The genus Amycolatopsis: Indigenous plasmids, cloning vectors and gene transfer systems

Abstract

The genus Amycolatopsis is a member of the phylogenetic group nocardioform actinomycetes. Most of the members of the genus Amycolatopsis are known to produce antibiotics. Additionally, members of this genus have been reported to metabolize aromatic compounds as the sole sources of carbon and energy. Development of genetic manipulation in Amycolatopsis has progressed slowly due to paucity of genetic tools and methods. The occurrence of indigenous plasmids in different species of Amycolatopsis is not very common. Till date, only three indigenous plasmids viz., pMEA100, pMEA300 and pA387 have been reported in Amycolatopsis species. Various vectors based on the indigenous plasmids, pMEA100, pMEA300 and pA387, have been constructed. These vectors have proved useful for molecular genetics studies of actinomycetes. Molecular genetic work with Amycolatopsis strains is not easy, since transformation methods have to be developed, or at least optimized, for each particular strain. Nonetheless, methods for effi cient transformation (polyethyleneglycol (PEG) induced protoplast transformation, transformation by electroporation and direct transformation) have been developed and used successfully for the introduction of DNA into several Amycolatopsis species. The construction of plasmid cloning vectors and the development of gene transfer systems has opened up possibilities for studying the molecular genetics of these bacteria.

Identification of the chelocardin biosynthetic gene cluster from Amycolatopsis sulphurea: a platform for producing novel tetracycline antibiotics

“…The Amycolatopsis strains have often proven difficult to transform and genetically manipulate (Malhotra & Lal, 2007;Dhingra et al, 2003). We have invested very significant efforts to develop a transformation procedure for A. sulphurea, where we tested various growth media, numerous actinomycete plasmids (replicative and integrative) in combination with different procedures, such as protoplast transformation and E. coli-A.…”

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

“…9 Conjugation experiments are easier to conduct than electroporation, as they do not require an expensive electroporation apparatus.…”

“…pMEA100 and pMEA300, which exist in integrated or freely replicating forms, are not suitable for molecular genetic studies. 9 pULVK2A 10 and pDXM32, 11 which are derivatives of pA387, are replicable plasmids that are routinely used in gene cloning. Meanwhile, several methods for the introduction of DNA into different Amycolatopsis species were successfully developed.…”

“…Meanwhile, several methods for the introduction of DNA into different Amycolatopsis species were successfully developed. 9 These plasmids and transfer systems have greatly facilitated studies of Amycolatopsis species.…”

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

“…The genus Amycolatopsis belongs to the taxonomic group of nocardioform actinomycetes and includes bacteria which are producers of medically important secondary metabolites, such as antibiotics [1]. To date, three indigenous plasmids, viz., pMEA100 [2], pMEA300 [3] and pA387 [4], have been reported in this genus.…”