Alteration of Escherichia coli topoisomerase IV to novobiocin resistance

Abstract

DNA gyrase and topoisomerase IV (topo IV) are the two essential type II topoisomerases of Escherichia coli. Gyrase is responsible for maintaining negative supercoiling of the bacterial chromosome, whereas topo IV's primary role is in disentangling daughter chromosomes following DNA replication. Coumarins, such as novobiocin, are wide-spectrum antimicrobial agents that primarily interfere with DNA gyrase. In this work we designed an alteration in the ParE subunit of topo IV at a site homologous to that which confers coumarin resistance in gyrase. This parE mutation renders the encoded topo IV approximately 40-fold resistant to inhibition by novobiocin in vitro and imparts a similar resistance to inhibition of topo IV-mediated relaxation of supercoiled DNA in vivo. We conclude that topo IV is a secondary target of novobiocin and that it is very likely to be inhibited by the same mechanism as DNA gyrase.

FIG. 1.

(A) Alignment of the ParE and GyrB N-terminal amino acid sequences. Amino acids listed between the ParE and GyrB lines indicate sequence identity, and plus signs indicate amino acid similarity. Residues known to be important for binding of ATP to GyrB are colored green, and residues involved in direct binding of novobiocin to GyrB are indicated in red. Amino acids implicated in both novobiocin and ATP binding are colored blue. Colored residues correspond to contacts made with either 5′-adenylyl-β,γ-imidodiphosphate or novobiocin in crystal structures with the GyrBp24 fragment (29). Homologous positions in ParE are also highlighted for clarity. The asterisk indicates Gly-164, which is mutated to Val in gyrB234 strains, conferring novobiocin resistance and temperature sensitivity. (B) Crystal structure of the N-terminal amino acids (15 to 180) of GyrB (45). Residues important for binding of ATP and novobiocin are shown as stick models and colored according to the color scheme described in panel A. Gly-164 is colored black, and Arg-136 is indicated with an arrow.

FIG. 2.

Wild-type and altered topo IV activity assays in vitro. Relaxed (Rel) and negatively supercoiled [(-) sc] DNAs are indicated. (A) Titration of wild-type ParE. The indicated amount of wild-type ParE protein was incubated with 150 ng of negatively supercoiled pUC18 in the presence or absence of an excess (50 ng) of ParC. (B) Titration of ParE R132C. The amount of ParE R132C protein indicated is a corrected value based on the measured 90% purity of the protein preparation. ParE R132C activity was measured as in panel A. (C) Lack of a diffusible inhibitor of topo IV in the ParE R132C preparation. The indicated amounts of wild-type ParE alone, ParE R132C alone, or wild-type ParE and ParE R132C together were incubated with DNA in the presence of an excess of ParC. (D) Titration of novobiocin into topo IV activity assays. The indicated concentrations of novobiocin were included in activity assays with 4 ng of wild-type (wt) ParE or 18 ng of ParE R132C (labeled “R”).

FIG. 3.

Positive supercoiling assay for topo IV activity in vivo. (A and B) Gel electrophoresis of intrinsic pBR322 in the four acr strains indicated (gyrB^r represents gyrB234). Plasmid was isolated from each strain and run on a 1% agarose gel containing 5 μg of chloroquine/ml. The four strains were treated with the following concentrations of novobiocin (Novo): 10 μg/ml (lanes 2 and 7), 50 μg/ml (lanes 3 and 8), 200 μg/ml (lanes 4 and 9), and 1,000 μg/ml (lanes 5 and 10.) Lanes 1 and 6 are from strains not treated with novobiocin. Lanes labeled Rel contain relaxed pBR322 as markers. Nicked (N) and linear (L) species are indicated. The region in front of the relaxed markers where positively supercoiled pBR322 runs is labeled (+) sc.

FIG. 4.

Viability of gyrase and topo IV novobiocin-resistant strains in the presence of novobiocin. Cultures of the strains were diluted and plated onto increasing concentrations of novobiocin. The symbols correspond to the following acr strain genotypes: ▾, gyrB⁺ parE⁺; ▵, gyrB⁺ parE^R132C; ▪, gyrB234 parE⁺; ○, gyrB234 parE^R132C. Each point represents the mean of two independent experiments. For each experiment, the cells were plated in duplicate for all concentrations of novobiocin tested, and the number of colonies was averaged. Error bars correspond to the standard error of the mean between the two independent experiments. The curves indicated by dotted lines are marked with the gyrB allele, followed by the parE allele, of the strains to which they correspond. +, wild-type; r, gyrB234; R, parE^R132C.