GyrB Proteins Research Articles

A Gyrase Mutant with Low Activity Disrupts Supercoiling at the Replication Terminus

When a mutation in an essential gene shows a temperature-sensitive phenotype, one usually assumes that the protein is inactive at nonpermissive temperature. DNA gyrase is an essential bacterial enzyme composed of two subunits, GyrA and GyrB. The gyrB652 mutation results from a single base change that substitutes a serine residue for arginine 436 (R436-S) in the GyrB protein. At 42 degrees C, strains with the gyrB652 allele stop DNA replication, and at 37 degrees C, such strains grow but have RecA-dependent SOS induction and show constitutive RecBCD-dependent DNA degradation. Surprisingly, the GyrB652 protein is not inactive at 42 degrees C in vivo or in vitro and it doesn't directly produce breaks in chromosomal DNA. Rather, this mutant has a low k(cat) compared to wild-type GyrB subunit. With more than twice the normal mean number of supercoil domains, this gyrase hypomorph is prone to fork collapse and topological chaos near the terminus of DNA replication.

Mutations in gyrA and parC genes in nalidixic acid-resistant Escherichia coli strains from food products, humans and animals.

Mutations in quinolone targets were analysed in 80 unrelated nalidixic acid-resistant (NALR) Escherichia coli strains whose nalidixic acid and ciprofloxacin MICs ranged from 32 to >256 mg/L and 0.03-64 mg/L, respectively. These strains were isolated from food products (23) and faecal samples from humans (15) and healthy animals (42). Thirteen nalidixic acid-susceptible (NALS) E. coli strains were also analysed. Mutations in gyrA and parC genes were studied by PCR and sequencing. No amino acid changes were detected in GyrA or ParC proteins of the 13 NALS strains. A single change in the GyrA protein was detected in all 61 NALR strains with ciprofloxacin MICs </= 2 mg/L with the following substitutions (number of strains): Ser-83-->Leu (54), Ser-83-->Ala (one), Ser-83-->Val (one), Asp-87-->Asn (two), Asp-87-->Tyr (two) and Asp-87-->Gly (one). A double change in GyrA was found in 18 of 19 NALR strains with ciprofloxacin MICs >/= 4 mg/L. Amino acid substitutions were Ser-83-->Leu, with an additional change [Asp-87-->Asn (15), Asp-87-->Tyr (two) or Asp-87-->His (one)]. The remaining strain (ciprofloxacin MIC 4 mg/L) showed a single Ser-83-->Leu substitution. In respect of the ParC protein, a single change at Ser-80 or Glu-84 was found in 25 of 42 strains, with ciprofloxacin MICs ranging from 0.5 to 32 mg/L. A double substitution (Ser-80-->Ile and Glu-84-->Gly) was found in one strain (ciprofloxacin MIC 64 mg/L). No amino acid changes were detected in the GyrB protein of 18 NALR strains.

Active-site residues of Escherichia coli DNA gyrase required in coupling ATP hydrolysis to DNA supercoiling and amino acid substitutions leading to novobiocin resistance.

DNA gyrase is a bacterial type II topoisomerase which couples the free energy of ATP hydrolysis to the introduction of negative supercoils into DNA. Amino acids in proximity to bound nonhydrolyzable ATP analog (AMP. PNP) or novobiocin in the gyrase B (GyrB) subunit crystal structures were examined for their roles in enzyme function and novobiocin resistance by site-directed mutagenesis. Purified Escherichia coli GyrB mutant proteins were complexed with the gyrase A subunit to form the functional A(2)B(2) gyrase enzyme. Mutant proteins with alanine substitutions at residues E42, N46, E50, D73, R76, G77, and I78 had reduced or no detectable ATPase activity, indicating a role for these residues in ATP hydrolysis. Interestingly, GyrB proteins with P79A and K103A substitutions retained significant levels of ATPase activity yet demonstrated no DNA supercoiling activity, even with 40-fold more enzyme than the wild-type enzyme, suggesting that these amino acid side chains have a role in the coupling of the two activities. All enzymes relaxed supercoiled DNA to the same extent as the wild-type enzyme did, implying that only ATP-dependent reactions were affected. Mutant genes were examined in vivo for their abilities to complement a temperature-sensitive E. coli gyrB mutant, and the activities correlated well with the in vitro activities. We show that the known R136 novobiocin resistance mutations bestow a significant loss of inhibitor potency in the ATPase assay. Four new residues (D73, G77, I78, and T165) that, when changed to the appropriate amino acid, result in both significant levels of novobiocin resistance and maintain in vivo function were identified in E. coli.

