title: BEEP: An assay to detect bio-energetic and envelope permeability alterations in Pseudomonas aeruginosa.
abstract: We developed an effective and rapid assay to detect both bio-energetic and envelope permeability (BEEP) alterations of Pseudomonas aeruginosa. The assay is based on quantification of extracellular ATP in bacterial cultures using luciferase as a reporter. To demonstrate the validity of our assay we conducted a biased screen of a transposon insertion library in P. aeruginosa strain PAO1 in order to expedite the isolation of mutants with defects in bioenergetic pathways. We successfully isolated insertion mutants that were reduced for extracellular ATP accumulation and identified the corresponding mutations that caused the phenotype. Most of the genes identified from this analysis were associated with energy metabolism and several appeared to be potentially novel bioenergetic targets. In addition, we show that treatment of P. aeruginosa strain PAO1 with antibiotics that disrupt the bacterial cell envelope leads to greater extracellular ATP accumulation. In summary, increases in extracellular ATP accumulation above wild type levels indicated a perturbation of membrane permeability while decreases in extracellular ATP accumulation indicated defects in bioenergetics.
authors.affiliation: Department of Biological Sciences, Auburn University, AL 36849, United States. Electronic address: suhsang@auburn.edu.
authors.forename: Sang-Jin
authors.initials: SJ
authors.lastname: Suh
authors.affiliation: Division of Basic Medical Sciences, Mercer University School of Medicine, Macon, GA 31207, United States. Electronic address: shuman_j@mercer.edu.
authors.forename: Jon
authors.initials: J
authors.lastname: Shuman
authors.affiliation: Division of Basic Medical Sciences, Mercer University School of Medicine, Macon, GA 31207, United States. Electronic address: carroll_l@mercer.edu.
authors.forename: Leslie P
authors.initials: LP
authors.lastname: Carroll
authors.affiliation: Division of Basic Medical Sciences, Mercer University School of Medicine, Macon, GA 31207, United States. Electronic address: silo-suh_l@mercer.edu.
authors.forename: Laura
authors.initials: L
authors.lastname: Silo-Suh
mesh_terms: D000255:Adenosine Triphosphate / Q000032:analysis / Q000378:metabolism; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000235:genetics; D002462:Cell Membrane / Q000737:chemistry / Q000502:physiology*; D004251:DNA Transposable Elements; D004734:Energy Metabolism; D008156:Luciferases; D016296:Mutagenesis; D010539:Permeability; D011550:Pseudomonas aeruginosa / Q000187:drug effects / Q000235:genetics / Q000378:metabolism* / Q000502:physiology
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D004251:DNA Transposable Elements; D000255:Adenosine Triphosphate; D008156:Luciferases
keywords: Bioenergetics; Cell envelope; Extracelluar ATP; Gram negative; Pseudomonas aeruginosa