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

Abstract

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 ...

The Action of the Bacterial Toxin Microcin B17

“…We investigated the effect of MccB17 on ATP-independent relaxation by gyrase and found that the inhibition of this reaction by the toxin was also slow and incomplete. These results show that MccB17 inhibits supercoiling and relaxation of gyrase to a similar extent, and contrary to earlier reports (9,11), the action of MccB17 is not ATP-dependent. In contrast with MccB17, quinolones were shown to rapidly and completely inhibit the supercoiling and relaxation reactions of gyrase (36 -38).…”

“…It is expected that the effect of Ca 2ϩ is due to its substitution for Mg 2ϩ in the cleavage-religation process. Interaction of MccB17 with Gyrase-From our data and previous work (9,11), the action of MccB17 on gyrase is clearly distinct from that of quinolone drugs, such as ciprofloxacin, even if they both stabilize the cleavage complex. Quinolones completely inhibit the supercoiling and relaxation reactions of gyrase, and their effects can be rationalized from their proposed interaction with the distorted DNA (G segment) at the enzyme active site (44).…”

“…work on the effect of MccB17 on DNA supercoiling catalyzed by DNA gyrase suggested that MccB17 is an inefficient inhibitor of the enzyme (9,11). In this work we found that the inhibition by 37.5 M MccB17 is slow and incomplete, i.e.…”

“…Reactions-Previous work reported that MccB17 inhibits gyrase-catalyzed supercoiling slowly (11) or not at all (9) and that stabilization of the cleavage complex is ATP-dependent (9). To further investigate these effects, we have examined the gyrase supercoiling and relaxation reactions in the presence of MccB17 under a range of conditions.…”

“…Of the two pairs of tandem, fused 4,2-bisheterocycles in MccB17, the integrity of the second one (B-site tandem; Fig. 1A) was found to be crucial for the potency of MccB17 (10,11).…”