Nucleotide sequence of plasmid pA387 of <i>Amycolatopsis benzoatilytica</i> and construction of a conjugative shuttle vector

Malhotra, Shweta; Majumdar, S. K.; Kumar, Mukesh; Bhasin, V K; Gartemann, Karl-Heinz; Lal, Rup

doi:10.1002/jobm.200700326

Cited by 8 publications

(9 citation statements)

References 36 publications

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“…In addition, the 58‐bp sequence (positions 1347–1404) shared 79% identity with ORF9 of pA387. The genes ORF8–ORF10 were sufficient for autonomous replication in pA387, whereas ORF7 was not necessary . The nucleic acid sequence of AORIP_25 from 299 to 798 bp was 46% identical to ORF7 of pRL1 in Streptomyces sp .…”

Section: Resultsmentioning

confidence: 97%

“…A. benzoatilytica (formerly A. orientalis ) DSM 43387 contains a non‐integrative plasmid, pA387 (GenBank accession no. EF375609, 30.2 kb) . Various vectors based on the pA387 replicon have been constructed, and some have been applied in research on A. mediterranei and A. orientalis strains ; however, much work on the stability and operability of the vectors must be performed.…”

Section: Introductionmentioning

confidence: 99%

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Characterization of plasmid pXL100 from Amycolatopsis orientalis HCCB10007 and construction of a shuttle vector

Zhu

et al. 2014

J. Basic Microbiol.

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Many strains of Amycolatopsis, such as Amycolatopsis orientalis, A. balhimycina, and A. mediterranei, are important antibiotic producers. Three indigenous plasmids, pMEA100, pMEA300, and pA387, found in this genus have been sequenced. However, only some vectors based on pA387 have been widely applied in Amycolatopsis research. An indigenous plasmid, pXL100, was isolated from the vancomycin producer A. orientalis HCCB10007. Sequence analysis of pXL100 revealed its total length to be 33,499 bp and GC content to be 68.9%. A 2830-bp fragment containing three ORFs has been identified as essential for replication in A. orientalis, but it has no significant similarity to any known replicons. A vector, pLYZW7-3, was constructed based on the pXL100 replicon and could be transferred into A. mediterranei and A. orientalis by electroporation or conjugation with high frequency. A mutant with a disrupted gene was successfully complemented with the pLYZW7-3 vector, indicating that the vector is potentially useful in Amycolatopsis research.

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Section: Resultsmentioning

confidence: 97%

Section: Introductionmentioning

confidence: 99%

Characterization of plasmid pXL100 from Amycolatopsis orientalis HCCB10007 and construction of a shuttle vector

Zhu

et al. 2014

J. Basic Microbiol.

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“…20 Here, we established a conjugal transfer system for A. mediterranei U32 based on the integrative plasmid pDZL802. Integration of this single-copy plasmid was stable in the absence of selective pressure, which is a distinct advantage compared with the replicable vectors used in genetic studies and industrial strain improvement.…”

Section: Discussionmentioning

confidence: 99%

Conjugation of φBT1-derived integrative plasmid pDZL802 in Amycolatopsis mediterranei U32

Zhou

Wang

et al. 2017

Bioengineered

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The genus Amycolatopsis is well known for its ability to produce antibiotics, and an increasing number of valuable biotechnological applications, such as bioremediation, biodegradation, bioconversion, and potentially biofuel, that use this genus have been developed. Amycolatopsis mediterranei is an industrial-scale producer of the important antibiotic rifamycin, which plays a vital role in antimycobacterial therapy. Genetic studies of Amycolatopsis species have progressed slowly due to the lack of efficient transformation methods and stable plasmid vectors. In A. mediterranei U32, electroporation and replicable plasmid vectors have been developed. Here, we establish a simple and efficient conjugal system by transferring integrative plasmid pDZL802 from ET12567 (pUZ8002) to A. mediterranei U32, with an efficiency of 4 £ 10 ¡5 CFU per recipient cell. This integrative vector, based on the 'BT1 int-attP locus, is a stable and versatile tool for A. mediterranei U32, and it may also be applicable to various other Amycolatopsis species for strain improvement, heterologous protein expression, and synthetic biology experiments.

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“…Until now, few plasmids have been described in Amycolatopsis species (22), and only four of them (pMEA100, pMEA300, pA387 and pXL100) have been further studied. However, only the replicative vectors derived from the endogenous plasmids pA387 and pXL100 have been shown to replicate into several Amycolatopsis species (12, 23).…”

Section: Introductionmentioning

confidence: 99%

“…For some time the introduction of DNA into Amycolatopsis has been a limiting factor, until this problem could be circumvented by the development of a method of direct transformation of Amycolatopsis mycelia (10) or by using intergeneric conjugation from Escherichia coli to introduce single-stranded DNA into the Amycolatopsis host (11,12).…”

Section: Introductionmentioning

confidence: 99%

Marker-free genome engineering in Amycolatopsis using the pSAM2 site-specific recombination system

Santos

Caraty-Philippe

Darbon

et al. 2021

Preprint

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Actinobacteria belonging to the genus Amycolatopsis are important for antibiotic production and other valuable biotechnological applications such as biodegradation or bioconversion. Despite their industrial importance, tools and methods for the genetic manipulation of Amycolatopsis are less developed than in other actinobacteria such as Streptomyces. Moreover, most of the existing methods do not support convenient marker-free genome engineering. Here, we report the use of the pSAM2 site-specific recombination system for the efficient deletion of marker genes or large DNA regions in Amycolatopsis. For this purpose, we constructed a shuttle vector, replicating in Escherichia coli and Amycolatopsis, expressing the Xis and Int proteins from the Streptomyces integrative and conjugative element pSAM2. These proteins are sufficient for site-specific recombination between the attachment sites attL and attR. We also constructed two plasmids, replicative in E. coli but not in Amycolatopsis, for the integration of the recombination sites attL and attR on each side of a region targeted for deletion. We exemplified the use of these tools in Amycolatopsis mediterranei DSM 40773 by obtaining with high efficiency (>95%) a marker-free deletion of one single gene in the rifamycin biosynthetic gene cluster or of the entire 90-kb cluster.

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Nucleotide sequence of plasmid pA387 of Amycolatopsis benzoatilytica and construction of a conjugative shuttle vector

Cited by 8 publications

References 36 publications

Characterization of plasmid pXL100 from Amycolatopsis orientalis HCCB10007 and construction of a shuttle vector

Characterization of plasmid pXL100 from Amycolatopsis orientalis HCCB10007 and construction of a shuttle vector

Conjugation of φBT1-derived integrative plasmid pDZL802 in Amycolatopsis mediterranei U32

Marker-free genome engineering in Amycolatopsis using the pSAM2 site-specific recombination system

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