Detection and quantification of membrane-damaging antimicrobials using pHluorin2-based bacterial biosensors

Bacteriocins are ribosomally synthesized antimicrobial peptides of bacterial origin. Many known bacteriocins kill susceptible bacteria by interacting with cell surface receptors and disrupting membrane integrity, leading to the collapse of pH homeostasis. The fluorescent protein pHluorin2 is a GFP-derivative characterized by two distinct excitation peaks at 400 and 475 nm that change in relative fluorescence intensities in a ratiometric manner in response to changes in pH. Bacteria expressing pHluorin2 show fluorescence profiles characteristic for their intracellular pH. When placed in buffer at a pH that is different from the intracellular pH by +/- 1-2 units, these bacteria are able to maintain pH homeostasis. However, when treated with compounds that disrupt membrane integrity such as many bacteriocins, the intracellular pH rapidly changes to the pH of the buffer resulting in a change in the fluorescence profile of pHluorin2 within seconds. This change can be assessed with any method that is able to detect fluorescence intensities at the two excitation maxima of the protein. Hence, pHluorin2-expressing bacteria can be used to measure the activity of membrane-damaging antimicrobials by orders of magnitude faster than with conventional, growth-dependent assays. The methods presented here describe construction and use of live biosensor bacteria that express pHluorin2 for investigation of membrane-damaging compounds such as bacteriocins. Applications of these biosensors include characterization, screening, and quantification by various methods comprising spectrophotometry in microtiter plate readers, microscopy, and flow cytometry. Additionally, the protocol introduces a procedure for preparation of ready-to-use assay plates with freeze-dried pHluorin2 biosensor bacteria.