Quinolones are potent antibacterial agents that specifically target bacterial DNA gyrase and topoisomerase IV. Widespread use of these agents has contributed to the rise of bacterial quinolone resistance. Previous studies have shown that quinolone resistance arises by mutations in chromosomal genes. Recently, a multiresistance plasmid was discovered that encodes transferable resistance to quinolones. We have cloned the plasmid-quinolone resistance gene, termed qnr, and found it in an integron-like environment upstream from qacE⌬1 and sulI. The gene product Qnr was a 218-aa protein belonging to the pentapeptide repeat family and shared sequence homology with the immunity protein McbG, which is thought to protect DNA gyrase from the action of microcin B17. Qnr had pentapeptide repeat domains of 11 and 28 tandem copies, separated by a single glycine with a consensus sequence of A͞C D͞N L͞F X X. Because the primary target of quinolones is DNA gyrase in Gram-negative strains, we tested the ability of Qnr to reverse the inhibition of gyrase activity by quinolones. Purified Qnr-His6 protected Escherichia coli DNA gyrase from inhibition by ciprofloxacin. Gyrase protection was proportional to the concentration of Qnr-His6 and inversely proportional to the concentration of ciprofloxacin. The protective activity of Qnr-His6 was lost by boiling the protein and involved neither quinolone inactivation nor independent gyrase activity. Protection of topoisomerase IV, a secondary target of quinolone action in E. coli, was not evident. How Qnr protects DNA gyrase and the prevalence of this resistance mechanism in clinical isolates remains to be determined.

Although quinolone resistance usually results from chromosomal mutations, recent studies indicate that quinolone resistance can also be plasmid mediated. The gene responsible, qnr, is distinct from the known quinolone resistance genes and in previous studies seemed to be restricted to Klebsiella pneumoniae and Escherichia coli isolates from the University of Alabama in Birmingham, where this resistance was discovered. In Shanghai, the frequency of ciprofloxacin resistance in E. coli has exceeded 50% since 1993. Seventy-eight unique ciprofloxacin-resistant clinical isolates of E. coli from Shanghai hospitals were screened for the qnr gene by colony blotting and Southern hybridization of plasmid DNA. Conjugation experiments were done with azide-resistant E. coli J53 as a recipient with selection for plasmid-encoded antimicrobial resistance (chloramphenicol, gentamicin, or tetracycline) and azide counterselection. qnr genes were sequenced, and the structure of the plasmid DNA adjacent to qnr was analyzed by primer walking with a sequential series of outward-facing sequencing primers with plasmid DNA templates purified from transconjugants. Six (7.7%) of 78 strains gave a reproducible hybridization signal with a qnr gene probe on colony blots and yielded strong signals on plasmid DNA preparations. Quinolone resistance was transferred from all six probe-positive strains. Transconjugants had 16-to 250-fold increases in the MICs of ciprofloxacin relative to that of the recipient. All six strains contained qnr with a nucleotide sequence identical to that originally reported, except for a single nucleotide change (CTA3CTG at position 537) encoding the same amino acid. qnr was located in complex In4 family class 1 integrons. Two completely sequenced integrons were designated In36 and In37. Transferable plasmid-mediated quinolone resistance associated with qnr is thus prevalent in quinoloneresistant clinical strains of E. coli from Shanghai and may contribute to the rapid increase in bacterial resistance to quinolones in China.

Stem cells divide both to produce new stem cells and to generate daughter cells that can differentiate. The underlying mechanisms are not well understood, but conceptually are of two kinds. Intrinsic mechanisms may control the unequal partitioning of determinants leading to asymmetric cell divisions that yield one stem cell and one differentiated daughter cell. Alternatively, extrinsic mechanisms, involving stromal cell signals, could cause daughter cells that remain in their proper niche to stay stem cells, whereas daughter cells that leave this niche differentiate. Here we use Drosophila spermatogenesis as a model stem cell system to show that there are excess stem cells and gonialblasts in testes that are deficient for Raf activity. In addition, the germline stem cell population remains active for a longer fraction of lifespan than in wild type. Finally, raf is required in somatic cells that surround germ cells. We conclude that a cell-extrinsic mechanism regulates germline stem cell behaviour.

Quinolone resistance normally arises by mutations in the chromosomal genes for type II topoisomerases and by changes in the expression of proteins that control the accumulation of quinolones inside bacteria. A novel mechanism of plasmid-mediated quinolone resistance was recently reported that involves DNA gyrase protection by a pentapeptide repeat family member called Qnr. This family includes two other members, McbG and MfpA, that are also involved in resistance to gyrase inhibitors. Purified Qnr-His 6 was shown to protect Escherichia coli DNA gyrase directly from inhibition by ciprofloxacin. Here we have provided a biochemical basis for the mechanism of quinolone resistance. We have shown that Qnr can bind to the gyrase holoenzyme and its respective subunits, GyrA and GyrB. The binding of Qnr to gyrase does not require the presence of the complex of enzyme, DNA, and quinolone, since binding occurred in the absence of relaxed DNA, ciprofloxacin, or ATP. We hypothesize that the formation of Qnr-gyrase complex occurs before the formation of the cleavage complex. Furthermore, there was a decrease in DNA binding by gyrase when the enzyme interacted with Qnr. Therefore, it is possible that the reaction intermediate recognized by Qnr is one early in the gyrase catalytic cycle, in which gyrase has just begun to interact with DNA. Quinolones bind later in the catalytic cycle and stabilize a ternary complex consisting of the drug, gyrase, and DNA. By lowering gyrase binding to DNA, Qnr may reduce the amount of holoenzyme-DNA targets for quinolone inhibition.Quinolones are synthetic compounds that have been used extensively for treatment of a variety of infectious diseases (12). Increasing use of fluoroquinolones has triggered an increase in bacterial resistance. At present, resistance to fluoroquinolones has been observed even in pathogens such as Escherichia coli that had been originally highly susceptible to this class of antibiotics. Previous studies have shown that quinolone resistance arises by mutations in the chromosomal genes for type II topoisomerases, the targets of quinolone action (6), and by changes in expression of efflux pumps and porins that control the accumulation of these agents inside the bacterial cell (29). A novel mechanism of plasmid-mediated quinolone resistance was recently reported that involves DNA gyrase protection by a protein from the pentapeptide repeat family called Qnr.Topoisomerases are a large group of enzymes found in all organisms and are involved in maintaining the topological state of DNA. Type II topoisomerases such as DNA gyrase cleave both strands of DNA to allow one double-stranded DNA molecule to pass through another, followed by religation of the original strand (18). Gyrase is responsible for the maintenance of steady-state levels of negative supercoiling and is essential for chromosome condensation, transcription initiation, and enzyme complex movement in replication and transcription (2).Gyrase, first discovered and characterized in 1976 (9), is only found in bacteria, an...