The pUltra plasmid series: A robust and flexible tool for fluorescent labeling of Enterobacteria.

Fluorescent labeling has been an invaluable tool for the study of living organisms and bacterial species are no exception to this. Here we present and characterize the pUltra plasmids which express constitutively a fluorescent protein gene (GFP, RFP, YFP or CFP) from a strong synthetic promoter and are suitable for the fluorescent labeling of a broad range of Enterobacteria. The amount of expressed fluorophore from these genetic constructs is such, that the contours of the cells can be delineated on the basis of the fluorescent signal only. In addition, labeling through the pUltra plasmids can be used successfully for fluorescence and confocal microscopy while unambiguous distinction of cells labeled with different colors can be carried out efficiently by microscopy or flow cytometry. We compare the labeling provided by the pUltra plasmids with that of another plasmid series encoding fluorescent proteins and we show that the pUltra constructs are vastly superior in signal intensity and discrimination power without having any detectable growth rate effects for the bacterial population. We also use the pUltra plasmids to produce mixtures of differentially labeled pathogenic Escherichia, Shigella and Salmonella species which we test during infection of mammalian cells. We find that even inside the host cell, different strains can be distinguished effortlessly based on their fluorescence. We, therefore, conclude that the pUltra plasmids are a powerful labeling tool especially useful for complex biological experiments such as the visualization of ecosystems of different bacterial species or of enteric pathogens in contact with their hosts.