GyrB-225, a mutation of DNA gyrase that compensates for topoisomerase I deficiency: investigation of its low activity and quinolone hypersensitivity

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.

Brachyspira (Serpulina) hyodysenteriae gyrB mutants and interstrain transfer of coumermycin A(1) resistance.

To further develop genetic techniques for the enteropathogen Brachyspira hyodysenteriae, the gyrB gene of this spirochete was isolated from a lambdaZAPII library of strain B204 genomic DNA and sequenced. The putative protein encoded by this gene exhibited up to 55% amino acid sequence identity with GyrB proteins of various bacterial species, including other spirochetes. B. hyodysenteriae coumermycin A(1)-resistant (Cn(r)) mutant strains, both spontaneous and UV induced, were isolated by plating B204 cells onto Trypticase soy blood agar plates containing 0.5 microg of coumermycin A(1)/ml. The coumermycin A(1) MICs were 25 to 100 microg/ml for the resistant strains and 0.1 to 0.25 microg/ml for strain B204. Four Cn(r) strains had single nucleotide changes in their gyrB genes, corresponding to GyrB amino acid changes of Gly(78) to Ser (two strains), Gly(78) to Cys, and Thr(166) to Ala. When Cn(r) strain 435A (Gly(78) to Ser) and Cm(r) Km(r) strain SH (DeltaflaA1::cat Deltanox::kan) were cultured together in brain heart infusion broth containing 10% (vol/vol) heat-treated (56 degrees C, 30 min) calf serum, cells resistant to chloramphenicol, coumermycin A(1), and kanamycin could be isolated from the cocultures after overnight incubation, but such cells could not be isolated from monocultures of either strain. Seven Cn(r) Km(r) Cm(r) strains were tested and were determined to have resistance genotypes of both strain 435A and strain SH. Cn(r) Km(r) Cm(r) cells could not be isolated when antiserum to the bacteriophage-like agent VSH-1 was added to cocultures, and the numbers of resistant cells increased fivefold when mitomycin C, an inducer of VSH-1 production, was added. These results indicate that coumermycin resistance associated with a gyrB mutation is a useful selection marker for monitoring gene exchange between B. hyodysenteriae cells. Gene transfer readily occurs between B. hyodysenteriae cells in broth culture, a finding with practical importance. VSH-1 is the likely mechanism for gene transfer.

Mechanism of quinolone resistance in Staphylococcus aureus.

