PMID:11399091

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Citation

Heddle, JG, Lu, T, Zhao, X, Drlica, K and Maxwell, A (2001) gyrB-225, a mutation of DNA gyrase that compensates for topoisomerase I deficiency: investigation of its low activity and quinolone hypersensitivity. J. Mol. Biol. 309:1219-31

Abstract

The B subunit of DNA gyrase (GyrB) consists of a 43 kDa N-terminal domain, containing the site of ATP binding and hydrolysis, and a 47 kDa C-terminal domain that is thought to play a role in interactions with GyrA and DNA. In cells containing a deletion of topA (the gene encoding DNA topoisomerase I) a compensatory mutation is found in gyrB. This mutation (gyrB-225) results in a two amino acid insertion in the N-terminal domain of GyrB. We found that cells containing this mutation are more sensitive than wild-type cells to quinolone drugs with respect to bacteriostatic and lethal action. We have characterised the mutant GyrB protein in vitro and found it to have reduced DNA supercoiling, relaxation, ATPase, and cleavage activities. The mutant enzyme is up to threefold more sensitive to quinolones than wild-type. The mutation also increases the affinity of GyrB for GyrA and DNA, while the affinity of quinolone for the enzyme-DNA complex is unaffected. We propose that the loss in activity is due to misfolding of the GyrB-225 protein, providing an example in which misfolding of one protein, DNA gyrase, suppresses a deficiency of another, topoisomerase I. The increased quinolone sensitivity is proposed to be a consequence of an altered conformation of the protein that renders quinolones better able to disrupt, rather than generate, gyrase-drug-DNA complexes.

Links

PubMed Online version:10.1006/jmbi.2001.4733

Keywords

Adenosine Triphosphatases/antagonists & inhibitors; Adenosine Triphosphatases/chemistry; Adenosine Triphosphatases/genetics; Adenosine Triphosphatases/metabolism; Alleles; Amino Acid Substitution/genetics; Anti-Infective Agents/metabolism; Anti-Infective Agents/pharmacology; Ciprofloxacin/metabolism; Ciprofloxacin/pharmacology; DNA Gyrase; DNA Topoisomerases, Type I/deficiency; DNA Topoisomerases, Type I/genetics; DNA Topoisomerases, Type II/chemistry; DNA Topoisomerases, Type II/genetics; DNA Topoisomerases, Type II/metabolism; DNA, Superhelical/chemistry; DNA, Superhelical/genetics; DNA, Superhelical/metabolism; Drug Tolerance; Escherichia coli/drug effects; Escherichia coli/enzymology; Escherichia coli/genetics; Genetic Complementation Test; Kinetics; Models, Molecular; Nucleic Acid Conformation; Oxolinic Acid/metabolism; Oxolinic Acid/pharmacology; Plasmids/chemistry; Plasmids/genetics; Plasmids/metabolism; Protein Binding/drug effects; Protein Folding; Protein Structure, Tertiary; Suppression, Genetic/genetics; Surface Plasmon Resonance; Thermodynamics; Topoisomerase II Inhibitors

Main Points of the Paper

Please summarize the main points of the paper.

Materials and Methods Used

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Phenotype Annotations

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Phenotype of Taxon Information Genotype Information (if known) Condition Information OMP ID OMP Term Name ECO ID ECO Term Name Notes Status

a mutation or genetic difference within a strain

  • Taxon: Escherichia coli
  • Strain: K-12
  • Substrain: SD104
  • NCBI Taxon ID: 83333
  • Genotype of Reference Strain: gyrB
  • Genotype of Experimental Strain : gyrB-225 See figure 1
  • Reference Condition:

decreased resistance to Quinolone

ECO:0000178

in vivo assay evidence

Increase (2x) sensitivity to quinolone Ciprofloxacin Figure 2.

CHEBI:100241


a mutation or genetic difference within a strain

  • Taxon: Escherichia coli
  • Strain: K-12
  • Substrain: SD104
  • NCBI Taxon ID: 83333
  • Genotype of Reference Strain: gyrB
  • Genotype of Experimental Strain : gyrB-225 See figure 1
  • Reference Condition:

decreased resistance to Quinolone

ECO:0000178

in vivo assay evidence

Increase (2x) sensitivity to quinolone oxolinic acid Figure 2.

CHEBI:138856

a mutation or genetic difference within a strain

  • Taxon: Escherichia coli
  • Strain: K-12
  • Substrain: SD104
  • NCBI Taxon ID: 83333
  • Genotype of Reference Strain: gyrB
  • Genotype of Experimental Strain : gyrB-225 See figure 1
  • Reference Condition:
OMP:0000294

DNA supercoiling phenotype

ECO:0000267

enzyme-linked immunoabsorbent assay evidence

Decreased Supercoiling by 6 times. See table 1.

a mutation or genetic difference within a strain

  • Taxon: Escherichia coli
  • Strain: K-12
  • Substrain: SD104
  • NCBI Taxon ID: 83333
  • Genotype of Reference Strain: gyrB
  • Genotype of Experimental Strain : gyrB-225 See figure 1
  • Reference Condition:

DNA Relaxation phenotype

ECO:0000267

enzyme-linked immunoabsorbent assay evidence

Decreased DNA relaxation by 2 times. See table 1.


Notes

This OMP term needs to be more specific, Resistance to antimicrobial Quinolone. Waiting on term development.

  • Supercoiling Phenotype: Possibly need new term with increase decrease supercoiling (and negative supercoiling) under a new node since existing node implies state rather than a relative phenotype.
  • DNA cleavage Phenotype: Need to make term.
  • DNA relaxation phenotype: need to make term.

References

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