The Pseudomonas aeruginosa DNA gyrase gyrA gene was cloned and sequenced from strain PAO1. An open reading frame of 2,769 bp was found; it coded for a protein of 923 amino acids with an estimated molecular mass of 103 kDa. The derived amino acid sequence shared 67% identity with Escherichia coli GyrA and 54% identity with Bacillus subtUis GyrA, although conserved regions were present throughout the sequences, particularly toward the N terminus. Complementation of an E. coli mutant with a temperature-sensitive gyrA gene with the PAO1 gyrA gene showed that the gene is expressed in E. coli and is able to functionally complement the E. coli DNA gyrase B subunit. Expression of PAO1 gyrA in E. coli or P. aeruginosa with mutationally altered gyrA genes caused a reversion to wild-type quinolone susceptibility, indicating that the intrinsic susceptibility of the PAO1 GyrA to quinolones is comparable to that of the E. coli enzyme. PCR was used to amplify 360 bp of P. aeruginosa gyrA encompassing the so-called quinolone resistance-determining region from ciprofloxacin-resistant clinical isolates from patients with cystic fibrosis. Mutations were found in three of nine isolates tested; these mutations caused the following alterations in the sequence of GyrA Asp at position 87 (Asp-87) to Asn, Asp-87 to Tyr, and Thr-83 to Ile. The resistance mechanisms in the other six isolates are unknown. The results of the study suggested that mechanisms other than a mutational alteration in gyrA are the most common mechanism of ciprofloxacin resistance in P. aeruginosa from the lungs of patients with cystic fibrosis.The enzyme DNA gyrase is a type II DNA topoisomerase that is able to introduce negative superhelical coils into covalently closed bacterial DNA in an ATP-dependent process. Gyrase also plays a role in DNA replication, recombination, decatenation, and transcriptional regulation of some supercoiling-sensitive promoters (for a review, see reference 24). The gyrase holoenzyme is a tetramer of two A and two B subunits, the products of the gyrA and gyrB genes, respectively. The A subunits are responsible for DNA breakage and reunion, while the B subunits are the site of ATP hydrolysis. The enzyme has proven to be a good target for antimicrobial agents; the actions of the A subunits are inhibited by quinolones such as nalidixic acid and ciprofloxacin, while those of the B subunits are inhibited by coumarins such as coumermycin Al and novobiocin. The peptide antibiotic microcin B17 and the glycocinnamoylspermidine agent cinodine have also been shown to be inhibitors of DNA gyrase function (22,33). The contiguous gyrB-gyrA locus has been cloned and sequenced in Bacillus subtilis (19), Staphylococcus aureus (10), Mycoplasma pneumoniae (1), and Haloferax spp. (9). In Escherichia coli (32, 37), Klebsiella pneumoniae (3), Pseudomonas putida (23), Neisseria gonorrhoeae (31), and Campylobacter jejuni (34), the cloned gyrA and gyrB genes are not contiguous. The deduced amino acid sequences of the A and B subunits are very well conserved with...

Examination of morphological and biochemical changes in Escherichia coli KL-16 after exposure to ciprofloxacin revealed distinct concentration-dependent responses. At levels close to the MIC extensive filamentation was seen, whereas at the most bactericidal concentration cells were elongated but not filamented. At higher concentrations ovoid cells were seen. Some cells showed surface vacuoles, but no significant leakage of cell contents was detected. Filamentation and vacuolation were shown to make only a minor contribution to ciprofloxacin-induced cell death, and the data indicate that there are two or more mechanisms involved in the lethal action of ciprofloxacin.

Light-scattering diagrams (phase functions) from single living cells and beads suspended in an optical trap were recorded with 30-ms time resolution. The intensity of the scattered light was recorded over an angular range of 0.5-179.5 degrees using an optical setup based on an elliptical mirror and rotating aperture. Experiments revealed that light-scattering diagrams from biological cells exhibit significant and complex time dependence. We have attributed this dependence to the cell's orientational dynamics within the trap. We have also used experimentally measured phase function information to calculate the time dependence of the optical radiation pressure force on the trapped particle and show how it changes depending on the orientation of the particle. Relevance of these experiments to potential improvement in the sensitivity of label-free flow cytometry is discussed.

The effects of nalidixic acid and four fluoroquinolones on DNA, RNA, and protein synthesis in the presence and absence of 20 mg of chloramphenicol per liter were examined by comparing the killing kinetics, MIC, morphological response, and maximum concentration to induce recA in Eschenichia coli. All agents demonstrated paradoxical killing kinetics, in that above an optimum concentration the rate of bactericidal action was slower. Filamentation of E. coli AB1157 was observed with all quinolones up to the optimum bactericidal concentration. Addition of chloramphenicol reduced the bactericidal activity, inhibited filamentation, and abolished recA induction, but it had no effect on DNA synthesis inhibition by any of the agents. Excellent correlation was obtained between the concentration required to inhibit DNA synthesis by 50%, the MIC, the maximum concentration to induce recA, and the optimum bactericidal concentration. Evidence from this study and previously published data suggest that the primary mechanism of action of quinolones is independent of the SOS response and does not require active protein synthesis; however, induction of recA and SOS responses is consequential and enhances cell death.The precise mechanisms of action of quinolone (including fluoroquinolone) antimicrobial agents have yet to be determined; however, one of the major interactions of quinolones is that with the enzyme DNA gyrase (topoisomerase II). It has been shown by two assays (one that measures the conversion of relaxed plasmid DNA to its native supercoiled form and the other that measures the production of linear DNA in a cleavage reaction) that quinolones inhibit the supercoiling activity of DNA gyrase (10,15,32,36). It has been postulated that nalidixic acid inhibits the resealing of DNA that occurs at the replication fork catalyzed by DNA gyrase, thereby preventing supercoiling (11). It is proposed that the complex of quinolone plus DNA gyrase is bound to the DNA, forming a replication fork barrier and allowing the accumulation of gapped or single-stranded DNA (25), which has been shown to accumulate in nalidixic acid-treated Escherichia coli (7). Quinolones induce the SOS response (DNA repair mechanism [4,14,22,23]), the inducing signal for which is thought to be gapped DNA (25). While there is a large body of evidence that DNA gyrase is the major target of quinolones, measurements of the concentration of a quinolone required to inhibit in vitro supercoiling activity often produce values that are 10-fold higher than the MIC (10,16,27). The poor correlation is unlikely to be due to a partially denatured DNA gyrase preparation (A. Maxwell, personal communication).The concentration of a quinolone needed to inhibit in vivo DNA synthesis by 50% (IC50) and quinolone MICs show an excellent correlation (2; J. M. Diver, L. J. V. Piddock, and R. Wise, Proc. 15th Int. Congr. Chemother., abstr. 985, 1987), and it has therefore been hypothesized that the initial reaction in the cascade of events causing quinolone-induced * Corresponding author...

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