The resistance mechanisms to fluoroquinolones in Staphylococcus aureus were clarified by analyzing mutations in the genes encoding target enzymes, and examining the expression of the efflux pump, and determining the inhibitory activities of fluoroquinolones against the altered enzymes. Mutations in the grlA and gyrA genes of 344 clinical strains of S. aureus isolated in 1994 in Japan were identified by combinations of methods - single-strand conformation polymorphism analysis, restriction fragment length analysis, and direct sequencing - to identify possible relationships with fluoroquinolone resistance. Five types of single-point mutations and four types of double mutations were observed in the grlA gene in 204 strains (59.3%). Four types of single-point mutations and four types of double mutations were found in the gyrA gene in 188 strains (54.7%). Among these mutations, the grlA mutation of TCC --> TTC or TAC (Ser-80 --> Phe or Tyr) and the gyrA mutation of TCA --> TTA (Ser-84 --> Leu) were the principal ones, being detected in 137 (39.8%) and 121 (35.2%) isolates, respectively. A total of 15 types of mutation combinations within both genes were related to ciprofloxacin resistance (MIC greater than or equal 3.13 microg/ml) and were present in 193 mutants (56.1%). Strains containing mutations in both genes were highly resistant to ciprofloxacin (MIC50 =50 microg/ml). Those strains with the Ser-80 --> Phe or Tyr alteration in grlA, but wild type in gyrA showed a lower level of ciprofloxacin resistance (MIC50 less than or equal 12.5 microg/ml). Levofloxacin was active against 68 of 193 isolates (35.2%) with mutations at codon 80 of grlA in the presence or absence of concomitant mutations at codons 73, 84, or 88 in gyrA (MIC less than or equal 6.25 microg/ml). Sitafloxacin (DU-6859a) showed good activity in 186 of 193 isolates (96.4%), with an MIC of less than or equal 6.25 microg/ml. The contribution of membrane-associated multidrug efflux protein (NorA) expression to fluoroquinolone resistance was clarified by the checker-board titration method for determining the MIC of norfloxacin alone and in combination with carbonyl cyanide m-chlorophenylhydrazone. Among 344 clinical isolates, 139 strains (40.4%), in which the MIC of norfloxacin varied from 1.56 to >800 microg/ml, overexpressed the NorA protein. GrlA and GrlB proteins of topoisomerase IV, and GyrA and GyrB proteins of DNA gyrase encoded by genes with or without mutations were purified separately. The inhibitory activities of fluoroquinolones against the topoisomerase IV which contained a single amino acid change (Ser --> Phe at codon 80, Glu --> Lys at codon 84 of grlA, and Asp --> Asn at codon 432 of grlB) were from 5 to 95 times weaker than the inhibitory activities against the non-altered enzyme. These results suggest that the mutations in the corresponding genes may confer quinolone resistance; the active efflux pump, NorA, was considered to be the third quinolone-resistance mechanism. The numerous and complicated mutations seen may explain the rapid and widespread development of quinolone resistance described in S. aureus. Sitafloxacin showed good antibacterial activity against ciprofloxacin- or levofloxacin-resistant mutants because of its high inhibitory activity against both topoisomerase IV and DNA gyrase.

Streptococcus pneumoniae DNA gyrase and topoisomerase IV: overexpression, purification, and differential inhibition by fluoroquinolones.

Streptococcus pneumoniae gyrA and gyrB genes specifying the DNA gyrase subunits have been cloned into pET plasmid vectors under the control of an inducible T7 promoter and have been separately expressed in Escherichia coli. Soluble 97-kDa GyrA and 72-kDa GyrB proteins bearing polyhistidine tags at their respective C-terminal and N-terminal ends were purified to apparent homogeneity by one-step nickel chelate column chromatography and were free of host E. coli topoisomerase activity. Equimolar amounts of the gyrase subunits reconstituted ATP-dependent DNA supercoiling with comparable activity to gyrase of E. coli and Staphylococcus aureus. In parallel, S. pneumoniae topoisomerase IV ParC and ParE subunits were similarly expressed in E. coli, purified to near homogeneity as 93- and 73-kDa proteins, and shown to generate efficient ATP-dependent DNA relaxation and DNA decatenation activities. Using the purified enzymes, we examined the inhibitory effects of three paradigm fluoroquinolones-ciprofloxacin, sparfloxacin, and clinafloxacin-which previous genetic studies with S. pneumoniae suggested act preferentially through topoisomerase IV, through gyrase, and through both enzymes, respectively. Surprisingly, all three quinolones were more active in inhibiting purified topoisomerase IV than gyrase, with clinafloxacin showing the greatest inhibitory potency. Moreover, the tested agents were at least 25-fold more effective in stabilizing a cleavable complex (the relevant cytotoxic lesion) with topoisomerase IV than with gyrase, with clinafloxacin some 10- to 32-fold more potent against either enzyme, in line with its superior activity against S. pneumoniae. The uniform target preference of the three fluoroquinolones for topoisomerase IV in vitro is in apparent contrast to the genetic data. We interpret these results in terms of a model for bacterial killing by quinolones in which cellular factors can modulate the effects of target affinity to determine the cytotoxic pathway.

