Dataset Information

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

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.

SUBMITTER: Beilharz K

PROVIDER: S-EPMC4579452 | biostudies-literature | 2015 Oct

REPOSITORIES: biostudies-literature

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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.

Beilharz Katrin K van Raaphorst Renske R Kjos Morten M Veening Jan-Willem JW

Applied and environmental microbiology 20150807 20

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, ...[more]

PMID: 26253684

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Project description:Streptococcus pneumoniae is a human pathogen responsible for the majority of childhood pneumonia and media otitis cases worldwide. The diversity of its capsular polysaccharides (CPS) results in more than 91 serotypes of which at least 23 are virulent. Various CPS conjugated to immunogenic carrier proteins are currently licensed and provide protection against the infection caused by the respective serotypes but not against new and emerging virulent serotypes. In this study, we considered the conserved protein antigen PsaA, the pneumococcal surface adhesin A, in order to overcome the limitations of CPS antigens. The PsaA was translationally fused to a polyhydroxybutyrate (PHB) synthase which mediated production of PsaA displayed on PHB inclusions in recombinant Escherichia coli. This suggested that the PsaA fusion to the PHB synthase did not interfere with PHB synthase activity and its ability to mediate formation of nano-sized inclusions composed of a PHB core surrounded by the PHB synthase fused to PsaA. Isolated PHB beads showed a negative surface charge. Transmission electron microscopy analysis suggested that the PsaA fusion to the PHB synthase reduced the size of PHB beads from about 500 nm to 100 nm. The integrity and antigenicity of the fusion protein attached to isolated PHB beads was confirmed by SDS-PAGE, tryptic peptide fingerprinting analysis using MALDI-TOF-MS/MS and immunoblotting using a monoclonal anti-PsaA antibody. Mice immunized with PsaA displaying PHB beads produced high and specific IgG levels dominated by IgG1 isotype. While IgG1 titer were similar between soluble and insoluble PsaA, the IgG2 titers were strongly increased upon vaccination with insoluble PsaA i.e. PsaA displayed on PHB beads. Particulate PsaA-PHB beads elicited IgG antibodies recognizing PsaA in whole cell lysates of seven different serotypes of S. pneumoniae. This study suggested that PHB beads are suitable carriers for PsaA in order to induce a significant and specific Th-2-type immune response.

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Dataset Information

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

Publications

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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