Topological information embodied in local juxtaposition geometry provides a statistical mechanical basis for unknotting by type-2 DNA topoisomerases.

Topoisomerases may unknot by recognizing specific DNA juxtapositions. The physical basis of this hypothesis is investigated by considering single-loop conformations in a coarse-grained polymer model. We determine the statistical relationship between the local geometry of a juxtaposition of two chain segments and whether the loop is knotted globally, and ascertain how the knot/unknot topology is altered by a topoisomerase-like segment passage at the juxtaposition. Segment passages at a "free" juxtaposition tend to increase knot probability. In contrast, segment passages at a "hooked" juxtaposition cause more transitions from knot to unknot than vice versa, resulting in a steady-state knot probability far lower than that at topological equilibrium. The reduction in knot population by passing chain segments through a hooked juxtaposition is more prominent for loops of smaller sizes, n, but remains significant even for larger loops: steady-state knot probability is only approximately 2%, and approximately 5% of equilibrium, respectively, for n=100 and 500 in the model. An exhaustive analysis of approximately 6000 different juxtaposition geometries indicates that the ability of a segment passage to unknot correlates strongly with the juxtaposition's "hookedness". Remarkably, and consistent with experiments on type-2 topoisomerases from different organisms, the unknotting potential of a juxtaposition geometry in our polymer model correlates almost perfectly with its corresponding decatenation potential. These quantitative findings suggest that it is possible for topoisomerases to disentangle by acting selectively on juxtapositions with "hooked" geometries.

Inferring global topology from local juxtaposition geometry: interlinking polymer rings and ramifications for topoisomerase action.

Lattice modeling is applied to investigate how the configurations of local chain juxtapositions may provide information about whether two ring polymers (loops) are topologically linked globally. Given a particular juxtaposition, the conditional probability that the loops are linked is determined by exact enumeration and extensive Monte Carlo sampling of conformations satisfying excluded volume constraints. A discrimination factor fL, defined as the ratio of linked to unlinked probabilities, varies widely depending on which juxtaposition is presumed. /log fL/s that are large for small loop size n tend to decrease, signaling diminishing topological information content of the juxtapositions, with increasing n. However, some juxtaposition geometries can impose sufficient overall conformational biases such that /log fL/ remains significant for large n. Notably, for two loops as large as n=200 in the model, the probability that passing the segments of a hooked juxtaposition would unlink an originally linked configuration is remarkably high, approximately 85%. In contrast, segment-passage of a free juxtaposition would link the loops from an originally unlinked configuration more than 90% of the time. The statistical mechanical principles emerging from these findings suggest that it is physically possible for DNA topoisomerases to decatenate effectively by acting selectively on juxtapositions with specific "hooked" geometries.

Quorum sensing and multidrug transporters in Escherichia coli.

Previously, we found that the quorum sensing transcription factor SdiA up-regulates AcrAB. Others found that a 4-quinolone was a quorum-sensing signal in Pseudomonas aeruginosa. In Escherichia coli, there are at least three multidrug transporters (AcrAB/TolC, MdfA, and NorE) that exude fluoroquinolones. Here, we show that DeltaacrAB, tolC210, or DeltanorE mutants have the same growth rate as WT cells in exponential phase but grow to higher cell density in stationary phase. Overproduction of either pump caused cells to reach lower density. mdfA had no effect. Conditioned medium (CM) from cells overexpressing acrAB represses cell growth more than CM from WT cells. CM from pump mutant cells represses cell growth less than CM from WT cells. These results were not affected by the deletion of luxS, which synthesizes the quorum-sensing signal autoinducer 2 (AI-2). Expression of the rpoS gene encoding the stationary phase sigma factor is induced earlier in cells overexpressing acrAB and later in acrAB mutant cells. These results support a model in which a natural function of AcrAB/TolC and NorE is to export signals for cell-cell communication. Drugs exported by pumps may resemble communication molecules normally exuded.

