Concurrent assessment of inner and outer membrane permeabilization and bacteriolysis in E. coli by multiple-wavelength spectrophotometry.
We developed a dual wavelength spectrophotometric assay that permitted beta-lactamase and beta-galactosidase activities to be measured concurrently in a single sample. We also constructed a target cell, E. coli ML-35p, that was substantially cryptic for its periplasmic beta-lactamase and cytoplasmic beta-galactosidase unless outer membrane (beta-lactamase) or inner membrane (beta-galactosidase) permeabilization occurred. By applying the spectrophotometric assay to whole target cells, we could ascertain the kinetics of inner and outer membrane permeabilization by biological agents, including serum, polymyxin B and mellitin. By monitoring the reactions at an additional wavelength, we could also follow the kinetics of serum-mediated bacteriolysis. These experiments illustrate the principle of multiple wavelength spectrophotometry and provide examples of its use to monitor and dissect the action of biological agents on a gram-negative bacterium.
D001433:Bacteriolysis / Q000187:drug effects; D001790:Blood Physiological Phenomena; D002463:Cell Membrane Permeability / Q000187:drug effects; D002473:Cell Wall / Q000187:drug effects / Q000502:physiology*; D002511:Cephalosporins / Q000378:metabolism; D002863:Chromogenic Compounds; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology / Q000502:physiology*; D006801:Humans; D008555:Melitten / Q000494:pharmacology; D009598:Nitrophenylgalactosides / Q000378:metabolism; D011112:Polymyxin B / Q000494:pharmacology; D013053:Spectrophotometry; D013379:Substrate Specificity; D001616:beta-Galactosidase / Q000378:metabolism
D002511:Cephalosporins; D002863:Chromogenic Compounds; D008555:Melitten; D009598:Nitrophenylgalactosides; C034313:pyridine-2-azo-4-dimethylaniline cephalosporin; D001616:beta-Galactosidase; D011112:Polymyxin B