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Antimicrob Agents Chemother. 2016 Dec 27;61(1). pii: e00790-16. doi: 10.1128/AAC.00790-16. Print 2017 Jan.

Competitive Growth Enhances Conditional Growth Mutant Sensitivity to Antibiotics and Exposes a Two-Component System as an Emerging Antibacterial Target in Burkholderia cenocepacia.

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Department of Microbiology, University of Manitoba, Winnipeg, Manitoba, Canada.
Beijing Institute of Radiation Medicine, State Key Laboratory of Proteomics, Cognitive and Mental Health Research Center, Beijing, China.
Department of Mathematics and Statistics, University of Minnesota Duluth, Duluth, Minnesota, USA.
Department of Microbiology, University of Manitoba, Winnipeg, Manitoba, Canada
Department of Medical Microbiology and Infectious Disease, University of Manitoba, Winnipeg, Canada.


Chemogenetic approaches to profile an antibiotic mode of action are based on detecting differential sensitivities of engineered bacterial strains in which the antibacterial target (usually encoded by an essential gene) or an associated process is regulated. We previously developed an essential-gene knockdown mutant library in the multidrug-resistant Burkholderia cenocepacia by transposon delivery of a rhamnose-inducible promoter. In this work, we used Illumina sequencing of multiplex-PCR-amplified transposon junctions to track individual mutants during pooled growth in the presence of antibiotics. We found that competition from nontarget mutants magnified the hypersensitivity of a clone underexpressing gyrB to novobiocin by 8-fold compared with hypersensitivity measured during clonal growth. Additional profiling of various antibiotics against a pilot library representing most categories of essential genes revealed a two-component system with unknown function, which, upon depletion of the response regulator, sensitized B. cenocepacia to novobiocin, ciprofloxacin, tetracycline, chloramphenicol, kanamycin, meropenem, and carbonyl cyanide 3-chlorophenylhydrazone, but not to colistin, hydrogen peroxide, and dimethyl sulfoxide. We named the gene cluster esaSR for enhanced sensitivity to antibiotics sensor and response regulator. Mutational analysis and efflux activity assays revealed that while esaS is not essential and is involved in antibiotic-induced efflux, esaR is an essential gene and regulates efflux independently of antibiotic-mediated induction. Furthermore, microscopic analysis of cells stained with propidium iodide provided evidence that depletion of EsaR has a profound effect on the integrity of cell membranes. In summary, we unraveled a previously uncharacterized two-component system that can be targeted to reduce antibiotic resistance in B. cenocepacia.


Burkholderia; Gram-negative bacteria; Illumina; antibiotic profiling; antibiotic resistance; drug efflux; drug targets; essential genes; transposon mutant; two-component regulatory systems

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