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.