Development of a Cloning System for Streptomyces

Abstract

Two potentially useful plasmid cloning vectors for Streptomyces are described. SCP2 and its high fertility variant, SCP2*, are self-transmissible plasmids which are capable of promoting chromosomal recombination. Although genetically distinguishable, no physical differences between SCP2 and SCP2* have been detected. Velocity sedimentation analyses indicate the plasmids to be of molecular weight 18-20 × 106 and restriction endonuclease cleavage maps of both plasmids indicate the presence of many restriction sites which ought to be amenable to the insertion of DNA without damaging functions essential for plasmid replication and maintenance. Although first isolated from Streptomyces coelicolor A3(2), SCP2* has been transferred by mating to S. parvulus ATCC 12434 and S. lividans JI1326 whereupon stable replication was observed. A series of self-transmissible plasmids, SLP1.1-6, have been isolated from S. lividans JI1326 which range in molecular weight from 6.25 to 8.2 × 104. Some of these plasmids contain unique restriction sites which are known to be present within regions of the plasmid genome that are not essential for plasmid replication or maintenance, a useful characteristic for any potential cloning vehicle. Essential to the development of a cloning system is a means of introducing the recombinant DNA into the host organism. SCP2, SCP2,* and all of the SLP1 series of plasmids exhibit a property termed "lethal zygosis," which allows for the recognition of plasmid-containing spores at very high resolution. A procedure has been developed which involves the uptake of covalently closed circular plasmid DNA by protoplasts in the presence of polyethylene glycol and the visual detection of transformants after regeneration of the protoplasts using the plasmid determined phenotype of lethal zygosis. Frequencies of transformation of up to 85% of the regenerated protoplasts are obtained.