Mutations in Bartonella bacilliformis gyrB confer resistance to coumermycin A1.

This study describes the first isolation and characterization of spontaneous mutants conferring natural resistance to an antibiotic for any Bartonella species. The Bartonella bacilliformis gyrB gene, which encodes the B subunit of DNA gyrase, was cloned and sequenced. The gyrB open reading frame (ORF) is 2,079 bp and encodes a deduced amino acid sequence of 692 residues, corresponding to a predicted protein of approximately 77.5 kDa. Sequence alignment indicates that B. bacilliformis GyrB is most similar to the GyrB protein from Bacillus subtilis (40.1% amino acid sequence identity) and that it contains the longest N-terminal tail (52 residues) of any GyrB characterized to date. The cloned B. bacilliformis gyrB was expressed in an Escherichia coli S30 cell extract and was able to functionally complement a temperature-sensitive E. coli Cour gyrB mutant (strain N4177). We isolated and characterized spontaneous mutants of B. bacilliformis resistant to coumermycin A1, an antibiotic that targets GyrB. Sequence analysis of gyrB from 12 Cour mutants of B. bacilliformis identified single nucleotide transitions at three separate loci in the ORF. The predicted amino acid substitutions resulting from these transitions are Gly to Ser at position 124 (Gly124-->Ser), Arg184-->Gln, and Thr214-->Ala or Thr214-->Ile, which are analogous to mutated residues found in previously characterized resistant gyrB genes from Borrelia burgdorferi, E. coli, Staphylococcus aureus, and Haloferax sp. The Cour mutants are three to five times more resistant to coumermycin A1 than the wild-type parental strain.

Molecular analysis of the DNA gyrB gene from Myxococcus xanthus.

DNA gyrase, an essential type II topoisomerase, mediates negative supercoiling of the bacterial chromosome, thereby affecting the processes of DNA replication, transcription, recombination and repair. The gyrB gene from the Gram-negative soil bacterium Myxococcus xanthus was sequenced. The sequence predicts a protein of 815 amino acid residues displaying significant homology to all known GyrB proteins. A 6-His-GyrB fusion protein was overexpressed in Escherichia coli and purified to near homogeneity using affinity chromatography on Ni-nitrilotriacetic acid-agarose and novobiocin-Sepharose columns. The fusion protein bound novobiocin and cross-reacted with anti-E. coli GyrB antibodies, indicating structural and functional similarities to the E. coli DNA GyrB. The gene was mapped to the region of the origin of replication (oriC) of M. xanthus.

IL-10 inhibits macrophage activation and proliferation by distinct signaling mechanisms: evidence for Stat3-dependent and -independent pathways.

Interleukin-10 (IL-10) limits inflammatory responses by inhibiting macrophage activation. In macrophages, IL-10 activates Stat1 and Stat3. We characterized IL-10 responses of the J774 mouse macrophage cell line, and of J774 cells expressing wild-type hIL-10R, mutant hIL-10R lacking two membrane-distal tyrosines involved in recruitment of Stat3 (hIL-10R-TyrFF), a truncated Stat3 (DeltaStat3) which acts as a dominant negative, or an inducibly active Stat3-gyraseB chimera (Stat3-GyrB). A neutralizing anti-mIL-10R monoclonal antibody was generated to block the function of endogenous mIL-10R. IL-10 inhibited proliferation of J774 cells and of normal bone marrow-derived macrophages, but not J774 cells expressing hIL-10RTyrFF. Dimerization of Stat3-GyrB by coumermycin mimicked the effect of IL-10, and expression of DeltaStat3 blocked the anti-proliferative activity of IL-10. For macrophage de-activation responses, hIL10R-TyrFF could not mediate inhibition of lipopolysaccharide-induced TNFalpha, IL-1beta or CD86 expression, while DeltaStat3 did not interfere detectably with these IL-10 responses. Thus signals mediating both anti-proliferative and macrophage de-activation responses to IL-10 require the two membrane-distal tyrosines of IL-10R, but Stat3 appears to function only in the anti-proliferative response.

