Changed properties of the A subunit in DNA gyrase with a B subunit mutation

Summary To investigate the interaction of subunits A and B of DNA gyrase during DNA supercoiling, a Cou^r mutant of Escherichia coli was obtained and the effect of nalidixic acid on the supercoiling of DNA by wild-type and mutant enzymes was assayed. The enzyme of the Cou^r strain proved to be more sensitive to nalidixic acid than the wild-type DNA gyrase. Hence the mutation affecting the B subunit can also change the properties of the A subunit, which fact suggests that the two subunits of DNA gyrase are in contact during DNA supercoiling.     

DNA gyrase inhibitors block development of Bacillus subtilis bacteriophage SP01

SP01 development was inhibited by nalidixic acid and novobiocin in the sensitive host Bacillus subtilis 168M. Inhibition by novobiocin was prevented by a Novr mutation in the cellular DNA gyrase gene. Nalidixic acid inhibition persisted in hosts carrying a Nalr gyrase, but could be overcome by phage mutation. We conclude that SP01 requires for its development subunit B of the host DNA gyrase, but replaces or modifies subunit A.     

Involvement of host DNA gyrase in growth of bacteriophage T5.

Bacteriophage T5 did not grow at the nonpermissive temperature of 42 degrees C in Escherichia coli carrying a temperature-sensitive mutation in gyrB [gyrB(Ts)], but it did grow in gyrA(Ts) mutants at 42 degrees C. These findings indicate that the A subunit of host DNA gyrase is unnecessary, whereas the B subunit is necessary for growth of T5. The necessity for the B subunit was confirmed by a strong inhibition of T5 growth by novobiocin and coumermycin A1, which interfere specifically with the function of the B subunit of host DNA gyrase. However, T5 growth was also strongly inhibited by nalidixic acid, which interferes specifically with the function of the A subunit. This inhibition was due to the interaction of nalidixic acid with the A subunit and not just to its binding to DNA, because appropriate mutations in the gyrA gene of the host conferred nalidixic acid resistance to the host and resistance to T5 growth in such a host. The inhibition by nalidixic acid was also not due to a cell poison formed between nalidixic acid and the A subunit (K. N. Kreuzer and N. R. Cozzarelli, J. Bacteriol. 140:424-435, 1979) because nalidixic acid inhibited growth of T5 in a gyrA(Ts) mutant (KNK453) at 42 degrees C. We suggest that T5 grows in KNK453 at 42 degrees C because its gyrA(Ts) mutation is leaky for T5. Inhibition of T5 growth due to inactivation of host DNA gyrase was caused mainly by inhibition of T5 DNA replication. In addition, however, late T5 genes were barely expressed when host DNA gyrase was inactivated.     

Interactions of subunits of DNA gyrase

Interaction of DNA gyrase A- and B-subunits during the process of DNA supercoiling was studied. For this purpose a E. coli Cour-1 mutant resistant to coumermycin and containing a mutation in the B-subunit of DNA gyrase was isolated and the influence of the DNA gyrase A-subunit specific inhibitor-nalidixic acid-on DNA supercoiling by wild-type and mutant enzymes was investigated. It turned out that the enzyme from the Cour-1 mutant strain was more sensitive to nalidixic acid than the DNA gyrase from the wild-type strain. Hence, the mutation affecting the B-subunit is capable to change A-subunit properties. That makes it possible to draw the conclusion about a close structural interaction of DNA gyrase subunits during DNA supercoiling.     

The epsilon subunit of DNA polymerase III Is involved in the nalidixic acid-induced SOS response in Escherichia coli

Quinolone antibacterial drugs such as nalidixic acid target DNA gyrase in Escherichia coli. These inhibitors bind to and stabilize a normally transient covalent protein-DNA intermediate in the gyrase reaction cycle, referred to as the cleavage complex. Stabilization of the cleavage complex is necessary but not sufficient for cell killing--cytotoxicity apparently results from the conversion of cleavage complexes into overt DNA breaks by an as-yet-unknown mechanism(s). Quinolone treatment induces the bacterial SOS response in a RecBC-dependent manner, arguing that cleavage complexes are somehow converted into double-stranded breaks. However, the only proteins known to be required for SOS induction by nalidixic acid are RecA and RecBC. In hopes of identifying additional proteins involved in the cytotoxic response to nalidixic acid, we screened for E. coli mutants specifically deficient in SOS induction upon nalidixic acid treatment by using a dinD::lacZ reporter construct. From a collection of SOS partially constitutive mutants with disruptions of 47 different genes, we found that dnaQ insertion mutants are specifically deficient in the SOS response to nalidixic acid. dnaQ encodes DNA polymerase III epsilon subunit, the proofreading subunit of the replicative polymerase. The deficient response to nalidixic acid was rescued by the presence of the wild-type dnaQ gene, confirming involvement of the epsilon subunit. To further characterize the SOS deficiency of dnaQ mutants, we analyzed the expression of several additional SOS genes in response to nalidixic acid using real-time PCR. A subset of SOS genes lost their response to nalidixic acid in the dnaQ mutant strain, while two tested SOS genes (recA and recN) continued to exhibit induction. These results argue that the replication complex plays a role in modulating the SOS response to nalidixic acid and that the response is more complex than a simple on/off switch.     

