Cloning and analysis of a locus (mcr) involved in mitomycin C resistance in Streptomyces lavendulae

August, Paul R.; Flickinger, Michael C.; Sherman, David H.

doi:10.1128/jb.176.14.4448-4454.1994

Cited by 57 publications

(54 citation statements)

References 25 publications

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“…DrrC may bind DNR (and DXR) specifically, because it did not also confer mitomycin resistance to the E. coli uvrA mutant. (Mitomycin resistance involves a novel type of drug modification in the producing strain, Streptomyces lavendulae [3].) Since DrrC was unable to protect the E. coli uvrA mutant from the lethality of UV or mitomycin, classic factors that induce the UvrABC excision repair system in E. coli (16), it is unlikely that DrrC facilitates the repair of DNA damage resulting from the free-radicalpromoting effects of DNR and DXR.…”

Section: Discussionmentioning

confidence: 99%

The Streptomyces peucetius drrC gene encodes a UvrA-like protein involved in daunorubicin resistance and production

et al. 1996

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The drrC gene, cloned from the daunorubicin (DNR)-and doxorubicin-producing strain of Streptomyces peucetius ATCC 29050, encodes a 764-amino-acid protein with a strong sequence similarity to the Escherichia coli and Micrococcus luteus UvrA proteins involved in excision repair of DNA. Expression of drrC was correlated with the timing of DNR production in the growth medium tested and was not dependent on the presence of DNR. Since introduction of drrC into Streptomyces lividans imparted a DNR resistance phenotype, this gene is believed to be a DNR resistance gene. The drrC gene could be disrupted in the non-DNR-producing S. peucetius dnrJ mutant but not in the wild-type strain, and the resulting dnrJ drrC double mutant was significantly more sensitive to DNR in efficiency-of-plating experiments. Expression of drrC in an E. coli uvrA strain conferred significant DNR resistance to this highly DNR-sensitive mutant. However, the DrrC protein did not complement the uvrA mutation to protect the mutant from the lethal effects of UV or mitomycin even though it enhanced the UV resistance of a uvrA ؉ strain. We speculate that the DrrC protein mediates a novel type of DNR resistance, possibly different from the mechanism of DNR resistance governed by the S. peucetius drrAB genes, which are believed to encode a DNR antiporter.Microorganisms require one or more self-resistance determinants to produce antibiotics, except in cases in which they are insensitive to the antibiotic's effect(s). The resistance genes usually are clustered with the structural (biosynthetic) and regulatory genes and encode proteins that either inactivate the antibiotic, facilitate its export, or modify the host to render it insensitive to the antibiotic (9). Multiple rather than single resistance mechanisms are often found; in this case, it is not known whether any one resistance mode is sufficient to ensure survival or antibiotic production. We address this question here.Streptomyces peucetius, which produces the important antitumor drugs daunorubicin (DNR) (4, 10) and doxorubicin (DXR) (2), contains the drrAB (17) and ric2 (8) resistance genes, which are assumed to provide self-resistance to these two antibiotics because they confer DNR and DXR resistance when introduced into Streptomyces lividans. The DrrA protein strongly resembles bacterial proteins that transport compounds by an ATP-dependent process (20), as well as the Mdr1 P glycoprotein responsible for DNR-DXR resistance of human cancer cells and known to act as an ATP-dependent transporter (19). DrrA thus is a candidate for a DNR-DXR binding and transport protein, whereas the hydrophobic DrrB protein could be responsible for binding DrrA to the bacterial membrane. The ric2 locus, which unlike drrAB is not part of the cluster of DXR production genes (8), may also be important for self-resistance. A third gene, drrC, in the same cluster of genes as is drrAB, encodes the DrrC protein described here. S. lividans drrC ϩ transformants display a DNR resistance phenotype similar to that of drrAB tran...

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Section: Discussionmentioning

confidence: 99%

The Streptomyces peucetius drrC gene encodes a UvrA-like protein involved in daunorubicin resistance and production

et al. 1996

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“…S. lavendulae genomic DNA was isolated by the lysozyme-2ϫ Kirby mix method described by Hopwood et al (20). General DNA manipulation and single-stranded DNA isolation were done as described previously (2). Oligonucleotides for PCR, primer extension, and sequencing were obtained from Gibco BRL (Gaithersburg, Md.).…”

Section: Methodsmentioning

confidence: 99%

“…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).…”

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confidence: 99%

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

et al. 1997

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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...

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“…A number of ESTs related to drug resistance and remediation were identified. These included a transcriptional activator containing a Zn 2 C 6 -type zinc finger motif required for the induction of fluconazole resistance (Talibi & Raymond, 1999), a mitomycin oxidase (August et al, 1994), a multidrug ABC transporter and a salicylate hydroxylase, which catalyses the formation of catechol from salicylate (You et al, 1991), a key intermediate in naphthalene catabolism.…”

Section: Functional Analysis Of the Est Librariesmentioning

confidence: 99%

EST analysis of cDNA libraries from the entomopathogenic fungus Beauveria (Cordyceps) bassiana. I. Evidence for stage-specific gene expression in aerial conidia, in vitro blastospores and submerged conidia

et al. 2006

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The entomopathogenic fungus Beauveria (Cordyceps) bassiana holds much promise as a pest biological control agent. B. bassiana produces at least three in vitro single cell infectious propagules, including aerial conidia, vegetative cells termed blastospores and submerged conidia, that display different morphological, biochemical and virulence properties. Populations of aerial conidia, blastospores and submerged conidia were produced on agar plates, rich liquid broth cultures and under conditions of nutrient limitation in submerged cultures, respectively. cDNA libraries were generated from mRNA isolated from each B. bassiana cell type and ∼2500 5′ end sequences were determined from each library. Sequences derived from aerial conidia clustered into 284 contigs and 963 singlets, with those derived from blastospores and submerged conidia forming 327 contigs with 788 singlets, and 303 contigs and 1079 contigs, respectively. Almost half (40–45 %) of the sequences in each library displayed either no significant similarity (e value >10−4) or similarity to hypothetical proteins found in the NCBI database. The expressed sequence tag dataset also included sequences representing a significant portion of proteins in cellular metabolism, information storage and processing, transport and cell processes, including cell division and posttranslational modifications. Transcripts encoding a diverse array of pathogenicity-related genes, including proteases, lipases, esterases, phosphatases and enzymes producing toxic secondary metabolites, were also identified. Comparative analysis between the libraries identified 2416 unique sequences, of which 20–30 % were unique to each library, and only ∼6 % of the sequences were shared between all three libraries. The unique and divergent representation of the B. bassiana transcriptome in the cDNA libraries from each cell type suggests robust differential gene expression profiles in response to environmental conditions.

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Cloning and analysis of a locus (mcr) involved in mitomycin C resistance in Streptomyces lavendulae

Cited by 57 publications

References 25 publications

The Streptomyces peucetius drrC gene encodes a UvrA-like protein involved in daunorubicin resistance and production

The Streptomyces peucetius drrC gene encodes a UvrA-like protein involved in daunorubicin resistance and production

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

EST analysis of cDNA libraries from the entomopathogenic fungus Beauveria (Cordyceps) bassiana. I. Evidence for stage-specific gene expression in aerial conidia, in vitro blastospores and submerged conidia

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