Expression inEscherichia coliof Y5 Mutant and N-Terminal Domain-Deleted DNA Gyrase B Proteins Affects Strongly Plasmid Maintenance

Escherichia coliDNA gyrase B subunit (GyrB) is composed of a 43-kDa N-terminal domain containing an ATP-binding site and a 47-kDa C-terminal domain involved in the interaction with the gyrase A subunit (GyrA). Site-directed mutagenesis was used to substitute, in both the entire GyrB subunit and its 43-kDa N-terminal fragment, the amino acid Y5 by either a serine (Y5S) or a phenylalanine residue (Y5F). Under standard conditions, cells bearing Y5S or Y5F mutant GyrB expression plasmids produced significantly less recombinant proteins than cells transformed with the wild-type plasmid. This dramatic decrease in expression of mutant GyrB proteins was not observed when the corresponding N-terminal 43 kDa mutant plasmids were used. Examination of the plasmid content of the transformed cells after induction showed that the Y5F and Y5S GyrB protein level was correlated with the plasmid copy number. By repressing tightly the promoter activity encoded by these expression vectors during cell growth, it was possible to restore the normal level of the mutant GyrB encoding plasmids in the transformed bacteria. Treatment with chloramphenicol before protein induction enabled large overexpression of the GyrB mutant Y5F and Y5S proteins. In addition, the decrease in plasmid copy number was also observed when the 47-kDa C-terminal fragment of the GyrB subunit was expressed in bacteria grown under standard culture conditions. Analysis of DNA supercoiling and relaxation activities in the presence of GyrA demonstrated that purified Y5-mutant GyrB proteins were deficient for ATP-dependent gyrase activities. Taken together, these results show that Y5F and Y5S mutant GyrB proteins, but not the corresponding 43-kDa N-terminal fragments, competein vivowith the bacterial endogenous GyrB subunit of DNA gyrase, thereby reducing the plasmid copy number in the transformed bacteria by probably acting on the level of negative DNA supercoilingin vivo.This competition could be mediated by the presence of the intact 47-kDa C-terminal domain in the Y5F and Y5S mutant GyrB subunits. This study demonstrates also that the amino acid Y5 is a crucial residue for the expression of the gyrase B activityin vivo.Thus, ourin vivoapproach may also be useful for detecting other important amino acids for DNA gyrase activity, as mutations affecting the ATPase activity or GyrB/GyrB, or GyrB/GyrA protein interactions.

Expression inEscherichia coliof Y5-Mutant and N-terminal Domain-Deleted DNA Gyrase B Proteins Affects Strongly Plasmid Maintenance

