Selective Proteomic Analysis of Cellular Subpopulations in Bacterial Biofilms

Fluorescence confocal microscopy images of a biofilm formed by the bacterial pathogen Pseudomonas aeruginosa. The red fluorescence comes from the dye SYTO Red, which stains all of the bacterial cells, and shows the tufted structure characteristic of many bacterial biofilms. The green fluorescence arises from an alkyne dye that reacts with proteins that contain the non-canonical amino acid azidonorleucine (Anl). Anl is incorporated into protein only in cells that contain a mutant form of the methionyl-tRNA synthetase, which is expressed under control of the stress promoter rpoS. Because Anl-labeled proteins can be separated from the rest of the protein pool and identified by mass spectrometry, the method represented here allows targeted proteomic analysis of sub-populations of stressed cells in complex biofilm structures. Scale bar: 50 microns.