Microcin B17 Blocks DNA Replication and Induces the SOS System in Escherichia coli

Herrero, Marta; Moreno, Felipe

doi:10.1099/00221287-132-2-393

Cited by 51 publications

(39 citation statements)

References 28 publications

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“…1) involves posttranslational modification of a 69-residue polypeptide-precursor to generate two thiazoles, two oxazoles, and two 4,2-fused bisheterocycles (3,4), followed by cleavage of the 26-residue leader sequence by an unidentified protease (5,6). Exposure of sensitive bacteria to MccB17 results in inhibition of DNA synthesis, induction of the SOS response in cells with active replication, mutagenic effects, and DNA degradation (7). These observations suggest that MccB17 is either a DNAdamaging agent or targets a key DNA-processing pathway such as replication.…”

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confidence: 99%

In vitro characterization of DNA gyrase inhibition by microcin B17 analogs with altered bisheterocyclic sites

Zamble

Miller

Heddle

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

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M icrocins are a class of small antibiotics (Ͻ10 kDa) produced and excreted by some strains of Enterobacteriaceae at the stationary phase of growth (1). Microcin B17 (MccB17) is a ribosomally synthesized compound that is produced in Escherichia coli harboring the plasmid-borne mcb operon, which contains the genes necessary for antibiotic synthesis, dedicated export, and immunity (2). The production of mature MccB17 ( Fig. 1) involves posttranslational modification of a 69-residue polypeptide-precursor to generate two thiazoles, two oxazoles, and two 4,2-fused bisheterocycles (3, 4), followed by cleavage of the 26-residue leader sequence by an unidentified protease (5, 6). Exposure of sensitive bacteria to MccB17 results in inhibition of DNA synthesis, induction of the SOS response in cells with active replication, mutagenic effects, and DNA degradation (7). These observations suggest that MccB17 is either a DNAdamaging agent or targets a key DNA-processing pathway such as replication.Genetic experiments designed to identify the target of MccB17 resulted in the isolation and characterization of a mutation in the B subunit of DNA gyrase (8). Strains carrying this mutation, a Trp-Arg substitution at position 751, exhibited increased resistance to MccB17 and a decrease in MccB17-induced DNA cleavage. Gyrase is a prokaryotic type II topoisomerase that introduces negative supercoils into DNA (9, 10). During the reaction, the enzyme passes through a transient intermediate in which two tyrosine hydroxyl groups form phosphodiester bonds with the exposed 5Ј phosphates of doublestrand cleaved DNA. This intermediate allows the enzyme to pull the ends of the DNA break apart to form a gate through which another DNA segment is passed. The quinolone family of drugs traps this covalent intermediate, known as the cleavable complex, and the accumulation of this complex is monitored in vitro by treatment with SDS and proteinase K, which releases linear DNA (11-13). The ternary complex blocks passage of the replication fork (14, 15), which may initiate the bactericidal activity of the quinolones, although the exact irreversible lethal event has not been firmly established (16). Recent in vitro analysis of the interaction between gyrase and MccB17 revealed that this antibiotic also causes accumulation of the cleavable complex in a manner similar to that of the quinolone drugs (17).Gyrase is the target of several classes of clinically successful drugs, including the quinolones and the coumarins (11, 13); however, the emergence of resistant strains is an incentive to elucidate the mechanism of action of existing drugs and develop new candidates. The identification of gyrase as a target of MccB17 provides an opportunity to analyze the structurefunction relationship of this unusual antibiotic. The experiments described here investigate the effects of modifying MccB17 at the sites of the tandem heterocycles, labeled A and B (Fig. 1). The interaction of MccB17 analogs with DNA gyrase was evaluated by measuring the rate of formation of the ...

