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. 2015 Oct;81(20):7244-52.
doi: 10.1128/AEM.02033-15. Epub 2015 Aug 7.

Red Fluorescent Proteins for Gene Expression and Protein Localization Studies in Streptococcus Pneumoniae and Efficient Transformation With DNA Assembled via the Gibson Assembly Method

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Red Fluorescent Proteins for Gene Expression and Protein Localization Studies in Streptococcus Pneumoniae and Efficient Transformation With DNA Assembled via the Gibson Assembly Method

Katrin Beilharz et al. Appl Environ Microbiol. .
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Abstract

During the last decades, a wide range of fluorescent proteins (FPs) have been developed and improved. This has had a great impact on the possibilities in biological imaging and the investigation of cellular processes at the single-cell level. Recently, we have benchmarked a set of green fluorescent proteins (GFPs) and generated a codon-optimized superfolder GFP for efficient use in the important human pathogen Streptococcus pneumoniae and other low-GC Gram-positive bacteria. In the present work, we constructed and compared four red fluorescent proteins (RFPs) in S. pneumoniae. Two orange-red variants, mOrange2 and TagRFP, and two far-red FPs, mKate2 and mCherry, were codon optimized and examined by fluorescence microscopy and plate reader assays. Notably, protein fusions of the RFPs to FtsZ were constructed by direct transformation of linear Gibson assembly (isothermal assembly) products, a method that speeds up the strain construction process significantly. Our data show that mCherry is the fastest-maturing RFP in S. pneumoniae and is best suited for studying gene expression, while mKate2 and TagRFP are more stable and are the preferred choices for protein localization studies. The RFPs described here will be useful for cell biology studies that require multicolor labeling in S. pneumoniae and related organisms.

Figures

FIG 1
FIG 1
Inducible RFP expression in Streptococcus pneumoniae. The scheme for RFP gene expression strains after integration of the PZn-rfp promoter fusion at the nonessential bgaA locus by double crossover is shown. Strains were constructed by transformation with plasmids pKB01-PZn-rfp that carry PZn-rfp (mCherry, mOrange2, tagRFP, or mKate2) and a tetracycline resistance marker between the homologous recombination sites (shown in gray; ′gatC and ′bgaA). Lollipops depict transcriptional terminators. Similar constructs where the PZn promoter was replaced with the PssbB promoter were also made (see Fig. S1 in the supplemental material).
FIG 2
FIG 2
Fluorescence intensities of the different red fluorescent proteins. (A) Western blot analysis of expression levels of RFPs in whole-cell extracts detected using anti-RFP antibody. (B) Mean fluorescence on the population level determined using a plate reader equipped with a standard RFP filter set. See Materials and Methods for details of the filters used. (C and D)Average fluorescence intensities on the single-cell level measured by fluorescence microscopy using an mCherry filter set and polychroic mirror QUAD2 (C) or a TRITC filter set and polychroic mirror QUAD1 (D). Values are in arbitrary units (A.U.). Single-cell fluorescence levels were determined using MicrobeTracker (20). Error bars depict the standard error of the mean. mOrange2 values are not shown in the plots since signals above background level could not be detected.
FIG 3
FIG 3
Fast generation of RFP fusions to FtsZ in S. pneumoniae. (A) Gibson assembly procedure (14) for construction of FtsZ-RFP-expressing strains. (B) Images show localization of FtsZ fused to TagRFP, mKate2, and mCherry. Phase-contrast (PC), RFP, and overlay (merge) images are shown. Scale bar, 2 μm. (C) Immunodetection of FtsZ-RFP fusion proteins in whole-cell extract by anti-RFP antibody. Asterisks indicate degradation products.
FIG 4
FIG 4
Double labeling with RFP and GFP in S. pneumoniae. (A) Localization of FtsZ-RFP (different RFP variants in the different panels) and GFP-StkP. (B) Localization of HlpA-RFP (different RFP variants in the different panels) and GFP-StkP. Phase-contrast (PC), RFP, and GFP images are shown, in addition to an overlay of GFP and RFP (merge). Scale bars, 2 μm.

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