Mutants Suppressing Novobiocin Hypersensitivity of amukBNull Mutation

Abstract

The mukB gene is essential for the partitioning of sister chromosomes in Escherichia coli. A mukB null mutant is hypersensitive to the DNA gyrase inhibitor novobiocin. In this work, we isolated mutants suppressing the novobiocin hypersensitivity of the mukB null mutation. All suppressor mutations are localized in or near the gyrB gene, and the four tested clones have an amino acid substitution in the DNA gyrase beta subunit. We found that in the mukB mutant, the process of sister chromosome segregation is strikingly hypersensitive to novobiocin; however, the effect of novobiocin on growth, which was measured by culture turbidity, is the same as that of the wild-type strain.The smtA-mukF-mukE-mukB operon (22) is located at the 21-min map position of the Escherichia coli chromosome. Deletion mutants of each muk gene show common phenotypes, such as frequent production of anucleate cells upon cell division, chromosome guillotining (cutting) by septum closure, and temperature-sensitive growth; however, they are nearly normal in chromosome replication, homologous recombination, mutation frequency, and sensitivity to UV irradiation (10, 11, 22; for a review, see reference 4). MukF, MukE, and MukB form a large complex (22). MukB is a member of the SMC superfamily (9). A MukB-green fluorescent protein fusion protein is localized as one, two, or four foci at regular cellular positions in the presence of both MukF and MukE (12). To examine the function of the MukB protein, many suppressor mutants of mukB mutations have been analyzed (for a review, see reference 4). Mutations of the topA gene encoding topoisomerase I suppress temperature-sensitive growth and anucleate-cell production (16). It has been demonstrated in synchronized cultures that replicated sister chromosomes are associated with one another for substantial times (6, 18). In contrast, results in randomly growing cultures in enriched medium appear to be consistent with the model in which replicated sister copies of oriC separate immediately after replication (8). However, it is hard to determine which model is right from the results of such random cultures, because the actual time of initiation of chromosome replication is not clear in this type of experiment.A mukB null mutant is hypersensitive to novobiocin, an inhibitor (20). Deletion mutants of each of the mukB, mukF, and mukE genes and a deletion mutant lacking all three muk genes are hypersensitive to novobiocin (14). Weitao et al. (20) showed that a seqA null mutation suppressed temperaturesensitive growth, anucleate-cell production, and novobiocin hypersensitivity in the mukB null mutation. Inconsistently, Onogi et al. (14) reported that a seqA or dam null mutation partially suppressed temperature-sensitive growth but failed to suppress the anucleate-cell production and novobiocin hypersensitivity of these muk null mutants.It is not yet clear what the mechanism of the novobiocin hypersensitivity of these muk mutants is. What is the target protein that is hypersensitive to novobiocin in these muk null...

DX-619, a Novel Des-Fluoro(6) Quinolone Manifesting Low Frequency of Selection of Resistant Staphylococcus aureus Mutants: Quinolone Resistance beyond Modification of Type II Topoisomerases

“…It is noteworthy that mutants DX-619-C and DX-619-E manifested a four-to eightfold decrease in the MIC of novobiocin relative to that of wild-type ISP794. The Arg136Leu mutation in E. coli gyrB and the Asn470Gln mutation in S. aureus parE mutants are known to confer novobiocin hypersusceptibility (1,8). Although we did not find these mutations in DX-619-C or DX-619-E, a novel 215 to 217 deletion mutation in ParE was found in the latter mutant, but, as determined from the allelic exchange strain DX-619-E-⌬E-AE, this deletion mutation was not responsible for this phenotype.…”

ATP-Induced Shrinkage of DNA with MukB Protein and the MukBEF Complex of Escherichia coli

“…While the MukB homodimer has a long, rod-hinge-rod, V-shaped structure with small globular domains at both ends, the MukBEF complex has a similar structure but larger globular domains than those of the MukB homodimer (10). A mutant with a deletion of the mukB gene exhibits hypersensitivity to novobiocin, which inhibits DNA gyrase, suggesting that MukB relates to superhelicity or the three-dimensional structure of chromosomal DNA (1,18,19). Understanding how MukBEF operates may answer decades-old questions on how chromosomes are organized on a global scale.…”

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

“…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).…”

“…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).…”