Escherichia coliDNA gyrase B subunit (GyrB) is composed of a 43-kDa N-terminal domain containing an ATP-binding site and a 47-kDa C-terminal domain involved in the interaction with the gyrase A subunit (GyrA). Site-directed mutagenesis was used to substitute, in both the entire GyrB subunit and its 43-kDa N-terminal fragment, the amino acid Y5 by either a serine (Y5S) or a phenylalanine residue (Y5F). Under standard conditions, cells bearing Y5S or Y5F mutant GyrB expression plasmids produced significantly less recombinant proteins than cells transformed with the wild-type plasmid. This dramatic decrease in expression of mutant GyrB proteins was not observed when the corresponding N-terminal 43-kDa mutant plasmids were used. Examination of the plasmid content of the transformed cells after induction showed that the Y5F and Y5S GyrB protein level was correlated with the plasmid copy number. By repressing tightly the promoter activity encoded by these expression vectors during cell growth, it was possible to restore the normal level of the mutant GyrB encoding plasmids in the transformed bacteria. Treatment with chloramphenicol before protein induction enabled large overexpression of the GyrB mutant Y5F and Y5S proteins. In addition, the decrease in plasmid copy number was also observed when the 47-kDa C-terminal fragment of the GyrB subunit was expressed in bacteria grown under standard culture conditions. Analysis of DNA supercoiling and relaxation activities in the presence of GyrA demonstrated that purified Y5-mutant GyrB proteins were deficient for ATP-dependent gyrase activities. Taken together, these results show that Y5F and Y5S mutant GyrB proteins, but not the corresponding 43-kDa N-terminal fragments, competein vivowith the bacterial endogenous GyrB subunit of DNA gyrase, thereby reducing the plasmid copy number in the transformed bacteria by probably acting on the level of negative DNA supercoilingin vivo.This competition could be mediated by the presence of the intact 47-kDa C-terminal domain in the Y5F and Y5S mutant GyrB subunits. This study demonstrates also that the amino acid Y5 is a crucial residue for the expression of the gyrase B activityin vivo.Thus, ourin vivoapproach may also be useful for detecting other important amino acids for DNA gyrase activity, as mutations affecting the ATPase activity or the GyrB/GyrB or GyrB/GyrA protein interactions.

Identification and characterization of the gyrB gene from Treponema pallidum subsp. pallidum

The nucleotide sequence of a DNA gyrase B subunit gene ( gyrB) from Treponema pallidum has been determined. Southern blot analysis of T. pallidum chromosomal DNA indicated that this gene is present as a single copy. The organization of genes flanking the gyrB gene is unique in comparison to that of other bacteria. The gyrB gene encodes a 637 amino acid protein whose deduced sequence has a high degree of homology with type-II topoisomerase ATPase subunits (GyrB and ParE). Five type-II topoisomerase motifs, an ATP-binding site (Walker A), and amino acid residues that putatively interact with ATP, are highly conserved in the T. pallidum GyrB protein.

Involvement of topoisomerase IV and DNA gyrase as ciprofloxacin targets in Streptococcus pneumoniae.

Ciprofloxacin-resistant mutants of Streptococcus pneumoniae 7785 were generated by stepwise selection at increasing drug concentrations. Sequence analysis of PCR products from the strains was used to examine the quinolone resistance-determining regions of the GyrA and GyrB proteins of DNA gyrase and the analogous regions of the ParC and ParE subunits of DNA topoisomerase IV. First-step mutants exhibiting low-level resistance had no detectable changes in their topoisomerase quinolone resistance-determining regions, suggesting altered permeation or another novel resistance mechanism. Nine of 10 second-step mutants exhibited an alteration in ParC at Ser-79 to Tyr or Phe or at Ala-84 to Thr. Third- and fourth-step mutants displaying high-level ciprofloxacin resistance were found to have, in addition to the ParC alteration, a change in GyrA at residues equivalent to Escherichia coli GyrA resistance hot spots Ser-83 and Asp-87 or in GyrB at Asp-435 to Asn, equivalent to E. coli Asp-426, part of a highly conserved EGDSA motif in GyrB. No ParE changes were observed. Complementary analysis of two S. pneumoniae clinical isolates displaying low-level resistance to ciprofloxacin revealed a ParC change at Ser-79 to Phe or Arg-95 to Cys but no changes in GyrA, GyrB, or ParE. A highly resistant isolate, in addition to a ParC mutation, had a GyrA alteration at the residue equivalent to E. coli Asp-87. Thus, in both laboratory strains and clinical isolates, ParC mutations preceded those in GyrA, suggesting that topoisomerase IV is a primary topoisomerase target and gyrase is a secondary target for ciprofloxacin in S. pneumoniae.

