ABC Transporter Genes, kasKLM, Responsible for Self-resistance of a Kasugamycin Producer Strain

• Published in: Journal of antibiotics 2000/04/25, Vol.53(4), pp.373-384
• Main Authors: IKENO, SOUICHI, YAMANE, YASUHIRO, OHISHI, YOKO, KINOSHITA, NAOKO, HAMADA, MASA, TSUCHIYA, KAYOKO S., HORI, MAKOTO
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Aminoglycosides; Anti-Bacterial Agents - biosynthesis; Antibacterial agents; Antibiotics. Antiinfectious agents. Antiparasitic agents; ATP-Binding Cassette Transporters - genetics; Base Sequence; beta-Galactosidase - metabolism; Biological and medical sciences; Cloning, Molecular - methods; DNA, Bacterial - genetics; Drug Resistance, Microbial; Escherichia coli - genetics; Gene Expression; kasK gene; kasL gene; kasM gene; kasugamycin; Medical sciences; Molecular Sequence Data; Open Reading Frames; Pharmacology. Drug treatments; Plasmids - genetics; Streptomyces - genetics; Streptomyces kasugaensis
• ISSN: 0021-8820; 1881-1469
• DOI: 10.7164/antibiotics.53.373

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 5-15-kb upstream of kac338, 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 JM109, 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.