Mutational Analysis of Escherichia coliTopoisomerase IV

Bahng, Soon; Mossessova, Elena; Nurse, Pearl; Marians, Kenneth J.

doi:10.1074/jbc.275.6.4112

Cited by 12 publications

(5 citation statements)

References 19 publications

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“…GyrB has an N-terminal ATPase domain (residues 2 to 393 in Escherichia coli) and a C-terminal domain (residues 394 to 804) designated as BЈ (7). Recent X-ray crystallographic and mutational analyses of type II topoisomerase structures suggest that the BЈ domain of GyrB and the homologous region of ParE play an active part in the catalysis of DNA cleavage (3,12). In the model of topoisomerase activity proposed by Fass et al (12), ATP binding by the N-terminal domain of GyrB results in a conformational change that rotates the BЈ domain to expose the DNA-binding groove.…”

Section: Discussionmentioning

confidence: 99%

Genetic Analyses of Mutations Contributing to Fluoroquinolone Resistance in Clinical Isolates of Streptococcus pneumoniae

Weigel¹,

Anderson²,

Facklam

et al. 2001

Antimicrob Agents Chemother

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Twenty-one clinical isolates of Streptococcus pneumoniae showing reduced susceptibility or resistance to fluoroquinolones were characterized by serotype, antimicrobial susceptibility, and genetic analyses of the quinolone resistance-determining regions (QRDRs) of gyrA, gyrB, parC, and parE. Five strains were resistant to three or more classes of antimicrobial agents. In susceptibility profiles for gatifloxacin, gemifloxacin, levofloxacin, moxifloxacin, ofloxacin, sparfloxacin, and trovafloxacin, 14 isolates had intermediate- or high-level resistance to all fluoroquinolones tested except gemifloxacin (no breakpoints assigned). Fluoroquinolone resistance was not associated with serotype or with resistance to other antimicrobial agents. Mutations in the QRDRs of these isolates were more heterogeneous than those previously reported for mutants selected in vitro. Eight isolates had amino acid changes at sites other than ParC/S79 and GyrA/S81; several strains contained mutations in gyrB, parE, or both loci. Contributions to fluoroquinolone resistance by individual amino acid changes, including GyrB/E474K, ParE/E474K, and ParC/A63T, were confirmed by genetic transformation of S. pneumoniae R6. Mutations in gyrB were important for resistance to gatifloxacin but not moxifloxacin, and mutation of gyrA was associated with resistance to moxifloxacin but not gatifloxacin, suggesting differences in the drug-target interactions of the two 8-methoxyquinolones. The positions of amino acid changes within the four genes affected resistance more than did the total number of QRDR mutations. However, the effect of a specific mutation varied significantly depending on the agent tested. These data suggest that the heterogeneity of mutations will likely increase as pneumococci are exposed to novel fluoroquinolone structures, complicating the prediction of cross-resistance within this class of antimicrobial agents.

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Section: Discussionmentioning

confidence: 99%

Genetic Analyses of Mutations Contributing to Fluoroquinolone Resistance in Clinical Isolates of Streptococcus pneumoniae

Weigel¹,

Anderson²,

Facklam

et al. 2001

Antimicrob Agents Chemother

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“…Some of these amino acid residues have been mutated in other studies. Those results will be discussed in comparison to our observations as reported in the accompanying articles (12,13).…”

Section: Resultsmentioning

confidence: 48%

“…We report in this and the accompanying articles (12,13) the detailed analysis of the biochemical properties of Topo IV reconstituted with wild-type ParC (the DNA cleavage subunit; Refs. 14 and 15) and six mutant ParE subunits.…”

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confidence: 99%

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Mutational Analysis of Escherichia coliTopoisomerase IV

