Novobiocin Susceptibility of MukBEF-Deficient Escherichia coli Is Combinatorial with Efflux and Resides in DNA Topoisomerases

Abstract

Condensins play a key role in the global organization of bacterial chromosomes. In Escherichia coli, the inactivation of its sole condensin MukBEF induces severe growth defects and renders cells hypersusceptible to novobiocin. We report here that this hypersusceptibility can be observed in TolC-deficient cells and is therefore unrelated to multidrug efflux. We further show that mutations in MukE that impair its focal subcellular localization potentiate novobiocin and that the extent of the potentiation correlates with the residual activity of MukE. Finally, both DNA gyrase and topoisomerase IV might partially complement novobiocin susceptibility in a temperature-dependent manner. These data indicate that the observed antibiotic susceptibility resides in both type II DNA topoisomerases and is efflux independent. Furthermore, novobiocin susceptibility is associated with the activity of MukBEF and can be induced by its partial inactivation, which makes the protein a plausible target for inhibition. Condensins are essential for the global folding of bacterial chromosomes (reviewed in references 1, 2, and 3). These multisubunit complexes act as macromolecular clamps that bridge distant DNA segments and thereby stabilize the looped architecture of the chromosome (4). Escherichia coli encodes a single condensin, MukBEF (5, 6). MukB belongs to the family of the characteristically V-shaped structural maintenance of chromosome (SMC) proteins (7) and is responsible for the ATP-modulated DNA binding and bridging activity of the complex (8). MukBEF forms dynamic clusters at the core of the daughter chromosomes, which can be found in the middle of short cells or at the quarter positions of longer cells (9-11). Such focal localization is essential for cell viability and requires the activity of the regulatory subunit, MukEF (6, 12).The inactivation of MukBEF leads to severe growth defects, including anucleate cell formation, chromosome decondensation and cutting, a marked decline in colony formation, and an increase in novobiocin susceptibility (13,14). These phenotypes are alleviated at low temperatures and can be partially complemented by the overexpression of cold shock protein CspE and camphor resistance proteins CrcA and CrcB (15). Partial suppression of MukBEF phenotypes can also be achieved by mutations in topoisomerase I and DNA gyrase that increase the supercoiling of intracellular DNA (16). Such mutations increase the compactness of the chromosome and are expected to compensate for the chromosomal disorganization caused by the deletion of mukB. Besides the aforementioned genetic interaction with DNA gyrase, MukB forms a complex with the ParC subunit of the other type II DNA topoisomerase, topoisomerase IV (topo IV), and modulates its activity (17, 18).The mechanism of novobiocin susceptibility of MukB-deficient cells remains somewhat enigmatic. Spontaneous suppressors of novobiocin susceptibility of ⌬mukB cells have been mapped to GyrB, the ATPase subunit of DNA gyrase (13). However, no increase in susceptibility was...

Small Molecule Condensin Inhibitors

“…These cells were then supplemented with NSC260594, and PC 50 of novobiocin was measured. In agreement with previous studies, 14 susceptibility of cells to novobiocin declined with the increasing dosage of MukB (Figure 4H). This decline was accompanied by an increase in susceptibility to NSC260594.…”

“…The assay for screening employed novobiocin-dependent inhibition of growth of TolC deficient E. coli strain ETBW, which lacks the major outer membrane component of multidrug efflux pumps. 14 This choice of the strain helped eliminate the main source of false positives during screening. Indeed, many compounds potentiate antibiotics by improving their penetration into the cell, by either inhibiting drug efflux or disrupting the cell membrane.…”

“…18–21 The interaction between MukBEF and the two topoisomerases operationally defines the MukBEF pathway which might include other factors. 14…”

SMC and the bactofilin/PadC scaffold have distinct yet redundant functions in chromosome segregation and organization in Myxococcus xanthus

“…coli (Weitao et al, 1999;Adachi and Hiraga, 2003;Petrushenko et al, 2016). In immunoblots, Smc-mCherry accumulated as a full-length protein in the complementation strain ( Figure S4a), supporting that the full-length fusion protein is active.…”

“…Because some species lacking SMC are hypersensitive to DNA gyrase inhibition (Nolivos and Sherratt, 2014), we tested the Δ smc and Δ scpAB mutants for sensitivity to the DNA gyrase inhibitor novobiocin. Both mutants were hypersensitive to novobiocin compared to WT (Figure 2b) as previously observed for Deinococcus radiodurans (Bouthier de la Tour et al ., 2009) and E. coli (Weitao et al ., 1999; Adachi and Hiraga, 2003; Petrushenko et al ., 2016). In immunoblots, Smc‐mCherry accumulated as a full‐length protein in the complementation strain (Figure a), supporting that the full‐length fusion protein is active.…”