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. 2014 Dec 2;9(12):e113796. doi: 10.1371/journal.pone.0113796

Figure 5. Expression of Citrine derivatives containing N-terminal tags is dependent on ribosome-binding site accessibility.

Figure 5

(A) Partial sequence of plasmid pBCSJC001 highlights the consensus promoter region, the ribossome-binding site, the proposed transcription start site (+1) and translation start site (AUG) [18] [19]. (B) The mean fluorescence measured for the different constructs where Citrine has been fused to different tags at its N-terminal end is plotted relatively to the predicted thermal stability (minimum free energy in kcal/mol) of the 5′ end of the mRNA (+1 to +45) structures. The values for some Citrine derivatives are highlighted: Citrine (expressed in strain BCSMH033), iCitrine (strain BCSJC001), WchA(1-10)-Citrine (strain BCSMH063), MurN(1-10)-Citrine (strain BCSJC012) and Wze(1-10)-Citrine with mutated leucine (CTC) and glutamate (GAG) codons (strain BCSJC028). (C) Replacing the wild-type CTC leucine codon in pBCSJC006 for the alternative TTG and TTA codons in the tag derived from the N-terminal end of MurN resulted in increased cell fluorescence. Median fluorescence, with 25% and 75% inter-quartile range (black lines) emitted by unencapsulated R36A S. pneumoniae strains expressing Citrine (used as a negative control, strain BCSMH033), MurN(1-10)-Citrine with CTC codon (strain BCSJC013), with TTG codon (strain BCSJC035) and with TTA (strain BCSJC036). At least 100 cells of each strain were quantified. (Left) Representative images of each strain as well as the peptide and nucleotide sequences of the N-terminal tag. Scale bar: 1 µm. (Right) Representation of the 5′-end mRNA structure. Ribosome binding site (Red), AUG codon (Green) and mutated nucleotides (black) are highlighted. (D) Mutating the ATA isoleucine codon in pBCSJC028 to the AGA arginine codon in the tag derived from the N-terminal end of Wze resulted in increased cell fluorescence. Median fluorescence, with 25% and 75% inter-quartile range (black lines) emitted by unencapsulated R36A S. pneumoniae strains expressing iCitrine (used as a positive control, strain BCSJC011), Wze(1-10)-Citrine with CTC leucine and glutamate GAG codons (strain BCSJC028), Wze(1-10)-Citrine with CTC leucine, glutamate GAG and arginine AGA codons (strain BCSJC047). At least 100 cells of each strain were quantified. (Left) Representative images of each strain as well as the peptide and nucleotide sequences of the N-terminal tag. Scale bar: 1 µm. (Right) Representation of the 5′-end mRNA structure. Ribosome binding site (Red), AUG codon (Green) and mutated nucleotides (black) are highlighted.