Electrostatics of DNA-DNA juxtapositions: consequences for type II topoisomerase function.

Type II topoisomerases resolve problematic DNA topologies such as knots, catenanes, and supercoils that arise as a consequence of DNA replication and recombination. Failure to remove problematic DNA topologies prohibits cell division and can result in cell death or genetic mutation. Such catastrophic consequences make topoisomerases an effective target for antibiotics and anticancer agents. Despite their biological and clinical importance, little is understood about how a topoisomerase differentiates DNA topologies in a molecule that is significantly larger than the topoisomerase itself. It has been proposed that type II topoisomerases recognize angle and curvature between two DNA helices characteristic of knotted and catenated DNA to account for the enzyme's preference to unlink instead of link DNA. Here we consider the electrostatic potential of DNA juxtapositions to determine the possibility of juxtapositions occurring through Brownian diffusion. We found that despite the large negative electrostatic potential formed between two juxtaposed DNA helices, a bulk counterion concentration as small as 50 mM provides sufficient electrostatic screening to prohibit significant interaction beyond an interhelical separation of 3 nm in both hooked and free juxtapositions. This suggests that instead of electrostatics, mechanical forces such as those occurring in anaphase, knots, catenanes, or the writhe of supercoiled DNA may be responsible for the formation of DNA juxtapositions.

Exploring writhe in supercoiled minicircle DNA.

Using λ-Int recombination in E. coli, we have generated milligram quantities of supercoiled minicircle DNA. Intramolecular Int recombination was efficient down to lengths ~254 bp. When nicked and religated in the presence of ethidium bromide, 339 bp minicircles adopted at least seven unique topoisomers that presumably correspond to ΔLk ranging from 0 to -6, which we purified individually. We used these minicircles, with unique ΔLk, to address the partition into twist and writhe as a function of ΔLk. Gel electrophoresis and atomic force microscopy revealed progressively higher writhe conformations in the presence of 10 mM CaCl(2) or MgCl(2). From simplistic calculations of the bending and twisting energies, we predict the elastic free energy of supercoiling for these minicircles to be lower than if the supercoiling was partitioned mainly into twist. The predicted writhe corresponds closely with that which we observed experimentally in the presence of divalent metal ions. However, in the absence of divalent metal ions only limited writhe was observed, demonstrating the importance of electrostatic effects on DNA structure, when the screening of charges on the DNA is weak. This study represents a unique insight into the supercoiling of minicircle DNA, with implications for DNA structure in general.

A role for topoisomerase III in a recombination pathway alternative to RuvABC.

The physiological role of topoisomerase III is unclear for any organism. We show here that the removal of topoisomerase III in temperature sensitive topoisomerase IV mutants in Escherichia coli results in inviability at the permissive temperature. The removal of topoisomerase III has no effect on the accumulation of catenated intermediates of DNA replication, even when topoisomerase IV activity is removed. Either recQ or recA null mutations, but not helD null or lexA3, partially rescued the synthetic lethality of the double topoisomerase III/IV mutant, indicating a role for topoisomerase III in recombination. We find a bias against deleting the gene encoding topoisomerase III in ruvC53 or DeltaruvABC backgrounds compared with the isogenic wild-type strains. The topoisomerase III RuvC double mutants that can be constructed are five- to 10-fold more sensitive to UV irradiation and mitomycin C treatment and are twofold less efficient in transduction efficiency than ruvC53 mutants. The overexpression of ruvABC allows the construction of the topoisomerase III/IV double mutant. These data are consistent with a role for topoisomerase III in disentangling recombination intermediates as an alternative to RuvABC to maintain the stability of the genome.

A mutation in Escherichia coli DNA gyrase conferring quinolone resistance results in sensitivity to drugs targeting eukaryotic topoisomerase II.