Transformation of Neisseria gonorrhoeae: physical requirements of the transforming DNA

The 1600-bp (base pair) fragment encoding a portion of the nalidixic acid resistant DNA gyrase, subunit B, was characterized to determine what parameters effect transformation in the gonococcus. When this DNA (pSY2) was isolated from Escherichia coli, it was able to transform a variety of gonococcal strains to resistance to nalidixic acid via DNA-mediated transformation, irrespective of their restriction-modification phenotype. Nalidixic acid resistant transformants contained no plasmid DNA sequences that corresponded to the vector, as measured by plasmid screening procedures and colony hybridization techniques. Supercoiled and linear DNA transformed the gonococcus at the same efficiency. DNA fragments as small as 615 bp were able to transform the gonococcus. The presence of a 10-bp uptake sequence enhanced a DNA fragment's ability to transform the gonococcus by four orders of magnitude. When the fragment encoding the nalidixic acid resistant DNA gyrase was subcloned into M13mp18, both the replicative form and the single-stranded form of the phage were able to transform the gonococcus to nalidixic acid resistance.     

WQ-3810 exerts high inhibitory effect on quinolone-resistant DNA gyrase of Salmonella Typhimurium

ABSTRACT

The inhibitory effect of WQ-3810 on DNA gyrase was assayed to evaluate the potential of WQ-3810 as a candidate drug for the treatment of quinolone resistant Salmonella Typhymurium infection. The inhibitory effect of WQ-3810, ciprofloxacin and nalidixic acid was compared by accessing the drug concentration that halves the enzyme activity (IC₅₀) of purified S. Typhimurium wildtype and mutant DNA gyrase with amino acid substitution at position 83 or/and 87 in subunit A (GyrA) causing quinolone resistance. As a result, WQ-3810 reduced the enzyme activity of both wildtype and mutant DNA gyrase at a lower concentration than ciprofloxacin and nalidixic acid. Remarkably, WQ-3810 showed a higher inhibitory effect on DNA gyrase with amino acid substitutions at position 87 than with that at position 83 in GyrA. This study revealed that WQ-3810 could be an effective therapeutic agent, especially against quinolone resistant Salmonella enterica having amino acid substitution at position 87.     

Some properties of subunits of DNA gyrase from Pseudomonas aeruginosa PAO1 and its nalidixic acid-resistant mutant.

Subunits A and B of DNA gyrase were purified from Pseudomonas aeruginosa PAO1 and its mutant, which was resistant to nalidixic acid. Inhibition tests of DNA gyrases reconstituted with a combination of subunits from the two strains showed that an alteration of subunit A but not subunit B caused bacteria to resist fluoroquinolones.     

DNA gyrase (Topoisomerase II) from Pseudomonas aeruginosa

DNA gyrase (Topoisomerase II) has been purified from Pseudomonas aeruginosa strain PAO. This enzyme is inhibited by novobiocin and nalidixic acid. DNA gyrase from P. aeruginosa is resistant to a much higher level of nalidixic acid than is Escherichia coli DNA gyrase. This increased level of resistance may explain, at least in part, the higher levels of natural resistance exhibited by P. aeruginosa toward nalidixic acid.     

Prophage induction by DNA topoisomerase II poisons and reactive-oxygen species: role of DNA breaks.

