Abstract
Differential fluorescent labeling of bacteria has become instrumental for many aspects of microbiological research, such as the study of biofilm formation, bacterial individuality, evolution, and bacterial behavior in complex environments. We designed a variety of plasmids, each bearing one of eight unique, constitutively expressed fluorescent protein genes in conjunction with one of four different antibiotic resistance combinations. The fluorophores mTagBFP2, mTurquoise2, sGFP2, mClover3, sYFP2, mOrange2, mScarlet-I, and mCardinal, encoding for blue, cyan, green, green–yellow, yellow, orange, red, and far-red fluorescent proteins, respectively, were combined with selectable markers conferring tetracycline, gentamicin, kanamycin, and/or chloramphenicol resistance. These constructs were cloned into three different plasmid backbones: a broad host-range plasmid, a Tn5 transposon delivery plasmid, and a Tn7 transposon delivery plasmid. The utility of the plasmids and transposons was tested in bacteria from the phyla Actinobacteria, Proteobacteria, and Bacteroidetes. We were able to tag representatives from the phylum Proteobacteria at least via our Tn5 transposon delivery system. The present study enables labeling bacteria with a set of plasmids available to the community. One potential application of fluorescently-tagged bacterial species is the study of bacteria–bacteria, bacteria–host, and bacteria–environment interactions.
Highlights
Labeling bacterial strains using fluorescent proteins has been used in manifold investigations, such as the study of biofilm formation, bacterial individuality and evolution, and bacterial behavior in complex environments (Tolker-Nielsen and Molin, 2000; Monier and Lindow, 2005; Kroupitski et al, 2009; Tecon and Leveau, 2012; Diard et al, 2013; Remus-Emsermann et al, 2013b; Whitaker et al, 2017; Remus-Emsermann and Schlechter, 2018)
We show the heterologous expression of fluorescent proteins at the singlecell resolution in those environmental bacterial strains using fluorescence microscopy
E. coli carrying pMRE or pMRE-Tn5 plasmids were cultivated at 37◦C, whilst pMRE-Tn7 plasmid-carrying cells were cultivated at 30◦C to prevent plasmid loss
Summary
Labeling bacterial strains using fluorescent proteins has been used in manifold investigations, such as the study of biofilm formation, bacterial individuality and evolution, and bacterial behavior in complex environments (e.g., plant surfaces, soil, or the mammalian gut) (Tolker-Nielsen and Molin, 2000; Monier and Lindow, 2005; Kroupitski et al, 2009; Tecon and Leveau, 2012; Diard et al, 2013; Remus-Emsermann et al, 2013b; Whitaker et al, 2017; Remus-Emsermann and Schlechter, 2018) Such studies require the equipment of bacteria with bright, stable, fast maturing, and highly. Individual cells experience heterogeneous environments, and owing to their location, they might either be exposed to or protected from antibiotic pressure (Stewart and Costerton, 2001)
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