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confidence: 99%

In vitro characterization of DNA gyrase inhibition by microcin B17 analogs with altered bisheterocyclic sites

Zamble

Miller

Heddle

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

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“…The effects of MccB17 on sensitive E. coli cells include inhibition of replication, induction of the SOS response, induction of double-strand breaks in DNA by trapping of DNA gyrase complexes, and ultimately cell death (24,53). An MccB17 resistance mutation (resulting in a Trp751 to Arg substitution) has been mapped to position 2251 of the gyrB gene (14,53).…”

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The Highly Conserved TldD and TldE Proteins of Escherichia coli Are Involved in Microcin B17 Processing and in CcdA Degradation

et al. 2002

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Microcin B17 (MccB17) is a peptide antibiotic produced by Escherichia coli strains carrying the pMccB17 plasmid. MccB17 is synthesized as a precursor containing an amino-terminal leader peptide that is cleaved during maturation. Maturation requires the product of the chromosomal tldE (pmbA) gene. Mature microcin is exported across the cytoplasmic membrane by a dedicated ABC transporter. In sensitive cells, MccB17 targets the essential topoisomerase II DNA gyrase. Independently, tldE as well as tldD mutants were isolated as being resistant to CcdB, another natural poison of gyrase encoded by the ccd poison-antidote system of plasmid F. This led to the idea that TldD and TldE could regulate gyrase function. We present in vivo evidence supporting the hypothesis that TldD and TldE have proteolytic activity. We show that in bacterial mutants devoid of either TldD or TldE activity, the MccB17 precursor accumulates and is not exported. Similarly, in the ccd system, we found that TldD and TldE are involved in CcdA and CcdA41 antidote degradation rather than being involved in the CcdB resistance mechanism. Interestingly, sequence database comparisons revealed that these two proteins have homologues in eubacteria and archaebacteria, suggesting a broader physiological role.

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“…Microcin B17 (MccB17) is a bactericidal peptide of about 3,200 Da which inhibits DNA replication, induces the SOS repair system, and finally causes DNA degradation (15,30). Both microcins are plasmid encoded and are produced when cultures cease exponential growth and enter the stationary phase (19,28).…”

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mprA, an Escherichia coli gene that reduces growth-phase-dependent synthesis of microcins B17 and C7 and blocks osmoinduction of proU when cloned on a high-copy-number plasmid

1990

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Microcins B17 and C7 are plasmid-determined, peptide antibiotics produced by Escherichia coli when cells enter the stationary phase of growth. Microcinogenic strains are immune to the action of the microcin they synthesize. A well-characterized deficient-immunity phenotype is exhibited by microcin B17-producing cells in the absence of the immunity gene mcbG (M.C. Garrido, M. Herrero, R. Kolter, and F. Moreno, EMBO J. 7:1853-1862, 1988). A 14.6-kilobase-pair EcoRI chromosomal fragment was isolated by its ability to suppress this phenotype when cloned into a multicopy vector. This fragment was mapped to 57.5 min on the E. coli genetic map. The position of the gene responsible for suppression, designated mprA, was determined by insertional mutagenesis and deletion analysis. mprA was shown to be transcribed clockwise on the E. coli chromosome, and its product was identified as a 19-kilodalton polypeptide. Suppression was shown to be achieved by decreasing microcin B17 production. Increased mprA gene dosage also caused a decrease in microcin C7 production and blocked the osmoinduction of the proU locus in high-osmolarity media. Our results suggest that the mprA gene product could play a regulatory role on expression of several E. coli genes, this control being exerted at the transcriptional level.

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Microcin B17 Blocks DNA Replication and Induces the SOS System in Escherichia coli

Cited by 51 publications

References 28 publications

In vitro characterization of DNA gyrase inhibition by microcin B17 analogs with altered bisheterocyclic sites

In vitro characterization of DNA gyrase inhibition by microcin B17 analogs with altered bisheterocyclic sites

The Highly Conserved TldD and TldE Proteins of Escherichia coli Are Involved in Microcin B17 Processing and in CcdA Degradation

mprA, an Escherichia coli gene that reduces growth-phase-dependent synthesis of microcins B17 and C7 and blocks osmoinduction of proU when cloned on a high-copy-number plasmid

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