Selective toxicity alteration of a highly toxic antibiotic by an enzyme catalyzing antibiotic modification

Streptothricins (STs) (Fig. 1) are broad-spectrum antibiotics that were first isolated from Streptomyces lavendulae in 1943 (Waksman, 1943). All STs consist of a carbamoylated D-gulosamine to which the -lysine homopolymer (1 to 7 residues) and the amide form of the unusual amino acid ‘‘streptolidine lactam’’ are attached. STs inhibit protein biosynthesis in prokaryotic cells; in addition, they strongly inhibit the growth of eukaryotes such as yeasts (Goldstein & McCusker, 1999; Hentges et al., 2005; Shen et al., 2005), fungi (Idnurm et al., 2004), protozoa (Joshi et al., 1995), insects (Takemoto et al., 1980) and plants (Chamberlain et al., 1994). Therefore, STs are used as effective selective agents for recombinant DNA work in some of these organisms. However, STs are not currently used therapeutically due to their nephrotoxicity (Hoffmann et al., 1986a and 1986b; Hartl et al.). To date, many ST-resistance genes have been identified in transposons such as Tn1825 and Tn1826, which have been isolated from bacteria that are resistant to ST (Partridge & Hall, 2005); such transposons have also been isolated from human pathogens such as Shiga toxinproducing Escherichia coli (Singh et al., 2005) and the Shigella strain (Peirano et al., 2005). Bacterial resistance to antibiotics that inhibit protein biosynthesis (e.g., aminoglycosides) can occur as a result of decreased antibiotic uptake and accumulation, modification of 16S RNA or ribosomal proteins, or enzymatic modification of the antibiotics (Vakulenko & Mobashery, 2003). However, in the case of bacterial resistance to STs, only one resistance mechanism has yet been identified: the resistance is due to a modification of the ST molecule by monoacetylation at the -amino group of -lysine(s). In fact, in ST producers such as S. lavendulae (Horinouchi et al., 1987), S. rochei (Fernández-Moreno et al., 1997) and S. noursei (Krugel et al., 1988; Grammel et al., 2002), the ST-resistance genes encoding N-acetyltransferase (NAT) have been identified and their role in self-resistance against their own STs has been investigated. Based on this resistance mechanism and the fact that streptothricin D (ST-D, Fig. 1) is a more effective antibiotic than streptothricin F (ST-F, Fig. 1), the moiety of -lysine(s) has been shown to play a crucial role in antibiotic activity. On the other hand, Inamori et al. (Inamori et al., 1988) and Taniyama et al. (Taniyama et al., 1971) have independently reported that ST-F-acid (Fig. 1, termed as racenomycin-A-acid in their studies)—chemically prepared from ST-F—did not exhibit antibiotic activity against bacteria, fungi, and plants; however, the biological activity of ST-D-acid was not tested. This result confirmed that streptolidine lactam is essential for antibiotic activity. We therefore hypothesized that microorganisms showing resistance to STs through alternative resistance mechanisms might produce an enzyme that hydrolyzes streptolidine lactam, thereby inactivating STs. Actinomycetes are known to produce many natural products with structural diversity occurring due to the unique substrate specificities of the enzymes. Therefore, we focused on Streptomyces, the representative strains belonging to actinomycetes, to efficiently identify our target enzyme. Here, we describe the cloning of a gene whose product confers ST resistance through the modification of streptolidine lactam, as expected (Hamano et al., 2006). Additionally, we used the recombinant enzyme of this gene product to investigate its functions and properties. We also discuss an interesting observation regarding the selective toxicity of an ST compound that was converted by the gene product.