Rainbow Vectors for Broad-Range Bacterial Fluorescence Labeling

Abstract

Since their discovery, fluorescent proteins have been widely used to study protein function, localization or interaction, promoter activity and regulation, drug discovery or for non-invasive imaging. They have been extensively modified to improve brightness, stability, and oligomerization state. However, only a few studies have focused on understanding the dynamics of fluorescent proteins expression in bacteria. In this work, we developed a set plasmids encoding 12 fluorescent proteins for bacterial labeling to facilitate the study of pathogen-host interactions. These broad-spectrum plasmids can be used with a wide variety of Gram-negative microorganisms including Escherichia coli, Pseudomonas aeruginosa, Burkholderia cepacia, Bordetella bronchiseptica, Shigella flexneri or Klebsiella pneumoniae. For comparison, fluorescent protein expression and physical characteristics in Escherichia coli were analyzed using fluorescence microscopy, flow cytometry and in vivo imaging. Fluorescent proteins derived from the Aequorea Victoria family showed high photobleaching, while proteins form the Discosoma sp. and the Fungia coccina family were more photostable for microscopy applications. Only E2-Crimson, mCherry and mKeima were successfully detected for in vivo applications. Overall, E2-Crimson was the fastest maturing protein tested in E. coli with the best overall performance in the study parameters. This study provides a unified comparison and comprehensive characterization of fluorescent protein photostability, maturation and toxicity, and offers general recommendations on the optimal fluorescent proteins for in vitro and in vivo applications.

Fig 1. Radial Neighbor Joining tree of the fluorescent protein sequences selected in this study.

Fluorescent protein sequence alignments were analyzed using a Neighbor Joining radial tree. Analysis was performed using CLC Main Workbench v7.6.1. The distance between the nodes is indicated below the graph.

Fig 2. Fluorescent protein expression in E. coli: effects on bacterial fitness and protein maturation.

(A) E. coli strain E. cloni 10G harboring the plasmids listed in Table 1 were grown for 13 H in LB under constant shaking in a flat bottom clear 96 well plate. Absorbance at 600 nm was measured every 15 minutes using a Spectramax I3 fluorescence microplate reader (Molecular Devices, Sunnyvale, CA). (B) E. coli strain JM109 harboring the rainbow plasmids listed in Table 1 were grown in culture flasks under constant shaking for 12 H in LB. Fluorescent protein expression was then induced by adding 40 μM IPTG or PBS as control. Induction was maintained until the end of the experiment. Changes in absorbance and fluorescence were monitored using a Spectramax I3. Data are represented as percentage of the maximal fluorescence value reached during induction. Data were normalized using the absorbance at 600nm and analyzed with one sample t-test and the software Prizm 6.0. Significant changes in fluorescence compared to the non-induced control are denoted with an asterisk (*: p<0.05; **: p<0.01; ***: p<0.001).

Fig 3. Photobleaching during fluorescence microscopy imaging.

E. coli JM109 harboring the pUCP20T derived plasmids described in Table 1 were smeared on a microscopy slide, covered with a coverslip and imaged at x100 with immersion oil using a Zeiss Axioscop fluorescence microscope. Slides were excited continuously and images were acquired at regular intervals. Experiments were performed in triplicate and representative images are presented here.

Fig 4. Sensitivity to photobleaching.

E. coli JM109 harboring the pUCP20T derived plasmids described in Table 1 were smeared on a microscopy slide, covered with a coverslip and imaged at x100 with immersion oil using a Zeiss Axioscop fluorescence microscope. Slides were excited continuously and images were acquired at regular intervals. Fluorescence intensity was measured using ImageJ. Experiments were performed in triplicate and data statistically analyzed using unpaired two-tailed Student t-tests and the software Prizm 6.0.

Fig 5. Flow cytometry analysis of E. coli labeled with fluorescent proteins.

Bacterial suspensions of E. coli JM109 harboring the plasmids described in Table 1 were analyzed by flow cytometry using an LSRFortessa (BD). A. Bacterial fluorescence was measured with a constant voltage at the excitation wavelengths of 405 nm, 488 nm, 561 nm and 628 nm and the emission wavelengths of 450/50 nm, 515/30 nm, 525/50 nm, 585/15 nm, 610/20 nm, 620/20 nm, 670/30 nm, 695/40 nm, 730/45 nm and 780/60 nm. Data were analyzed with an unpaired two-tailed t-test and the software Prizm 6.0. Fluorescence values significantly higher than those of the unlabeled control bacteria are labeled in blue. No significant changes are shown in red. B. Spectral overlap represented as percentage of compensation was measured for each fluorescent protein at the excitation and emission wavelengths indicated in bold.

Fig 6. Bacterial fluorescence detection using an in vivo imaging system (IVIS).

E. coli strain JM109 harboring the plasmids listed in Table 1 were growth for 18 H on LA with 40 μM IPTG and 100 μg/ml carbenicillin. Plates were imaged using an IVIS Spectrum at the excitation and emission wavelength indicated on the figure. Fluorescence intensity is indicated on the right. The location of the strains harboring each plasmid on the plate is indicated on the bottom left.

Fig 7. Determination of fluorescence detection limits in vivo using a phantom mouse.

6.25x10⁵ to 1x10⁷ CFUs of E. coli strain E. cloni 10G were loaded into a capillary tube and inserted into the phantom mouse. The mouse was imaged at excitation and emission wavelengths ranging from 430 nm to 760 nm for each fluorescent protein tested using the IVIS spectrum (A). The scale for the number of photons detected is indicated on the right. (B). Photons detected in the regions of interest for each amount of bacteria. Measurement were performed in triplicate and data were analyzed using an unpaired two-tailed t-test and the software Prizm 6.0. (*: p<0.05; **: p<0.01; ***: p<0.001).