| The issue of antibiotic resistance has received considerable attention due to the problem of the emergence and rapid expansion of antibiotic-resistant pathogenic bacteria. Pseudomonas aeruginosa (P. aeruginosa) is a gram-negative opportunistic pathogen that continues to be a major cause of opportunistic nosocomial infections. It is also the important cause of chronic lung infections contributing to the death of patients with cystic fibrosis. One of the most important reasons for its prominence as a pathogen is that P. aeruginosa has high intrinsic resistance characteristics to many antibiotics. It is important to study insights into the mechanisms of resistance and develop new approaches to combat resistance of P. aeruginosa.To find such genes, a P. aeruginosa transposon insertion library of c.a.17000 clones was constructed and screened for resistant and hyper-sensitive mutants using seven antibiotics. The screen was initially carried out on agar plates containing different concentrations of these antibiotics. Colonies grown at MIC were collected as the resistant mutants, and those unable to grow at 1/2 MIC were considered hyper-susceptible mutants. Further tests were done in liquid medium with series of antibiotic concentrations and the exact MICs of these mutants were defined. Transposon insertions in 43 genes were found to cause a 3-fold or higher hypersusceptibility to at least one antibiotic. Some of the mutants exhibited at least threefold change in susceptibility to three antibiotics; they are mutants in PA4024, PA4342, and PA4456. Two mutants increased the bacterial resistance to carbenicillin by 128-fold. Among the disrupted genes identified in the mutants, two genes PA0426, PA4207 which encodes a Resistance-Nodulation-Cell Division multidrug efflux transporter and another one PA0424 which encodes multidrug resistance operon repressor MexR have an apparent role in P. aeruginosa resistance. Others show less obvious involvement in antibiotic resistance. The results indicate many genes previously unknown to be involved in antibiotic resistance are important for the intrinsic antibiotic resistance in P. aeruginosa, suggesting mechanisms other than membrane permeability and efflux pump play important roles in bacterial resistance to antibiotics. These mechanisms may be exploited as new antibiotic targets.ABC transporters are widespread among P. aeruginosa and comprise one of the largest protein families. It is clear that ATP-binding cassette (ABC) transporters play an important role in bacteria and living organisms. In order to find new target to eliminate the antibiotic resistance, we identified and characterized a mutant PA4456 encoding a putative ATP-binding component of ABC transporter essential for the PAO intrinsic resistance. Furthermore, the mutant deficient in this putative ATP binding component was analyzed. Conclusions withdrawn from the results were:(1) higher sensitive to tetracycline and greater accumulation of tetracycline compared to the WT using the florescence technique. (2) also sensitive to chloramphenicol, trimethoprim, ciprofloxacin, xylene, dimethy-formamide, and toluene. Complementation analyses indicated that PA4456 is important for the putative operon to involve in antibiotics transporter. These results strongly suggested that the predicted ATP-binding component (PA4456) is involved in antibiotic and organic transporter.In order to find out the network of genes, a genome-wide search of regulators for the transporter operon identified PhoQ as a potential repressor. More than 8-fold increase of PA4456 expression was observed in a phoQ mutant. These results indicate that this putative ABC transporter is functionally active in P. aeruginosa and regulated by the two component system of PhoQ.It has already been show that antibiotic resistance can produce specific bacterial ways of life and changes in bacterial metabolism. It is thus conceivable that resistance has integrated into global regulatory networks and might be controlled by the metabolic condition of bacteria.
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