Fluoroquinolones are broad-spectrum antimicrobial agents that target type II topoisomerases. Many fluoroquinolones are highly specific for bacterial type II topoisomerases and act against both DNA gyrase and topoisomerase IV. In Escherichia coli, mutations causing quinolone resistance are often found in the gene that encodes the A subunit of DNA gyrase. One common site for resistance-conferring mutations alters Ser83, and mutations to Leu or Trp result in high levels of resistance to fluoroquinolones. In the present study we demonstrate that the mutation of Ser83 to Trp in DNA gyrase (Gyr(S83W)) also results in sensitivity to agents that are potent inhibitors of eukaryotic topoisomerase II but that are normally inactive against prokaryotic enzymes. Epipodophyllotoxins, such as etoposide, teniposide and amino-azatoxin, inhibited the DNA supercoiling activity of Gyr(S83W), and the enzyme caused elevated levels of DNA cleavage in the presence of these agents. The DNA sequence preference for Gyr(S83W)-induced cleavage sites in the presence of etoposide was similar to that seen with eukaryotic type II topoisomerases. Introduction of the Gyr(S83W) mutation in E. coli strain RFM443-242 by site-directed mutagenesis sensitized it to epipodophyllotoxins and amino-azatoxin. Our results demonstrate that sensitivity to agents that target topoisomerase II is conserved between prokaryotic and eukaryotic enzymes, suggesting that drug interaction domains are also well conserved and likely occur in domains important for the biochemical activities of the enzymes.

DNA disentangling by type-2 topoisomerases.

A type-2 topoisomerase cleaves a DNA strand, passes another through the break, and then rejoins the severed ends. Because it appears that this action is as likely to increase as to decrease entanglements, the question is: how are entanglements removed? We argue that type-2 topoisomerases have evolved to act at "hooked" juxtapositions of strands (where the strands are curved toward each other). This type of juxtaposition is a natural consequence of entangled long strands. Our model accounts for the observed preference for unlinking and unknotting of short DNA plasmids by type-2 topoisomerases and well explains experimental observations.

Relative contributions of the AcrAB, MdfA and NorE efflux pumps to quinolone resistance in Escherichia coli.

Quinolones are widely used, broad-spectrum antimicrobial agents. In screens for genes that, when overexpressed, allow Escherichia coli to grow on otherwise lethal concentrations of the fluoroquinolone norfloxacin, the ydhE gene was identified. We have shown that ydhE encodes a multidrug efflux pump with a narrower substrate range than that of its closest homologue, encoded by norM, and named the gene norE. The relative contributions to drug resistance of NorE compared with the two other known E. coli quinolone pumps, AcrAB and MdfA, have been defined. Overexpression of each of the three pumps separately resulted in roughly similar levels of quinolone resistance, whereas simultaneous overexpression of norE or mdfA in combination with acrAB gave synergic increases in quinolone resistance. The level of quinolone resistance mediated by efflux pumps seems to be constrained to an approximately 10-fold maximum, even with increased production of the pumps. We measured the drug resistance of an isogenic set of strains containing the various permutations of single, double and triple drug efflux pump mutants. The DeltanorE and DeltamdfA mutants were somewhat more susceptible to fluoroquinolones than the parent strain, and acrAB mutants were four- to six-fold more susceptible. Mutants lacking two or all three efflux pumps were not significantly more susceptible to fluoroquinolones than those lacking only one of the three pumps.

Control of the AcrAB multidrug efflux pump by quorum-sensing regulator SdiA.

SdiA is an Escherichia coli protein that regulates cell division in a cell density-dependent, or quorum-sensing, manner. We report that SdiA also controls multidrug resistance by positively regulating the multidrug resistance pump AcrAB. Overproduction of SdiA confers multidrug resistance and increased levels of AcrAB. Conversely, sdiA null mutants are hypersensitive to drugs and have decreased levels of AcrB protein. Our findings provide a link between quorum sensing and multidrug efflux. Combined with previously published reports, our data support a model in which a role of drug efflux pumps is to mediate cell-cell communication in response to cell density. Xenobiotics expelled by pumps may resemble the communication molecules that they normally efflux.