A gyrB-like gene from the hyperthermophilic bacterion Thermotoga maritima

We have cloned and sequenced two overlapping DNA fragments (3236 bp) containing a gene encoding the ATPase subunit of a type II DNA topoisomerase from the hyperthermophilic bacterion Thermotoga maritima (Tm Top2B). The deduced protein is composed of 636 aa with a calculated molecular mass of 72 415 Da. It shares significant similarities with the ATPase subunits of mesophilic bacterial DNA topoisomerases II, either DNA gyrase (GyrB) or DNA topoisomerase IV (ParE). Although the highest similarity scores are obtained with GyrB proteins (55% identity with Bacillus subtilis DNA gyrase), a detailed phylogenetic analysis of all known DNA topoisomerases II does not allow us to determine if Tm Top2B corresponds to a DNA gyrase or a DNA topoisomerase IV. This hyperthermophilic Top2B protein exhibits a larger amount of charged amino acids than its mesophilic homologues, a feature which could be important for its thermostability. No gyrA-like gene has been found near top2B. A gene coding for a transaminase B-like protein was found in the upstream region of top2B.

Partner Switching Mechanisms in Inactivation and Rejuvenation of Escherichia coliDNA Gyrase by F Plasmid Proteins LetD (CcdB) and LetA (CcdA)

Escherichia coliDNA gyrase, as well as a free form of its A subunit (GyrA), exists in an inactivated form in cells that overproduce the F plasmid protein LetD (CcdB). We found that the inactivated DNA gyrase and GyrA protein can be rejuvenated in vitroby another F plasmid protein, LetA (CcdA). Using this rejuvenation as an assay, we purified the inactivated GyrA protein to near homogeneity and found it to be complexed with the LetD protein. The complex has a molecular mass of 230 kDa and was suggested to be a complex of two molecules each of GyrA and LetD proteins. The GyrA-LetD complex, in the presence of purified GyrB protein, does not cause DNA cleavage. Therefore, the LetD protein in the GyrA-LetD complex inhibits the gyrase action by a mechanism different from one that involves trapping a covalently linked gyrase-DNA complex. In as much as a free form of the LetD protein has been shown to induce DNA cleavage by gyrase, the LetD protein seems to have two distinct modes of action on DNA gyrase. Rejuvenation of the inactivated GyrA protein by the LetA protein was achieved in vitro, and mechanisms governing this process were examined using the purified proteins. The rejuvenated GyrA protein sediments through sucrose gradients as a single protein species of 190 kDa and is indistinguishable from a free form of GyrA protein. In the same sedimentation experiment, the LetD protein was seen to be complexed with the added LetA protein. Thus, the LetA protein apparently rejuvenates the GyrA protein by removing the bound LetD protein from the inactivated form, followed by formation of a LetA-LetD complex.

Mechanism of action of quinolones against Escherichia coli DNA gyrase.

The mechanism of action of quinolones was investigated by use of various DNA gyrases reconstituted from wild-type and mutant GyrA and GyrB proteins of Escherichia coli. The quinolone sensitivities of the DNA supercoiling activity of the gyrases were generally parallel to the quinolone susceptibilities of strains having the corresponding enzymes and depended on gyrase subunits but not on substrate DNA. [3H]Enoxacin did not bind to gyrase alone or DNA alone but bound to gyrase-DNA complexes when measured by a gel filtration method. There appeared to be two enoxacin binding phases, at low and high enoxacin concentrations, for the wild-type gyrase-DNA and type 2 GyrB (Lys-447 to Glu) mutant gyrase-DNA complexes but only one enoxacin binding phase at the concentrations used for the GyrA (Ser-83 to Leu) mutant gyrase-DNA and type 1 GyrB (Asp-426 to Asn) mutant gyrase-DNA complexes. New enoxacin binding sites appeared in the presence of enoxacin, and the enoxacin binding affinities for the sites, especially at low enoxacin concentrations, near the MICs for the strains having the corresponding gyrases, correlated well with the enoxacin sensitivities of the gyrases and the MICs. From the results obtained, we propose a quinolone pocket model as the mechanism of action of quinolones, in which quinolones exert their action through binding to a gyrase-DNA complex and the quinolone binding affinities for the complex are determined by both GyrA and GyrB subunits in concert.

