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Cloning and mapping of the genetic determinants for microcin C7 production and immunity
Abstract:
Microcin C7, a peptide antibiotic inhibitor of protein synthesis, is produced by Escherichia coli K-12 strains that carry the 43-kilobase low-copy-number plasmid pMccC7. Microcin C7 production and immunity determinants of this plasmid have been cloned into the vectors pBR322 and pACYC184. The resulting plasmids overproduce microcin C7 and express immunity against the microcin. Mcc- and Mcc- Imm- mutants have been isolated on recombinant plasmids by inserting transposable elements. Physical and phenotypic characterization of these mutants shows that a DNA region of 5 kilobases is required to produce microcin C7, and that two small regions located inside the producing region are also required to express immunity. Analysis of plasmids carrying mcc-lacZ gene fusions indicates that all microcin DNA is transcribed in the same direction. The results suggest that a structure like a polycistronic operon is responsible for microcin C7 production and immunity.
Insights
Researchers identified the genetic basis for microcin C7 (a protein synthesis inhibitor) production and immunity in Escherichia coli. A 5-kilobase DNA region on the pMccC7 plasmid is essential for microcin C7 synthesis and resistance.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Microcin C7 is a peptide antibiotic produced by Escherichia coli K-12.
- It functions as an inhibitor of protein synthesis.
- Production is linked to the low-copy-number plasmid pMccC7.
Purpose of the Study:
- To clone and characterize the genetic determinants of microcin C7 production and immunity.
- To identify the specific DNA regions responsible for microcin C7 synthesis and resistance.
- To elucidate the genetic organization of microcin C7 biosynthesis.
Main Methods:
- Cloning of microcin C7 production and immunity genes into expression vectors (pBR322, pACYC184).
- Isolation and characterization of microcin C7 (Mcc) and immunity (Mcc-Imm) mutants using transposable elements.
- Physical and phenotypic analysis of recombinant plasmids and mutants.
- Construction and analysis of mcc-lacZ gene fusions.
Main Results:
- Recombinant plasmids overproduced microcin C7 and conferred immunity.
- A 5-kilobase DNA region was identified as essential for microcin C7 production.
- Two smaller regions within the producing region were found necessary for immunity.
- Analysis of gene fusions indicated co-directional transcription of microcin DNA.
Conclusions:
- The genetic elements for microcin C7 production and immunity are located on a 5-kilobase DNA segment of the pMccC7 plasmid.
- Two distinct immunity regions are embedded within the larger production region.
- The genes involved in microcin C7 biosynthesis and immunity appear to be organized in a polycistronic operon structure.