Optimization of fluorescent tools for cell biology studies in Gram-positive bacteria.

The understanding of how Gram-positive bacteria divide and ensure the correct localization of different molecular machineries, such as those involved in the synthesis of the bacterial cell surface, is crucial to design strategies to fight bacterial infections. In order to determine the correct subcellular localization of fluorescent proteins in Streptococcus pneumoniae, we have previously described tools to express derivatives of four fluorescent proteins, mCherry, Citrine, CFP and GFP, to levels that allow visualization by fluorescence microscopy, by fusing the first ten amino acids of the S. pneumoniae protein Wze (the i-tag), upstream of the fluorescent protein. Here, we report that these tools can also be used in other Gram-positive bacteria, namely Lactococcus lactis, Staphylococcus aureus and Bacillus subtilis, possibly due to optimized translation rates. Additionally, we have optimized the i-tag by testing the effect of the first ten amino acids of other pneumococcal proteins in the increased expression of the fluorescent protein Citrine. We found that manipulating the structure and stability of the 5' end of the mRNA molecule, which may influence the accessibility of the ribosome, is determinant to ensure the expression of a strong fluorescent signal

The amino terminal end of different <i>S. pneumoniae</i> proteins ensures expression of the Citrine fluorescent signal when a conserved LE motif is present.

<p>(Left panel) Sequence of the first aminoacids of proteins WchA, MurM, MurN, Wze and Wzd that were linked to Citrine (shown as a white rectangle) are shown. Highlighted (black) are the conserved aminoacids L (leucine) and E (glutamic acid). (Right panel) Median fluorescence, with 25% and 75% inter-quartile range (black lines) of the fluorescence signal detected in <i>S. pneumoniae</i> R36A unencapsulated bacteria, in arbitrary units (A. U.), emitted by Citrine (BCSMH033), WchA_(1-10)-Citrine (BCSMH063), MurM_(1-10)-Citrine (BCSJC012), MurN_(1-10)-Citrine (BCSJC013), Wze_(1-10)-Citrine (BCSJC001) and Wzd_(1-10)-Citrine (BCSJC011). At least 100 cells of each strain were quantified. Representative images of each strain are shown. Scale bar: 1 µm.</p

Linking the i-tag to Citrine improves the expression of fluorescence in Gram-positive bacteria.

<p><b>A</b>) (Left panel) Median fluorescence, with 25% and 75% inter-quartile range (black lines) of the fluorescence signal detected in <i>S. pneumoniae</i> unencapsulated bacteria, in arbitrary units (A. U.), expressing Citrine (strain BCSMH033) or iCitrine (strain BCSJC001), and <i>L. lactis</i>, expressing Citrine (strain BCSJC039) and iCitrine (strain BCSJC040). At least 100 cells of each strain were quantified. Representative images of each strain are shown below the graph. Scale bar: 1 µm. (Right panel) Map of the pBCS plasmids. Fluorescent protein refers to Citrine and iCitrine. <i>repA</i>, <i>repB</i>, plasmid replication genes. <i>tet</i>, tetracycline resistance marker. T, transcription terminator. P, promoter. S2, stop codon. <b>B</b>) (Left panel) Median fluorescence, with 25% and 75% inter-quartile range (black lines) of the fluorescence signal emitted by Citrine and iCitrine detected in <i>S. aureus</i> bacteria, in arbitrary units (A. U.), expressing Citrine (strain BCSJC041) and iCitrine (strain BCSJC042), or <i>B. subtilis</i>, expressing Citrine (strain BCSJC043) or iCitrine (strain BCSJC044). At least 100 cells of each strain were quantified. Representative images of each strain are shown below the graph. Scale bar: 1 µm. (Right panel) Map of the pMAD plasmids. Fluorescent protein refers to Citrine and iCitrine. Unique restriction sites are indicated. <i>ermC</i>, erythromycin resistance marker.</p

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

<p>(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.</p

New plasmids for <i>S. pneumoniae</i> cell biology studies.

<p>(A) Map of the pBCS plasmids. Fluorescent protein refers to mCherry, Citrine, CFP or GFP, encoded by plasmids pBCSMH030, pBCSJC001, pBCSMH031 and pBCSMH032, respectively. <i>Apa</i>I and <i>Nae</i>I restriction sites, highlighted with an asterisk, are not available in plasmid pBCSMH030. <i>repA, repB</i>, plasmid replication genes. <i>tet</i>, 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.</p

Bacterial strains and plasmids.

<p>Bacterial strains and plasmids.</p

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

<p>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.</p

Applications of the developed tools for localization of <i>S. pneumoniae</i> proteins.

<p>(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.</p

Expression of the Citrine fluorescent signal is not dependent on the distance of the conserved LE motif to its N-terminal end.

<p>Aminoacid sequence of the different tags, containing the LE motif successively positioned 0 to 9 amino acids distant from the starting methionine, linked to Citrine (shown as a white rectangle) is shown. Median fluorescence, with 25% and 75% inter-quartile range (black lines) of the fluorescence signal emitted by the following unencapsulated bacteria <i>S. pneumoniae</i> R36A strains, in arbitrary units (A. U.): Empty plasmid (BCSMH030), Citrine (BCSMH033), i*(MLEPTIAQKKL)-Citrine (BCSJC014), i*(MPLETIAQKKL)-Citrine (BCSJC015), i*(MPTLEIAQKKL)-Citrine (BCSJC001), i*(MPTILEAQKKL)-Citrine (BCSJC016), i*(MPTIALEQKKL)-Citrine (BCSJC017), i*(MPTIAQLEKKL)-Citrine (BCSJC018), i*(MPTIAQKLEKL)-Citrine (BCSJC019), i*(MPTIAQKKLEL)-Citrine (BCSJC020), and i*(MPTIAQKKLLE)-Citrine (BCSJC021). The strains BCSMH030 and BCSMH033 were used as a negative control. At least 100 cells of each strain were quantified. Representative images of each strain are shown. Scale bar: 1 µm.</p

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

<p>(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.</p