Mossessova¹,

Levine²,

Peng³

et al. 2000

Journal of Biological Chemistry

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In order to define regions of ParE, one of the two subunits of topoisomerase IV, that are involved in catalysis during topoisomerization, we developed a selection procedure to isolate dominant-negative parE alleles. Both wild-type parC and mutagenized parE were expressed from a tightly-regulated lac promoter on a moderate-copy plasmid. Mutated parE alleles were rescued from those plasmids that caused IPTG-dependent cell death. The mutant ParE proteins could be divided into two groups when reconstituted with ParC to form topoisomerase IV, those that elicited hyper-DNA cleavage and those that affected covalent complex formation.Type II topoisomerases utilize the cycle of ATP binding, hydrolysis of the ␤-␥ phosphodiester bond, and release of ADP and P i to drive a series of conformational changes that allow the enzyme to pass one DNA helix through a transient proteinbridged, double-strand break in either another segment of the same DNA helix or a different DNA helix. This results in an alteration of the linking number of the DNA. In general, if the same DNA ring contains both the segment of DNA where the break is made and the segment that is passed through the break, the net result is the removal of supercoils. If these two segments of DNA are on different molecules, catenation or decatenation results. These properties make topoisomerases required for essentially all macromolecular processes that operate on DNA in the cell (1-3).The basic sequence of events necessary for one round of topoisomerization has been outlined and incorporated into the two-gate model (4). Capture of a segment of DNA (the T segment) to be transported through the DNA break (the DNA gate) is accomplished by ATP binding-dependent dimerization of two halves of the enzyme (the N gate). This initiates a concerted series of events where the T segment is then forced through the DNA gate, which then closes, resulting in the passage of the T segment to the interior of the enzyme. Release of the T segment from the enzyme occurs upon opening of the C gate. ATP hydrolysis results in re-opening of the N gate, thereby resetting the enzyme for another cycle.The prokaryotic and eukaryotic enzymes share extensive amino acid sequence similarity and are organized in a similar fashion (5, 6). The eukaryotic enzymes are homodimers of a single polypeptide chain that contain a N-terminal ATP-binding domain and a C-terminal DNA cleavage domain. In the prokaryotic enzymes, these domains are on separate subunits and the protein is a heterotetramer.Electron microscopic analysis (7,8) and the solution of several crystal structures (4, 9, 10) have begun to provide a picture of the detailed conformational changes required for topoisomerization and have offered some insight to the mechanism of covalent catalysis and drug resistance. However, little is known about the regions of the protein required for coupling ATP-binding and hydrolysis to operation of the DNA gate.In order to define these regions and to detect regions of the ATP-binding subunit that are involved in cova...

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“…Inactive ParE is unable to relax positive supercoils in DNA, which will impact the segregation of newly replicated chromosomes, leading to the accumulation of DNA at the center of the cell, inhibition of cell growth and eventually the generation of filamentous cells (Bahng et al, 2000). To test whether Ficmediated AMPylation of ParE affects the biological function of Topo IV, we expressed Fic proteins and their mutants in the E. coli strain BL21(DE3).…”

Section: Inhibition Of Topo IV By Fic Proteins Causes Cell Filamentatmentioning

confidence: 99%

Fic Proteins Inhibit the Activity of Topoisomerase IV by AMPylation in Diverse Bacteria

McCloskey

Chen

et al. 2020

Front. Microbiol.

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The Fic (filamentation induced by cyclic AMP) domain is a widely distributed motif with a conserved sequence of HPFx[D/E]GN[G/K]R, some of which regulate cellular activity by catalyzing the transfer of the AMP moiety from ATP to protein substrates. Some Fic proteins, including Fic-1 from the soil bacterium Pseudomonas fluorescens strain 2P24, have been shown to inhibit bacterial DNA replication by AMPylating the subunit B of DNA gyrase (GyrB), but the biochemical activity and cellular target of most Fic proteins remain unknown. Here, we report that Fic-2, which is another Fic protein from strain 2P24 and Fic-1 AMPylate the topoisomerase IV ParE at Tyr 109. We also examined Fic proteins from several phylogenetically diverse bacteria and found that those from Yersinia pseudotuberculosis and Staphylococcus aureus AMPylate ParE and GrlB, the counterpart of ParE in Gram-positive bacteria, respectively. Modification by Fic-1 of P. fluorescens and FicY of Y. pseudotuberculosis inhibits the relaxation activity of topoisomerase IV. Consistent with the inhibition of ParE activity, ectopic expression of these Fic proteins causes cell filamentation akin to the canonical par phenotype in which nucleoids are assembled in the center of elongated cells, a process accompanied by the induction of the SOS response. Our results establish that Fic proteins from diverse bacterial species regulate chromosome division and cell separation in bacteria by targeting ParE.

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Mutational Analysis of Escherichia coliTopoisomerase IV

Cited by 12 publications

References 19 publications

Genetic Analyses of Mutations Contributing to Fluoroquinolone Resistance in Clinical Isolates of Streptococcus pneumoniae

Genetic Analyses of Mutations Contributing to Fluoroquinolone Resistance in Clinical Isolates of Streptococcus pneumoniae

Mutational Analysis of Escherichia coliTopoisomerase IV

Fic Proteins Inhibit the Activity of Topoisomerase IV by AMPylation in Diverse Bacteria

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