Construction of improved tools for protein localization studies in Streptococcus pneumoniae

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.

Figure 1. Fluorescent signals emitted by mCherry, Citrine, CFP and GFP protein fusions are detectable in live S. pneumoniae bacteria.

(A) The septal localization of Wze protein fusions expressed in the encapsulated ATCC6314 strain is shown for Wze-mCherry (strain BCSMH015), Wze-Citrine (strain BCSMH016), Wze-CFP (strain BCSMH029), Wze-GFP (strain BCSMH030). No comparable signal was detected in cells containing an empty vector (strain BCSMH052) visualized using appropriate filters for each fluorescent protein. (B) Three cultures of unencapsulated R36A strain expressing Wze-mCherry (strain BCSMH006, red dots), Wze-Citrine (strain BCSMH007, yellow dots) and Wze-CFP (strain BCSMH035, blue dots) were prepared for fluorescence microscopy observation as described in the Materials and Methods section, mixed on the same slide, and visualized using appropriate filters for each fluorescent protein. Fluorescence intensity was plotted, showing that the signals from each protein do not overlap. A representative image is shown at the bottom of the figure. Exposure times were 5 sec. Scale bar: 2 µm.

Figure 2. Fluorescent signals emitted by untagged mCherry, Citrine, CFP and GFP proteins are not detectable.

The median intensity, with 25% (white error bars) and 75% (black error bars) inter-quartile range (in arbitrary units) of the fluorescence signal in unencapsulated bacteria expressing Wze-mCherry (strain BCSMH006), mCherry (strain BCSMH032), Wze-Citrine (strain BCSMH007), Citrine (strain BCSMH033), Wze-CFP (strain BCSMH033), CFP (strain BCSMH034), Wze-GFP (strain BCSMH035) and GFP (strain BCSMH036), is plotted. At least 100 cells of each strain were quantified. Representative images of each strain are shown at the bottom. Scale bar: 2 µm.

Figure 3. Linker region 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 BCSMH040–043) and mCherry alone (strain BCSMH032) is plotted. At least 100 cells of each strain were quantified.

Figure 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 constructs that still carry the first 10 aminoacids of Wze (P>0.05). A significant reduction of the fluorescent signal was observed in strains BCSJC004, BCSJC005 and BCSJC010 (*, P<0.01). (Bottom panel) A schematic representation of each Wze constructs is shown. White box represent the Citrine protein while gray boxes represent different regions of Wze protein (lighter grey – aminoacids 1 to 10, light grey – remaining N-terminus region between aminoacids 11 to 50, dark grew – central region between aminoacids 51–176, black box – C-terminus region between aminoacids 178–227. Strain name are indicated below.

Figure 5. Increased fluorescence resulting from the presence of the “i-tag” is due to increased protein levels.

(A) mRNA encoding Citrine was quantified by Real-time PCR in strains expressing specific aminoacid sequences from Wze fused to Citrine, relatively to the mRNA for the tetracycline resistance protein which is encoded in the plasmid backbone. Strains analyzed were BCSMH031 (transformed with an empty vector), BCSMH007 (expressing full Wze-Citrine), BCSMH033 (expressing Citrine), BCSJC003 (expressing Wze_(51–227)-Citrine), BCSJC004 (expressing Wze_{(1–50, 178–227)}-Citrine), BCSJC005 (expressing Wze_(1–177)-Citrine), BCSJC001 (expressing Wze_(1–10)-Citrine), BCSJC007 (expressing Wze_(11–50)-Citrine) and BCSJC002 (expressing Wze_(1–50)-Citrine). Cell extracts from these strains were separated by SDS-Page and analyzed using a Fluorescent Image Analyzer (B) and by Western-blot analysis using an antibody that recognizes Citrine protein (C), showing that fluorescence in strains containing the i-tag is due to increased protein levels and not to increased mRNA levels.

Figure 6. The nucleotide sequence of the i-tag determines the expression of fluorescence.

(Left panel) Median fluorescence, with 25% (white error bars) and 75% (black error bars) inter-quartile range (in arbitrary units) of the fluorescence emitted by Citrine from the following strains: BCSMH031 (empty plasmid), BCSMH033 (expressing Citrine), BCSJC001 (expressing Wze_(1–10)-Citrine), BCSJC006 (expressing Wze_(1–10)*-Citrine in which the sequence encoding for Leucine was changed from TTA to CTC). At least 100 cells of each strain were quantified. (Right panel) Sequence encoding aminoacids 3–6 of Wze present in Wze_(1–10)-Citrine and Wze_(1–10)*-Citrine.

Figure 7. New plasmids for S. pneumoniae cell biology studies.

(A) Map of the pBCS plasmids. Fluorescent protein refers to mCherry, Citrine, CFP or GFP, encoded by plasmids pBCSMH030, pBCSJC001, pBCSMH031 and pBCSMH032, respectively. ApaI and NaeI restriction sites, highlighted with an asterisk, are not available in plasmid pBCSMH030. repA, repB, plasmid replication genes. tet, tetracycline resistance marker. T, transcription terminator. P, promoter. S₁, stop codon in plasmid pBCSMH030. S₂, stop codon in plasmids pBCSJC001, pBCSMH031 and pBCSMH032. (B) Comparison of fluorescence emitted by strains expressing mCherry, Citrine, CFP and GFP alone, their improved i-tag versions and their Wze fusions. The median fluorescence, with 25% (white error bars) and 75% (black error bars) inter-quartile range (in arbitrary units) is plotted. At least 100 cells of each strain were quantified. Strain names are indicated below.

Figure 8. Applications of the developed tools for localization of S. pneumoniae proteins.

(A) Localization of the cell division protein FtsZ as a N-terminal fusion to CFP (CFP-FtsZ, strain BCSMH050) and to i-tagged CFP, (iCFP-FtsZ, strain BCSMH051). (B) Localization of the membrane Wzd protein as a N-terminal fusion to CFP (CFP-Wzd, strain BCSJF004) and to improved i-tagged CFP, (iCFP-Wzd, strain BCSJF003). (C) Localization of the Wze tyrosine kinase as a N-terminal fusion to Citrine (Citrine-Wze, strain BCSJF002) and to improved i-tag Citrine (iCitrine-Wze, BCSJF001). The i-tagged versions of the fluorescent reporters allowed the visualization of each protein at the expected sub-cellular region of bacteria, the division septum. Exposure times: 5 sec. Scale bar: 2 μm.