Construction of improved tools for protein localization studies in Streptococcus pneumoniae

PLoS One. 2013;8(1):e55049. doi: 10.1371/journal.pone.0055049. Epub 2013 Jan 22.

Abstract

We have constructed a set of plasmids that allow efficient expression of both N- and C-terminal fusions of proteins of interest to fluorescent proteins mCherry, Citrine, CFP and GFP in the Gram-positive pathogen Streptococcus pneumoniae. In order to improve expression of the fluorescent fusions to levels that allow their detection by fluorescence microscopy, we have introduced a 10 amino acid tag, named i-tag, at the N-terminal end of the fluorescent proteins. This caused increased expression due to improved translation efficiency and did not interfere with the protein localization in pneumococcal bacteria. Localizing fluorescent derivatives of FtsZ, Wzd and Wze in dividing bacteria validated the developed tools. The availability of the new plasmids described in this work should greatly facilitate studies of protein localization in an important clinical pathogen.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacterial Proteins / genetics
  • Genetic Vectors
  • Green Fluorescent Proteins / genetics
  • Humans
  • Luminescent Proteins / genetics
  • Microscopy, Fluorescence
  • Plasmids / genetics*
  • Recombinant Fusion Proteins* / genetics
  • Recombinant Fusion Proteins* / isolation & purification
  • Streptococcus pneumoniae / genetics*
  • Streptococcus pneumoniae / isolation & purification

Substances

  • Bacterial Proteins
  • Luminescent Proteins
  • Recombinant Fusion Proteins
  • red fluorescent protein
  • Green Fluorescent Proteins

Grant support

This study was funded by Fundação para a Ciência e Tecnologia (FCT), Lisbon, Portugal, through research grants PTDC/SAU-MII/75696/2006 and PTDC/BIA- MIC/100747/2008 awarded to SRF. The work performed at Instituto de Tecnologia Química e Biológica was supported additionally by FCT through grant PEst-OE/EQB/LA0004/2011. MXH and MJC were supported by FCT fellowships SFRH/BD/43797/2008 and SFRH/BPD/77758/2011, respectively. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.