Quinolone Mode of Action - New Aspects†

The interactions of quinolones with the complex of DNA gyrase and DNA have been elucidated by the sequencing of additional mutant gyrA and gyrB genes that produce altered quinolone susceptibility. Strong patterns have emerged in Escherichia coli in which amino acids between positions 67 and 106 of the gyrase A subunit (GyrA) and at positions 426 and 447 of the gyrase B subunit (GyrB) have been consistently identified as important for quinolone action. The susceptibility patterns and changes in amino acids 426 and 447 in mutant resistant GyrB proteins suggest direct electrostatic interactions with quinolones at these positions. The small size and the polar nature of the serine at position 83 of the E. coli GyrA protein are particularly important for determining enzyme sensitivity and bacterial susceptibility to quinolones. Norfloxacin and ciprofloxacin bind most stably to a complex of DNA gyrase and DNA rather than to either component alone, and reduction of norfloxacin binding to complexes containing resistant GyrA proteins confirms the biological relevance of this direct measure of quinolone interaction with the gyrase-DNA complex. Although recent crystallographic studies have expanded and refine information about gyrase structure at the atomic level, direct determination of the sites of quinolone binding within the gyrase-DNA complex awaits further studies. Although quinolones have little activity against E. coli topoisomerases I and III, topoisomerase IV, a recently described enzyme thought to be involved in chromosome segregation into daughter cells, has homology with GyrA and GyrB, particularly in regions important for quinolone action, and is inhibited by some quinolones in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification and characterization of DNA topoisomerase IV in Escherichia coli.

The subunits of topoisomerase IV (topo IV), the ParC and ParE proteins in Escherichia coli, were purified to near homogeneity from the respective overproducers. They revealed type II topoisomerase activity only when they were combined with each other. In the presence of Mg2+ and ATP, topo IV was capable of relaxing a negatively or positively supercoiled plasmid DNA or converting the knotted P4 phage DNA, whether nicked or ligated, to a simple ring. However, supercoiling activity was not detected. The topoisomerase activity was not detectable when the purified ParC and ParE proteins were combined with the purified GyrB and GyrA proteins, respectively. This is consistent with the result that neither a parC nor a parE mutation was compensated by transformation with a plasmid carrying either the gyrA or the gyrB gene. Simultaneous introduction of both the gyrA and gyrB plasmids corrected the phenotypic defect of parC and parE mutants. The results suggest that DNA gyrase can substitute for topo IV at least in some part of the function for chromosome partitioning. Antisera were prepared against the purified ParC, ParE, GyrA, and GyrB proteins and used to investigate cellular localization of these gene products. ParC protein was found to be specifically associated with inner membranes only in the presence of DNA. This result suggests that one of the functions of topo IV might be to anchor chromosomes on membranes as previously proposed for eukaryotic topoisomerase II.

GyrB mutations which confer coumarin resistance also affect DNA supercoiling and ATP hydrolysis by Escherichia coli DNA gyrase.

Coumarins are inhibitors of the ATP hydrolysis and DNA supercoiling reactions catalysed by DNA gyrase. Their target is the B subunit of gyrase (GyrB), encoded by the gyrB gene. The exact mode and site of action of the drugs is unknown. We have identified four mutations conferring coumarin resistance to Escherichia coli: Arg-136 to Cys, His or Ser and Gly-164 to Val. In vitro, the ATPase and supercoiling activities of the mutant GyrB proteins are reduced relative to the wild-type enzyme and show resistance to the coumarin antibiotics. Significant differences in the susceptibility of mutant GyrB proteins to inhibition by either chlorobiocin and novobiocin or coumermycin have been found, suggesting wider contacts between coumermycin and GyrB. We discuss the significance of Arg-136 and Gly-164 in relation to the notion that coumarin drugs act as competitive inhibitors of the ATPase reaction.