Characterization of a mitomycin-binding drug resistance mechanism from the producing organism, Streptomyces lavendulae

Abstract

In an effort to characterize the diversity of mechanisms involved in cellular self-protection against the antitumor antibiotic mitomycin C (MC), DNA fragments from the producing organism (Streptomyces lavendulae) were introduced into Streptomyces lividans and transformants were selected for resistance to the drug. Subcloning of a 4.0-kb BclI fragment revealed the presence of an MC resistance determinant, mrd. Nucleotide sequence analysis identified an open reading frame consisting of 130 amino acids with a predicted molecular weight of 14,364. Transcriptional analysis revealed that mrd is expressed constitutively, with increased transcription in the presence of MC. Expression of mrd in Escherichia coli resulted in the synthesis of a soluble protein with an M r of 14,400 that conferred high-level cellular resistance to MC and a series of structurally related natural products. Purified MRD was shown to function as a drug-binding protein that provides protection against cross-linking of DNA by preventing reductive activation of MC.Streptomyces species are gram-positive soil bacteria known for their ability to produce a wide range of biologically active metabolites. In addition, many resistance genes have been cloned from these bacteria. Mechanisms of cellular self-protection include drug inactivation, target site modification, reduction of intracellular concentration via efflux, and drug binding (8). The presence of multiple modes of self-protection toward a single antibiotic is well documented (9), often with one or more resistance determinants located adjacent to the corresponding biosynthetic genes.Mitomycin C (MC) was identified in 1956 as an antibiotic produced by Streptomyces lavendulae (18) and subsequently established as an important antitumor agent (19,21). MC functions as a prodrug and requires enzymatic or chemical reduction to become a highly reactive alkylating agent (19,40). The intracellular activation of MC is specified by endogenous flavoreductases (34) and proceeds by single electron reduction to the MC semiquinone radical. The relatively long-lived semiquinone species either rearranges to an alkylating intermediate (by further reduction) or transfers an electron to molecular oxygen to generate superoxide (32). Therefore, the ability of MC to inhibit bacterial and mammalian cell growth involves the combined action of DNA alkylation and the formation of reactive oxygen species. Other naturally occurring compounds within this class include bleomycin (37), enediynes (10), and the more recently discovered dihydrobenzoxazines (25,42).Recently, a locus, mcr, that confers high-level MC resistance in S. lavendulae has been reported (2, 3). The resistance gene, mcrA, encodes a flavoenzyme (MCRA) that reoxidizes reductively activated MC (23). In another example involving a DNA damaging agent, bleomycin self-resistance has been determined by drug modification (Bat) and binding (BLMA) proteins in Streptomyces verticillus (38). Beyond these examples, little is known about bacterial resistance to the growing cl...

Natural Antibiotic Resistance of Bacteria Isolated from Larvae of the Oil Fly, Helaeomyia petrolei

“…OF008 was identified as M. morganii (9), while the identity of OF013 remained ambiguous (9). they produce because they contain a resistance gene as part of the biosynthetic cluster (19). Therefore, one explanation for our finding that the aerobic bacteria obtained from oil fly larvae are predominantly Providencia and Acinetobacter species (9) is that these bacteria derive a selective advantage by excreting antibiotics that inhibit other microorganisms.…”

Characterization of a quinone reductase activity for the mitomycin C binding protein (MRD): Functional switching from a drug-activating enzyme to a drug-binding protein

“…In previous work that led to the identification of MRD as a MC-binding protein, relatively small amounts of protein and substrates (MC, NADH) were used in the in vitro DNA protection assay (7). Although this assay was sensitive enough to detect the protection of DNA from MC-mediated cross-linking, it was not suitable for the detection of potential enzymatic reaction products.…”

“…One locus (mcrA), which is located outside of the MC biosynthetic gene cluster, encodes a protein (MCRA) that reoxidizes the reductively activated MC species to the prodrug through a redox relay mechanism (5, 6). Two other loci (mrd and mct) were found within the MC biosynthetic gene cluster and encode a small soluble protein (MRD) and a membrane-associated protein (MCT), respectively (7,8). Whereas MCT has extensive amino acid sequence similarity with several actinomycete antibiotic export proteins, MRD does not significantly resemble any previously characterized protein.…”

“…Whereas MCT has extensive amino acid sequence similarity with several actinomycete antibiotic export proteins, MRD does not significantly resemble any previously characterized protein. DNA protection studies demonstrated that MRD can protect high G ϩ C DNA from MC-mediated cross-linking through a drug-binding activity that depends on the presence of NADH (7). Heterologous expression experiments also indicated that MRD can work together with MCT to form a highly efficient drug binding-export system (8).…”

Expression of the Streptomyces coelicolor SoxR Regulon Is Intimately Linked with Actinorhodin Production

“…The use of multiple resistance mechanisms against a single toxic product is not uncommon (10). Streptomyces lavendulae, for example, protects itself against the deleterious effects of mitomycin via three distinct modes: drug inactivation by a flarin adenine dinucleotide-dependent oxidoreductase (34), drug export (51), and drug sequestration (52). That S. coelicolor employs multiple mechanisms to prevent self-destruction by Act would account for the fact that the actII-ORF2/ORF3 actVA-ORF1 triple mutant (which is defective in exporting Act) is viable (5), as is the soxR mutant.…”