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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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Biosynthetic pathways of different types of nitrogenous phospholipids in actinomycetes. (A) The cell membrane of Amycolatopsis belongs to the type PII because PE is the dominant phospholipid detected. Two essential proteins (AORI_7345 and AORI_7346, labeled in red color) involved in the biosynthesis of PE were encoded by the A. orientalis genome, whereas the genes encoding enzymes involved in other types of nitrogenous phospholipids were not found (NF). Actinomycetes of type PI contain no nitrogenous phospholipids in their cell membrane, while type PII, type PIII, type PIV, and type PV actinomycetes contain the following characteristic phospholipids: PE, PC, GluNU, and PG, respectively. Panel (B) illustrates the analysis of isoprenyl diphosphate synthases from type strains of actinomycetes. The names and amino-acid sequences of the strains with different colors represent actinomycetes harboring different-length MKs: red, MK7 (C35); olive-green, MK8 (C40); blue, MK9 (C45). The amino-acid sequences of the chain-length determination (CLD) region are emphasized in green on the right of the panel. The protein sequences were obtained from NCBI at http://www.ncbi.nlm.nih.gov/protein/.
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Fig4: Biosynthetic pathways of different types of nitrogenous phospholipids in actinomycetes. (A) The cell membrane of Amycolatopsis belongs to the type PII because PE is the dominant phospholipid detected. Two essential proteins (AORI_7345 and AORI_7346, labeled in red color) involved in the biosynthesis of PE were encoded by the A. orientalis genome, whereas the genes encoding enzymes involved in other types of nitrogenous phospholipids were not found (NF). Actinomycetes of type PI contain no nitrogenous phospholipids in their cell membrane, while type PII, type PIII, type PIV, and type PV actinomycetes contain the following characteristic phospholipids: PE, PC, GluNU, and PG, respectively. Panel (B) illustrates the analysis of isoprenyl diphosphate synthases from type strains of actinomycetes. The names and amino-acid sequences of the strains with different colors represent actinomycetes harboring different-length MKs: red, MK7 (C35); olive-green, MK8 (C40); blue, MK9 (C45). The amino-acid sequences of the chain-length determination (CLD) region are emphasized in green on the right of the panel. The protein sequences were obtained from NCBI at http://www.ncbi.nlm.nih.gov/protein/.

Mentions: The cell membrane of actinomycetes is classified into five types according to the presence of certain nitrogenous phospholipids [35]. The Amycolatopsis cell membrane belongs to the PII type because only one nitrogenous phospholipid, namely phosphatidyl ethanolamine (PE), was usually detected in its cell membrane [35]. In prokaryotes, phosphatidylserine is first generated from CDP-diacylglycerol, a general intermediate for the synthesis of different types of phospholipids, catalyzed by phosphatidylserine synthase (PssA, EC: 2.7.8.8), and is then transformed into PE [36, 37] by phosphatidylserine decarboxylase (Psd, EC: 4.1.1.65) (Figure 4A). Orthologs of both pssA (AORI_7346) and psd (AORI_7345) could be identified in the A. orientalis genome. These two genes also exist in other actinomycetes with a type PII cell membrane, such as A. mediterranei, N. farcinica, S. coelicolor, and M. smegmatis, but are absent in actinomycetes with a type PI membrane (no nitrogenous phospholipids) or other types of cell membranes. Moreover, in the genomes of neither A. orientalis nor A. mediterranei did we identify genes encoding phosphatidylcholine synthase (Pcs, EC: 2.7.8.24), which catalyzes the formation of phosphatidyl choline (PC), the characteristic type PIII phospholipid, or the genes encoding phosphatidylglycerophosphatase A (PgpA, EC: 3.1.3.27), which catalyzes the formation of phosphatidyl glycerol (PG), the characteristic type PV phospholipid (Figure 4A). It is worth mentioning that the gene ept1, which encodes ethanolamine phosphotransferase (EPT1, EC: 2.7.8.1) that catalyzes the biosynthesis of PE from 1, 2-diacylglycerol and CDP-ethanolamine in eukaryotes, is also absent in any of the sequenced actinomycete genomes (Figure 4A).Figure 4


