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A second streptomycin resistance gene from Streptomyces griseus codes for streptomycin-3"-phosphotransferase.
P Heinzel1, O Werbitzky, J Distler
1Institut für Biochemie der Technischen Hochschule Darmstadt, Federal Republic of Germany.
Abstract:
Two genes, aphE and orf, coding for putative Mr 29,000 and Mr 31,000, proteins respectively, were identified in the nucleotide sequence of a 2.8 kbp DNA segment cloned from Streptomyces griseus N2-3-11. The aphE gene expressed streptomycin (SM) resistance and a SM phosphorylating enzyme in S. lividans strains. The two genes were found to be in opposite direction and seemed to share a common region of transcription termination. The aphE gene shows significant homology to the aph gene, encoding aminoglycoside 3'-phosphotransferase, APH(3'), from the neomycin-producing S. fradiae. The enzymatic specificity of the aphE gene product was identified to be SM 3"-phosphotransferase, APH(3"). The primary structure of the APH(3") protein is closely related to the members of the APH(3') family of enzymes. However, the APH(3") enzyme did not detectably phosphorylate neomycin or kanamycin. There is only low similarity of the protein to the APH(6) group of SM phosphotransferases. An evolutionary relationship between antibiotic and protein kinases is proposed.
Insights
Researchers identified two genes, aphE and orf, in Streptomyces griseus. The aphE gene confers streptomycin resistance by producing a specific phosphorylating enzyme, APH(3").
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- Streptomyces griseus is a source of antibiotics.
- Aminoglycoside resistance mechanisms are crucial in microbial studies.
- Gene cloning and characterization are key to understanding antibiotic resistance.
Purpose of the Study:
- To identify and characterize genes involved in streptomycin resistance in Streptomyces griseus.
- To determine the enzymatic activity and specificity of the identified streptomycin resistance gene product.
- To explore the evolutionary relationship of the identified enzyme with known phosphotransferases.
Main Methods:
- DNA sequencing of a cloned 2.8 kbp fragment from Streptomyces griseus.
- Gene expression analysis in Streptomyces lividans.
- Enzyme activity assays to determine substrate specificity.
- Bioinformatic analysis for protein homology and evolutionary relationships.
Main Results:
- Two genes, aphE and orf, were identified. The aphE gene confers streptomycin resistance and encodes a streptomycin 3"-phosphotransferase (APH(3")).
- The APH(3") enzyme specifically phosphorylates streptomycin but not neomycin or kanamycin.
- The aphE gene shows homology to aminoglycoside phosphotransferase genes, suggesting a conserved resistance mechanism.
Conclusions:
- The aphE gene provides a novel mechanism for streptomycin resistance in Streptomyces.
- The APH(3") enzyme represents a distinct member of the aminoglycoside phosphotransferase family.
- The findings suggest an evolutionary link between antibiotic phosphotransferases and protein kinases.