length of the non-core regions of A. orientalis. Furthermore, this difference is also enhanced to a certain extent (about 0.2 Mbp) by the smaller average size of the intergenic region (IR) both in the core and the non-core regions of the A. orientalis genome (Table 1), resulting in a more compact arrangement of genes (coding density of 90.4%) compared with that of A. mediterranei (89.1-89.3%).Figure 2

Bottom Line: Employing a customized PCR-targeting-based mutagenesis system along with the biochemical identification of vancomycin variants produced by the mutants, we were able to experimentally characterize a halogenase, a methyltransferase and two glycosyltransferases encoded in the vcm cluster.The broad substrate spectra characteristics of these modification enzymes were inferred.This study not only extended the genetic knowledge of the genus Amycolatopsis and the biochemical knowledge of vcm-related post-assembly tailoring enzymes, but also developed methodology useful for in vivo studies in A. orientalis, which has been widely considered as a barrier in this field.

View Article: PubMed Central - PubMed

Affiliation: Shanghai Laiyi Center for Biopharmaceutical R&D, Shanghai 200240, China. whjiang@sibs.ac.cn.

ABSTRACT

Background: Amycolatopsis orientalis is the type species of the genus and its industrial strain HCCB10007, derived from ATCC 43491, has been used for large-scale production of the vital antibiotic vancomycin. However, to date, neither the complete genomic sequence of this species nor a systemic characterization of the vancomycin biosynthesis cluster (vcm) has been reported. With only the whole genome sequence of Amycolatopsis mediterranei available, additional complete genomes of other species may facilitate intra-generic comparative analysis of the genus.

Results: The complete genome of A. orientalis HCCB10007 comprises an 8,948,591-bp circular chromosome and a 33,499-bp dissociated plasmid. In total, 8,121 protein-coding sequences were predicted, and the species-specific genomic features of A. orientalis were analyzed in comparison with that of A. mediterranei. The common characteristics of Amycolatopsis genomes were revealed via intra- and inter-generic comparative genomic analyses within the domain of actinomycetes, and led directly to the development of sequence-based Amycolatopsis molecular chemotaxonomic characteristics (MCCs). The chromosomal core/quasi-core and non-core configurations of the A. orientalis and the A. mediterranei genome were analyzed reciprocally, with respect to further understanding both the discriminable criteria and the evolutionary implementation. In addition, 26 gene clusters related to secondary metabolism, including the 64-kb vcm cluster, were identified in the genome. Employing a customized PCR-targeting-based mutagenesis system along with the biochemical identification of vancomycin variants produced by the mutants, we were able to experimentally characterize a halogenase, a methyltransferase and two glycosyltransferases encoded in the vcm cluster. The broad substrate spectra characteristics of these modification enzymes were inferred.

Conclusions: This study not only extended the genetic knowledge of the genus Amycolatopsis and the biochemical knowledge of vcm-related post-assembly tailoring enzymes, but also developed methodology useful for in vivo studies in A. orientalis, which has been widely considered as a barrier in this field.

Show MeSH
Related in: MedlinePlus