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A spectinomycin resistance determinant from the spectinomycin producer Streptomyces flavopersicus
Dimitrina Lyutzkanova1, Jurgen Distler2, Josef Altenbuchner1
1Institut für Industrielle Genetik, Universität Stuttgart, Allmandring 31, 70569 Stuttgart, Germany.
Abstract:
The spectinomycin (sp) resistance determinant from Streptomyces flavopersicus was cloned into Streptomyces lividans using the plasmid vector pIJ699. A plasmid, pDGL15, with a 3.65 kb insert from S. flavopersicus conferring resistance to Sp was isolated. DNA sequence analysis of the 3651 1 bp DNA insert revealed four open reading frames (ORFs). The amino acid sequence deduced from one ORF (SpcN) showed a high degree of similarity to an aminoglycoside phosphotransferase (StrN) and from a second one (SpcR) to a regulatory protein (StrR) of the streptomycin biosynthesis gene cluster from S. griseus. The two other ORFs were incomplete and the deduced amino acid sequences showed similarities to an amidinotransferase encoded in the streptomycin biosynthesis gene cluster of S. griseus and to the transposase of IS112, respectively. Expression of the spcN gene in E. coli under the control of tac promoter conferred Sp resistance to the cells. An enzymic assay confirmed that the gene product of spcN is an ATP-dependent aminoglycoside phosphotransferase which phosphorylates Sp and actinamine, the aminocyclitol moiety of Sp.
Insights
Researchers identified a spectinomycin resistance gene (spcN) from Streptomyces flavopersicus. This gene encodes an enzyme that inactivates spectinomycin, offering a potential new avenue for antibiotic resistance research.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- Antibiotic resistance is a growing global health concern.
- Streptomyces species are prolific producers of antibiotics and resistance determinants.
- Understanding the mechanisms of antibiotic resistance is crucial for developing new therapeutic strategies.
Purpose of the Study:
- To clone and characterize the spectinomycin resistance determinant from Streptomyces flavopersicus.
- To elucidate the function and mechanism of action of the identified resistance gene.
- To explore the potential applications of this resistance determinant.
Main Methods:
- Cloning of the resistance determinant into a plasmid vector (pIJ699) in Streptomyces lividans.
- DNA sequencing and analysis of the insert to identify open reading frames (ORFs).
- Expression of the putative resistance gene (spcN) in E. coli and subsequent enzymic assays.
Main Results:
- Isolation of a 3.65 kb DNA fragment conferring spectinomycin resistance.
- Identification of four ORFs, with spcN showing similarity to aminoglycoside phosphotransferases.
- Demonstration that the spcN gene product is an ATP-dependent aminoglycoside phosphotransferase that phosphorylates spectinomycin and actinamine.
- Expression of spcN in E. coli conferred spectinomycin resistance.
Conclusions:
- The spcN gene from S. flavopersicus encodes an aminoglycoside phosphotransferase responsible for spectinomycin resistance.
- The enzyme phosphorylates spectinomycin, inactivating the antibiotic.
- This finding contributes to the understanding of antibiotic resistance mechanisms in Streptomyces.