Various compounds were evaluated for their ability to induce prophage lambda in the Escherichia coli WP2s(lambda) microscreen assay. The inability of a DNA gyrase subunit B inhibitor (novobiocin) to induce prophage indicated that inhibition of the gyrase's ATPase was insufficient to elicit the SOS response. In contrast, poisons of DNA gyrase subunit A (nalidixic acid and oxolinic acid) were the most potent inducers of prophage among the agents examined here. This suggested that inhibition of the ligation function of subunit A, which also has a DNA nicking activity, likely resulted in DNA breaks that were available (as single-stranded DNA) to act as strong SOS-inducing signals, leading to prophage induction. Agents that both intercalated and produced reactive-oxygen species (the mammalian DNA topoisomerase II poisons, adriamycin, ellipticine, and m-AMSA) were the next most potent inducers of prophage. Agents that produced reactive-oxygen species only (hydrogen peroxide and paraquat) were less potent than adriamycin and ellipticine but more potent than m-AMSA. Agents that intercalated but did not generate reactive-oxygen species (actinomycin D) or that did neither (teniposide) were unable to induce prophage, suggesting that intercalation alone may be insufficient to induce prophage. These results illustrate the variety of mechanisms (and the relative effectiveness of these mechanisms) by which agents can induce prophage. Nonetheless, these agents may induce prophage by producing essentially the same type of DNA damage, i.e., DNA strand breaks. The potent genotoxicity of the DNA gyrase subunit A poisons illustrates the genotoxic consequences of perturbing an important DNA-protein complex such as that formed by DNA and DNA topoisomerase.     

Polyamines as modulators of gene expression under oxidative stress in Escherichia coli

Activity of enzymes of polyamine synthesis and contents of their products increased in E. coli cells in response to oxidative stress caused by addition of hydrogen peroxide to an exponentially growing culture. Putrescine and spermidine added to the culture medium in physiological concentrations significantly increased expression of genes oxyR and katG responsible for defense against oxidative stress, whereas cadaverine had no effect. The role of polyamines as modulators of the gene expression was confirmed by experiments with an inhibitor of polyamine synthesis, 1,3-diaminopropane, which decreased the level of cell polyamines and thus abolished the ability of the cell to induce oxyR expression under oxidative stress. A genetic method gave similar results: under oxidative stress mutants with disorders in polyamine synthesis displayed a significantly decreased level of induction of the oxyR and katG genes, and this level was recovered on addition of putrescine. In the presence of inhibitors of DNA-gyrase, nalidixic acid and novobiocin, the oxyR expression depended on the extent of DNA supercoiling. Putrescine decreased the inhibitory effects of nalidixic acid and novobiocin, and this confirmed its properties of a stimulator of DNA supercoiling. Resistance to rifampicin was studied to exemplify the mutation rate under oxidative stress. Putrescine decreased twofold the level of mutations and increased the number of viable cells in the culture exposed to oxidative stress.     

Requirement of DNA Gyrase for the initiation of chromosome replication in Escherichia coli K-12

Summary It has been found that strains carrying mutations in the dnaA gene are unusually sensitive to COU, NAL or NOV, which are known to inhibit DNA gyrase activities. The delay in the initiation of chromosome replication after COU treatment has been observed in cells with chromosomes synchronized by amino acid starvation or by temperature shift-up (dnaA46). The unusual sensitivity of growth to COU of the initiation mutant runs parallel to a higher sensitivity to the drug of the initiation of chromosome replication.The double mutant, dnaA46 cou-110 has been isolated and mutation cou-110 conferring resistance of growth, initiation and elongation of chromosome replication to COU was mapped in the gene coding for the subunit of DNA gyrase. The reduced frequency of appearance of the mutants resistant to COU, NAL or NOV in the initiation mutant suggests that some mutations in genes coding for DNA gyrase subunits cannot coexist with the dnaA46 mutation. The possible mechanisms of the requirement of DNA gyrase for dnaA-dependent initiation of E. coli chromosome are discussed.Abbreviations used COU coumermycin A₁ - NAL nalidixic acid - NOV novobiocin     

Effect of nalidixic acid and novobiocin on pBR322 genetic expression in Escherichia coli minicells.

The effects of two deoxyribonucleic acid (DNA) gyrase inhibitors, nalidixic acid and novobiocin, on the gene expression of plasmid pBR322 in Escherichia coli minicells were studied. Quantitative estimates of the synthesis of pBR322-coded polypeptides in novobiocin-treated minicells showed that the synthesis of a polypeptide of molecular weight of 34,000 (the tetracycline resistance protein) was reduced to 11 to 20% of control levels, whereas the amount of a polypeptide of 30,500 (the beta-lactamase precursor) was increased to as much as 200%. Nalidixic acid affected the synthesis of the tetracycline resistance protein similarly to novobiocin, although to a lesser extent. The effects of nalidixic acid were not observed in a nalidixic-resistant mutant; those induced by novobiocin were only partially suppressed in a novobiocin-resistant mutant. The synthesis of one of the inducible tetracycline-resistant proteins (34,000) coded by plasmid pSC101 was also reduced in nalidixic acid- and novobiocin-treated minicells. These results suggest that the gyrase inhibitors modified the interaction of ribonucleic acid polymerase with some promoters, either by decreasing the supercoiling density of plasmid DNA or by altering the association constant of the gyrase to specific DNA sites.     

