ABC transporter genes, kasKLM, responsible for self-resistance of a kasugamycin producer strain

We previously reported that a 7.6-kb DNA fragment from Streptomyces kasugae nsis M338-M1, a kasugamycin (KSM) producer, included KSM acetyltransferase gene (kac(338)) and some other genes possibly involved in KSM biosynthesis. As an extension of that study, a 10-kb SacI-KpnI DNA fragment, located 5 s imilar to 15-kb upstream of kac(338), was cloned and a 4.2-kb SacI-EcoRI fr agment therefrom was sequenced, revealing one incomplete (designated ORF J) and three complete open reading frames (designated kasK, kasL and kasM). T he coding frames of kasK, L and M overlap one another with terminator/initi ator ATGA sequence. RT-PCR analysis of a DNA region including kasKLM indica ted the presence of one transcript that is long enough to span the three ge nes. The kasK gene potentially encodes an ATP-binding protein of the ATP-bi nding cassette (ABC) transporter superfamily. Homology search for the deduc ed 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 havin g six putative transmembrane helices. An expression plasmid for kasKLM (pTV -KLM) was constructed and these genes were expressed in E. coli JM109, whic h had been sensitive to KSM. The transformant acquired resistance to KSM, s uggesting that KasK, L and M proteins as a set in S. kasugaensis M338-M1 pu mp out KSM to protect the producer from its toxic metabolite.