ABC transporter genes, kasKLM, responsible for self-resistance of a kasugamycin producer strain
Abstract:
We previously reported that a 7.6-kb DNA fragment from Streptomyces kasugaensis M338-M1, a kasugamycin (KSM) producer, included KSM acetyltransferase gene (kac338) and some other genes possibly involved in KSM biosynthesis. As an extension of that study, a 10-kb SacI-KpnI DNA fragment, located approximately 5-15-kb upstream of kac33, was cloned and a 4.2-kb SacI-EcoRI fragment therefrom was sequenced, revealing one incomplete (designated ORF J) and three complete open reading frames (designated kasK, kasL and kasM). The coding frames of kasK, L and M overlap one another with terminator/initiator ATGA sequence. RT-PCR analysis of a DNA region including kasKLM indicated the presence of one transcript that is long enough to span the three genes. The kasK gene potentially encodes an ATP-binding protein of the ATP-binding cassette (ABC) transporter superfamily. Homology search for the deduced KasK protein shows similarity to other ABC transporters involved in self-resistance of a mithramycin and possibly doxorubicin producer strain. The kasL and kasM genes encode different integral membrane proteins, both having six putative transmembrane helices. An expression plasmid for kasKLM (pTV-KLM) was constructed and these genes were expressed in E. coli JM 109, which had been sensitive to KSM. The transformant acquired resistance to KSM, suggesting that KasK, L and M proteins as a set in S. kasugaensis M338-M1 pump out KSM to protect the producer from its toxic metabolite.
Insights
Streptomyces kasugaensis M338-M1 utilizes a set of KasK, KasL, and KasM proteins to confer resistance to kasugamycin (KSM). These proteins form an ATP-binding cassette transporter system that exports KSM, protecting the producer strain from self-toxicity.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- Streptomyces kasugaensis M338-M1 produces the antibiotic kasugamycin (KSM).
- Previous studies identified the KSM acetyltransferase gene (kac338) and other potential biosynthesis genes.
- Understanding self-resistance mechanisms in antibiotic producers is crucial for metabolic engineering and drug discovery.
Purpose of the Study:
- To identify and characterize genes involved in kasugamycin (KSM) self-resistance in Streptomyces kasugaensis M338-M1.
- To elucidate the function of novel genes located upstream of the kac338 gene.
- To investigate the role of the identified genes in protecting the producer strain from KSM toxicity.
Main Methods:
- Cloning and sequencing of a 4.2-kb SacI-EcoRI DNA fragment upstream of kac338.
- Identification and characterization of open reading frames (ORFs) kasK, kasL, and kasM.
- Reverse transcription PCR (RT-PCR) to analyze kasKLM transcript.
- Bioinformatic analysis of deduced protein sequences (KasK, KasL, KasM).
- Heterologous expression of kasKLM genes in E. coli and assessment of KSM resistance.
Main Results:
- Sequencing revealed three complete ORFs: kasK, kasL, and kasM, with overlapping coding frames.
- KasK potentially encodes an ATP-binding protein belonging to the ATP-binding cassette (ABC) transporter superfamily.
- KasL and KasM encode integral membrane proteins with six putative transmembrane helices each.
- RT-PCR confirmed a single transcript spanning kasKLM, suggesting co-regulation.
- Expression of kasKLM in KSM-sensitive E. coli conferred resistance to KSM.
Conclusions:
- The KasK, KasL, and KasM proteins function as a set to confer kasugamycin (KSM) resistance in Streptomyces kasugaensis M338-M1.
- These proteins likely form an ABC transporter system responsible for exporting KSM out of the producer cell.
- This efflux mechanism protects the producing organism from the toxic effects of its own metabolite.
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