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Mutational cloning in Streptomyces and the isolation of antibiotic production genes
Abstract:
An attachment site-deleted derivative, phi C31KC400, of the Streptomyces temperate phage phi C31 was used to clone fragments of the genetic determinants (mmy) for the biosynthesis of an antibiotic, methylenomycin A. The vector carries a cloned viomycin resistance gene (vph), and can transduce a recipient to viomycin resistance when DNA sequences are common to the phage and the recipient: the phage integrates into the recipient's genome through a Campbell type of recombination at the site of the homology. For the cloning of mmy DNA, the homology was provided by the in vitro insertion into the vector of DNA from a methylenomycin A-producing streptomycete. Clones carrying mmy DNA could integrate into a methylenomycin-producing recipient's mmy genes, sometimes disrupting their expression: thus a search of viomycin-resistant transductants for methylenomycin non-producing derivatives identified lysogens which spontaneously released phi C31 phages carrying mmy DNA. Some of these lysogens participated in methylenomycin co-synthesis with previously isolated mmy mutants. At least 7 kb of mmy DNA was identified among the clones. Screening for mmy non-producers was simplified by exploiting the presence of the mmy genes on the (albeit unisolable) plasmid, SCP1. In the course of the experiment, SCP1, a low copy number plasmid in its primary host S. coelicolor A3(2), was shown to have a copy number of about 30 in the single S. parvulus SCP1+ transconjugant strain tested, and a molecular size probably greater than 200 kb.
Insights
Researchers cloned antibiotic biosynthesis genes (mmy) using a modified Streptomyces phage (phi C31KC400) and viomycin resistance marker. This method identified over 7 kb of methylenomycin A genetic determinants.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Streptomyces temperate phage phi C31 is a valuable tool for genetic manipulation.
- Methylenomycin A (mmy) is an antibiotic produced by certain Streptomyces species.
- Understanding the genetic basis of antibiotic biosynthesis is crucial for strain improvement and drug discovery.
Purpose of the Study:
- To clone and identify the genetic determinants (mmy) responsible for methylenomycin A biosynthesis.
- To develop a novel cloning strategy utilizing phage integration and antibiotic resistance markers.
Main Methods:
- Construction of an attachment site-deleted phi C31 derivative (phi C31KC400) carrying a viomycin resistance gene (vph).
- In vitro insertion of DNA from a methylenomycin A-producing Streptomyces into the phage vector.
- Transduction of recipients and selection for viomycin resistance to identify integrated clones.
- Screening for non-producing mutants to isolate phages carrying mmy DNA fragments.
Main Results:
- Successfully cloned fragments of the mmy gene cluster, identifying at least 7 kb of DNA.
- Demonstrated that phage integration could disrupt mmy gene expression, facilitating clone identification.
- Observed co-synthesis of methylenomycin A by some lysogens, indicating functional mmy gene fragments.
- Characterized the SCP1 plasmid, showing increased copy number and large molecular size in a transconjugant strain.
Conclusions:
- The developed cloning strategy is effective for isolating antibiotic biosynthesis genes in Streptomyces.
- The study provides significant insights into the genetic organization of methylenomycin A production.
- The findings contribute to the understanding of Streptomyces genetics and the engineering of antibiotic production pathways.