Lethal fragmentation of bacterial chromosomes mediated by DNA gyrase and quinolones

When DNA gyrase is trapped on bacterial chromosomes by quinolone antibacterials, reversible complexes form that contain DNA ends constrained by protein. Two subsequent processes lead to rapid cell death. One requires ongoing protein synthesis; the other does not. The prototype quinolone, nalidixic acid, kills wild-type Escherichia coli only by the first pathway; fluoroquinolones kill by both. Both lethal processes correlated with irreversible chromosome fragmentation, detected by sedimentation and viscosity of DNA from quinolone-treated cells. However, only fluoroquinolones fragmented purified nucleoids when incubated with gyrase purified from wild-type cells. A GyrA amino acid substitution (A67S) expected to perturb a GyrA-GyrA dimer interface allowed nalidixic acid to fragment chromosomes and kill cells in the absence of protein synthesis; moreover, it made a non-inducible lexA mutant hypersusceptible to nalidixic acid, a property restricted to fluoroquinolones with wild-type cells. The GyrA variation also facilitated immunoprecipitation of DNA fragments by GyrA antiserum following nalidixic acid treatment of cells. The ability of changes in both gyrase and quinolone structure to enhance protein synthesis-independent lethality and chromosome fragmentation is explained by drug-mediated destabilization of gyrase-DNA complexes. Instability of type II topoisomerase-DNA complexes may be a general phenomenon that can be exploited to kill cells.     

Development of a direct viable count procedure for some Gram-positive bacteria

The direct viable count (DVC) is a procedure for enumerating viable-nonculturable cells. It should be noted, however, that bacteria demonstrating the viable but nonculturable phase have to date included only Gram-negative species, mainly because the DVC procedure does not lend itself to the analysis of Gram-positive bacteria since the DVC procedure is dependent on the bacterium being sensitive to nalidixic acid. The authors report here concerning studies on an analogous procedure for the direct enumeration of viable-nonculturable Gram-positive bacteria.
To facilitate a differential DVC for Gram-positive bacteria, ciprofloxacin, enoxacin, norfloxacin or isopropyl cinodine were substituted for nalidixic acid. These antibiotics were chosen because, like nalidixic acid, they are DNA gyrase inhibitors. The concentrations used for each antibiotic were 1000 μg ml^-1, 100 μg ml^-1 and 10 mg ml^-1. Pure cultures of Staphylococcus aureus, Enterococcus faecalis, Streptococcus agalactiae, Listeria monocytogenes and Bacillus subtilis were obtained from the culture collection at the University of Wyoming and a faecal streptococcus was isolated from the Laramie wastewater treatment plant. An antibiotic and optimal concentration thereof was found which gave enlarged cells for all the organisms except the faecal streptococcus isolated from the wastewater plant for which no enlarged cells were ever seen. The antibiotic and concentration thereof which gave the optimal percent enlarged cells in the DVC procedure varied between organisms.     

Effects of inhibition of the B subunit of DNA gyrase on conjugation in Escherichia coli.

Antagonism of the DNA gyrase B subunit in the donor bacterium by coumermycin or thermal inactivation inhibited transfer of plasmid R64drd-11. Coumermycin also inhibited Hfr transfer, with kinetics after drug removal suggesting that transfer resumed from the point of inhibition, in contrast to inhibition with nalidixic acid, after which transfer reinitiated from the origin of transfer.     

Identification of DNA topoisomerases involved in immediate and transient DNA relaxation induced by heat shock inEscherichia coli

The linking number of plasmid DNA in exponentially growingEscherichia coli increases immediately and transiently after heat shock. The purpose of this study was to search for DNA topoisomerases that catalyze this relaxation of DNA. Neither introduction of atopA deletion mutation nor treatment of cells with DNA gyrase inhibitors affected the DNA relaxation induced by heat shock. Thus, DNA topoisomerase I and DNA gyrase are apparently not involved in the process. However, the reaction was inhibited by nalidixic acid or by oxolinic acid in thetopA mutant and the reaction was resistant to nalidixic acid in atopA mutant carrying, in addition, thenalA26 mutation. These results are interpreted as indicating that both DNA topoisomerase I and DNA gyrase are involved in the DNA relaxation induced by heat shock.