Of Wze resulted in proteins that were still fluorescent, although a significant decrease of about 40 in the intensity of the fluorescence signal was observed in both cases (Fig. 4). However, when the first 50 amino acids of the N-terminal region of Wze 22948146 were deleted, in strain BCSJC003, the expression of fluorescence was completely lost (Fig. 4). In order to determine the minimum size of the N-terminal region of Wze required for 
expression of fluorescence, S. pneumoniae R36A strain was transformed with plasmids encoding the first 3, 5, 7, 10 or 50 aminoacids of Wze, linked to the N-terminal of Citrine, and analyzed by fluorescence microscopy. Strains BCSJC008 and BCSJC009, expressing the Citrine protein fused to the first 3 or 5 amino acids of Wze, respectively, showed no fluorescence (Fig. 4). Strain BCSJC010, expressing the Citrine protein fused to the first 7 amino acids of Wze, showed a significant reduction in the fluorescent signal relative to that observed with the expression of the entire 57773-63-4 Wze-Citrine protein in strain BCSMH007 (Fig. 4). However, strains BCSJC001 and BCSJC002, expressing theCitrine protein fused to the first 10 or 50 amino acids of Wze, respectively, showed fluorescence levels similar to that obtained for strain BCSMH007, expressing the original Wze-Citrine fusion (Fig. 4). Expression of Wze aminoacids 11 to 50 fused to Citrine did not result in a fluorescent protein (Fig. 4), confirming that the first 10 amino acids of Wze were necessary and sufficient for expression of Citrine in S. pneumoniae. We have named this 10 amino acid tag, which improved protein expression in pneumococcal bacteria, “i-tag”. The increased fluorescence due to the presence of the i-tag fused to Citrine could be the result of higher mRNA or higher protein levels. We therefore quantified, by real-time PCR, the levels of mRNA encoding untagged Citrine, Wze-Citrine fusion and the various truncated forms of this fusion, in exponentially growing bacteria, relatively to the mRNA for the tetracycline resistance marker, encoded in the plasmid backbone. Fig. 5A shows that levels of the different mRNAs were not BTZ-043 site sufficiently different to explain the variability in fluorescence expression. However, analysis of Citrine protein levels in the same strains showed a correlation between strains in which Citrine protein could be detected and strains which were fluorescent, namely those encoding for Wze-Citrine (strain BCSMH007) and all that included the first 10 amino acids of the Wze fused to Citrine (strains BCSJC001, BCSJC002, BCSJC004 and BCSJC005). Taken together these results show that fusion of the i-tag to Citrine increased fluorescence levels due to increased protein levels and not increased mRNA levels. To determine if increased protein levels resulted from higher translation rates or increased protein stability, we generated a silent mutation in the sequence encoding the i-tag, fused to Citrine, and analyzed the fluorescence of the resulting constructs. We were able to identify mutations 
that did not alter the amino acid sequence of the tag, but resulted in loss of fluorescence, namely the substitution of UUA leucine codon by CUC (Fig. 6). Given that protein sequence was not altered, we can rule out the hypothesis that the itag acted by increasing stability of the fusion proteins. We also do not think that increased expression is due to the introduction of an additional ribosome-binding site, as previously reported by Halfmann and colleagues [22.Of Wze resulted in proteins that were still fluorescent, although a significant decrease of about 40 in the intensity of the fluorescence signal was observed in both cases (Fig. 4). However, when the first 50 amino acids of the N-terminal region of Wze 22948146 were deleted, in strain BCSJC003, the expression of fluorescence was completely lost (Fig. 4). In order to determine the minimum size of the N-terminal region of Wze required for expression of fluorescence, S. pneumoniae R36A strain was transformed with plasmids encoding the first 3, 5, 7, 10 or 50 aminoacids of Wze, linked to the N-terminal of Citrine, and analyzed by fluorescence microscopy. Strains BCSJC008 and BCSJC009, expressing the Citrine protein fused to the first 3 or 5 amino acids of Wze, respectively, showed no fluorescence (Fig. 4). Strain BCSJC010, expressing the Citrine protein fused to the first 7 amino acids of Wze, showed a significant reduction in the fluorescent signal relative to that observed with the expression of the entire Wze-Citrine protein in strain BCSMH007 (Fig. 4). However, strains BCSJC001 and BCSJC002, expressing theCitrine protein fused to the first 10 or 50 amino acids of Wze, respectively, showed fluorescence levels similar to that obtained for strain BCSMH007, expressing the original Wze-Citrine fusion (Fig. 4). Expression of Wze aminoacids 11 to 50 fused to Citrine did not result in a fluorescent protein (Fig. 4), confirming that the first 10 amino acids of Wze were necessary and sufficient for expression of Citrine in S. pneumoniae. We have named this 10 amino acid tag, which improved protein expression in pneumococcal bacteria, “i-tag”. The increased fluorescence due to the presence of the i-tag fused to Citrine could be the result of higher mRNA or higher protein levels. We therefore quantified, by real-time PCR, the levels of mRNA encoding untagged Citrine, Wze-Citrine fusion and the various truncated forms of this fusion, in exponentially growing bacteria, relatively to the mRNA for the tetracycline resistance marker, encoded in the plasmid backbone. Fig. 5A shows that levels of the different mRNAs were not sufficiently different to explain the variability in fluorescence expression. However, analysis of Citrine protein levels in the same strains showed a correlation between strains in which Citrine protein could be detected and strains which were fluorescent, namely those encoding for Wze-Citrine (strain BCSMH007) and all that included the first 10 amino acids of the Wze fused to Citrine (strains BCSJC001, BCSJC002, BCSJC004 and BCSJC005). Taken together these results show that fusion of the i-tag to Citrine increased fluorescence levels due to increased protein levels and not increased mRNA levels. To determine if increased protein levels resulted from higher translation rates or increased protein stability, we generated a silent mutation in the sequence encoding the i-tag, fused to Citrine, and analyzed the fluorescence of the resulting constructs. We were able to identify mutations that did not alter the amino acid sequence of the tag, but resulted in loss of fluorescence, namely the substitution of UUA leucine codon by CUC (Fig. 6). Given that protein sequence was not altered, we can rule out the hypothesis that the itag acted by increasing stability of the fusion proteins. We also do not think that increased expression is due to the introduction of an additional ribosome-binding site, as previously reported by Halfmann and colleagues [22.