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)

Biosynthetic pathways of different types of nitrogenous phospholipids in actinomycetes. (A) The cell membrane of Amycolatopsis belongs to the type PII because PE is the dominant phospholipid detected. Two essential proteins (AORI_7345 and AORI_7346, labeled in red color) involved in the biosynthesis of PE were encoded by the A. orientalis genome, whereas the genes encoding enzymes involved in other types of nitrogenous phospholipids were not found (NF). Actinomycetes of type PI contain no nitrogenous phospholipids in their cell membrane, while type PII, type PIII, type PIV, and type PV actinomycetes contain the following characteristic phospholipids: PE, PC, GluNU, and PG, respectively. Panel (B) illustrates the analysis of isoprenyl diphosphate synthases from type strains of actinomycetes. The names and amino-acid sequences of the strains with different colors represent actinomycetes harboring different-length MKs: red, MK7 (C35); olive-green, MK8 (C40); blue, MK9 (C45). The amino-acid sequences of the chain-length determination (CLD) region are emphasized in green on the right of the panel. The protein sequences were obtained from NCBI at http://www.ncbi.nlm.nih.gov/protein/.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig4: Biosynthetic pathways of different types of nitrogenous phospholipids in actinomycetes. (A) The cell membrane of Amycolatopsis belongs to the type PII because PE is the dominant phospholipid detected. Two essential proteins (AORI_7345 and AORI_7346, labeled in red color) involved in the biosynthesis of PE were encoded by the A. orientalis genome, whereas the genes encoding enzymes involved in other types of nitrogenous phospholipids were not found (NF). Actinomycetes of type PI contain no nitrogenous phospholipids in their cell membrane, while type PII, type PIII, type PIV, and type PV actinomycetes contain the following characteristic phospholipids: PE, PC, GluNU, and PG, respectively. Panel (B) illustrates the analysis of isoprenyl diphosphate synthases from type strains of actinomycetes. The names and amino-acid sequences of the strains with different colors represent actinomycetes harboring different-length MKs: red, MK7 (C35); olive-green, MK8 (C40); blue, MK9 (C45). The amino-acid sequences of the chain-length determination (CLD) region are emphasized in green on the right of the panel. The protein sequences were obtained from NCBI at http://www.ncbi.nlm.nih.gov/protein/.
Mentions: The cell membrane of actinomycetes is classified into five types according to the presence of certain nitrogenous phospholipids [35]. The Amycolatopsis cell membrane belongs to the PII type because only one nitrogenous phospholipid, namely phosphatidyl ethanolamine (PE), was usually detected in its cell membrane [35]. In prokaryotes, phosphatidylserine is first generated from CDP-diacylglycerol, a general intermediate for the synthesis of different types of phospholipids, catalyzed by phosphatidylserine synthase (PssA, EC: 2.7.8.8), and is then transformed into PE [36, 37] by phosphatidylserine decarboxylase (Psd, EC: 4.1.1.65) (Figure 4A). Orthologs of both pssA (AORI_7346) and psd (AORI_7345) could be identified in the A. orientalis genome. These two genes also exist in other actinomycetes with a type PII cell membrane, such as A. mediterranei, N. farcinica, S. coelicolor, and M. smegmatis, but are absent in actinomycetes with a type PI membrane (no nitrogenous phospholipids) or other types of cell membranes. Moreover, in the genomes of neither A. orientalis nor A. mediterranei did we identify genes encoding phosphatidylcholine synthase (Pcs, EC: 2.7.8.24), which catalyzes the formation of phosphatidyl choline (PC), the characteristic type PIII phospholipid, or the genes encoding phosphatidylglycerophosphatase A (PgpA, EC: 3.1.3.27), which catalyzes the formation of phosphatidyl glycerol (PG), the characteristic type PV phospholipid (Figure 4A). It is worth mentioning that the gene ept1, which encodes ethanolamine phosphotransferase (EPT1, EC: 2.7.8.1) that catalyzes the biosynthesis of PE from 1, 2-diacylglycerol and CDP-ethanolamine in eukaryotes, is also absent in any of the sequenced actinomycete genomes (Figure 4A).Figure 4

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