Eract with Wzd or WzeExpression of Fluorescent GNF-7 site proteins in S.pneumoniaeFigure 3. Linker region 23388095 present at the N-terminal of fluorescent proteins does not impair expression of fluorescence. (A) Partial sequence of plasmid pBCSMH001 highlights the linker region (grey), the GFP-like termini region (green) and the encoded mCherry (red). Amino acids between the first methionine and the aminoacids indicated by the arrows were removed to generate plasmids pBCSMH024-027 encoding truncated forms of mCherry. (B) The median intensity, with 25 (white error bars) and 75 (black error bars) inter-quartile range (in arbitrary units) of the fluorescence signal obtained from strains expressing Wze-mCherry (strain BCSMH006), truncated mCherry forms (strains BCSMH040?43) and mCherry alone (strain BCSMH032) is plotted. At least 100 cells of each strain were quantified. doi:10.1371/journal.pone.0055049.gproteins, normally localized at the dividing septum of encapsulated strains [14]. The developed tools were used to localize different proteins in S. pneumoniae, namely FtsZ, a central protein for bacterial division; Wzd, a membrane protein, and Wze, a cytoplasmic tyrosine kinase, both of which localize at the bacterial division septum when expressed together in pneumococcal cells. Figure 8 shows that fusions of these proteins to the C-terminal of CFP or Citrine only produced a fluorescent signal when the improved versions, containing the “i-tag”, of the fluorescent proteins were used, and not with the normal untagged versions. It should be highlighted that these transformants also express from the native chromosomal locus, the non-fluorescent versions of the FtsZ, Wzd or Wze proteins, Vasopressin site presumably increasing the cellular concentration of these proteins. Although we have not seen any difference in the localization pattern of fluorescent derivatives of Wzd-Citrine or Wze-Citrine expressed in the absence or in the presence of thenon-fluorescent original proteins [14], this may not be the case for fluorescent derivatives of other pneumococcal proteins.Final remarksWe were able to significantly improve the expression of four fluorescent proteins, mCherry, Citrine, CFP and GFP in the Gram-positive bacteria S. pneumoniae, by designing a tag that increases translation efficiency of heterologous proteins. The set of plasmids encoding improved versions of these fluorescent proteins allows 15857111 the expression of both N- and C-terminal fluorescent fusions of pneumococcal proteins and should greatly facilitate cell biology studies in this important pathogen.Materials and Methods Bacterial strains and growth conditionsBacterial strains and plasmids used in this study are listed in Table S1. S. pneumoniae was grown in C + Y liquid medium [24] atExpression of Fluorescent Proteins in S.pneumoniaeFigure 4. The first ten amino acids of Wze are required to obtain high levels of fluorescence. (Upper panel) Median fluorescence, with 25 (white error bars) and 75 (black error bars) inter-quartile range (in arbitrary units) emitted by Citrine fused to specific aminoacid sequences for Wze, as indicated below the graphic, in S. pneumoniae R36A strain. Strain BCSMH031 was used as a negative control. At least 100 cells of each strain were quantified. Exposure time was 5 sec. Kruskal-Wallis analysis with Dunn’s multiple post-test comparison did not reveal significant differences between the BCSMH007 strain, expressing Wze-Citrine, and strains BCSJC001 and BCSJC002, expressing Citrine const.Eract with Wzd or WzeExpression of Fluorescent Proteins in S.pneumoniaeFigure 3. Linker region 23388095 present at the N-terminal of fluorescent proteins does not impair expression of fluorescence. (A) Partial sequence of plasmid pBCSMH001 highlights the linker region (grey), the GFP-like termini region (green) and the encoded mCherry (red). Amino acids between the first methionine and the aminoacids indicated by the arrows were removed to generate plasmids pBCSMH024-027 encoding truncated forms of mCherry. (B) The median intensity, with 25 (white error bars) and 75 (black error bars) inter-quartile range (in arbitrary units) of the fluorescence signal obtained from strains expressing Wze-mCherry (strain BCSMH006), truncated mCherry forms (strains BCSMH040?43) and mCherry alone (strain BCSMH032) is plotted. At least 100 cells of each strain were quantified. doi:10.1371/journal.pone.0055049.gproteins, normally localized at the dividing septum of encapsulated strains [14]. The developed tools were used to localize different proteins in S. pneumoniae, namely FtsZ, a central protein for bacterial division; Wzd, a membrane protein, and Wze, a cytoplasmic tyrosine kinase, both of which localize at the bacterial division septum when expressed together in pneumococcal cells. Figure 8 shows that fusions of these proteins to the C-terminal of CFP or Citrine only produced a fluorescent signal when the improved versions, containing the “i-tag”, of the fluorescent proteins were used, and not with the normal untagged versions. It should be highlighted that these transformants also express from the native chromosomal locus, the non-fluorescent versions of the FtsZ, Wzd or Wze proteins, presumably increasing the cellular concentration of these proteins. Although we have not seen any difference in the localization pattern of fluorescent derivatives of Wzd-Citrine or Wze-Citrine expressed in the absence or in the presence of thenon-fluorescent original proteins [14], this may not be the case for fluorescent derivatives of other pneumococcal proteins.Final remarksWe were able to significantly improve the expression of four fluorescent proteins, mCherry, Citrine, CFP and GFP in the Gram-positive bacteria S. pneumoniae, by designing a tag that increases translation efficiency of heterologous proteins. The set of plasmids encoding improved versions of these fluorescent proteins allows 15857111 the expression of both N- and C-terminal fluorescent fusions of pneumococcal proteins and should greatly facilitate cell biology studies in this important pathogen.Materials and Methods Bacterial strains and growth conditionsBacterial strains and plasmids used in this study are listed in Table S1. S. pneumoniae was grown in C + Y liquid medium [24] atExpression of Fluorescent Proteins in S.pneumoniaeFigure 4. The first ten amino acids of Wze are required to obtain high levels of fluorescence. (Upper panel) Median fluorescence, with 25 (white error bars) and 75 (black error bars) inter-quartile range (in arbitrary units) emitted by Citrine fused to specific aminoacid sequences for Wze, as indicated below the graphic, in S. pneumoniae R36A strain. Strain BCSMH031 was used as a negative control. At least 100 cells of each strain were quantified. Exposure time was 5 sec. Kruskal-Wallis analysis with Dunn’s multiple post-test comparison did not reveal significant differences between the BCSMH007 strain, expressing Wze-Citrine, and strains BCSJC001 and BCSJC002, expressing Citrine const.
