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Cloning and characterization of the sisomicin-resistance gene from Micromonospora inyoensis
S L Goldberg1, J G Romero, Y M Deo
1Department of Microbiological and Cell Culture Development, Schering Corporation, Union, NJ 07083.
Abstract:
We cloned DNA fragments of sisomicin-producing Micromonospora inyoensis into Streptomyces plasmid vectors and identified Streptomyces lividans TK24 transformants expressing the M. inyoensis sisomicin-resistance (sisA) gene. The sisA gene was compared to the previously reported Micromonospora purpurea Kan-Gen (kanamycin-gentamicin)-resistance gene. While the restriction endonuclease digestion patterns of the two determinants appear to be divergent, the genes are nonetheless closely related, based on similar patterns and levels of aminoglycoside-resistance and their ability to cross-hybridize under stringent conditions. We have transformed recombinant plasmid pMD5-2, which carries the sisA gene, into our M. purpurea gentamicin-production strain and determined that gentamicin biosynthesis was not improved.
Insights
Researchers identified the sisomicin-resistance (sisA) gene in Micromonospora inyoensis. This gene, though related to the Micromonospora purpurea Kan-Gen resistance gene, did not enhance gentamicin production when introduced into the M. purpurea strain.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- The sisA gene confers resistance to sisomicin in Micromonospora inyoensis.
- Aminoglycoside resistance genes are crucial for understanding antibiotic biosynthesis and resistance mechanisms.
- Comparing resistance genes across different Micromonospora species can reveal evolutionary relationships and functional similarities.
Purpose of the Study:
- To clone and characterize the sisA gene from Micromonospora inyoensis.
- To compare the sisA gene with the kanamycin-gentamicin resistance gene from Micromonospora purpurea.
- To investigate the effect of the sisA gene on gentamicin biosynthesis in Micromonospora purpurea.
Main Methods:
- Cloning of DNA fragments from M. inyoensis into Streptomyces plasmid vectors.
- Transformation of Streptomyces lividans TK24 with recombinant plasmids.
- Analysis of gene expression and resistance patterns in transformants.
- Restriction endonuclease digestion and cross-hybridization studies.
- Transformation of the sisA gene into the M. purpurea gentamicin-production strain.
Main Results:
- The sisA gene from M. inyoensis was successfully cloned and expressed in S. lividans TK24.
- The sisA gene demonstrated close genetic relatedness to the M. purpurea Kan-Gen resistance gene, despite divergent restriction patterns.
- Both genes conferred similar levels of aminoglycoside resistance and showed cross-hybridization.
- Introduction of the sisA gene into the M. purpurea strain did not enhance gentamicin production.
Conclusions:
- The sisA gene is a novel aminoglycoside resistance determinant in M. inyoensis.
- The sisA gene and the M. purpurea Kan-Gen resistance gene share a common ancestry.
- The sisA gene does not play a direct role in enhancing gentamicin biosynthesis in M. purpurea.