Fluorescence intensity is not only dependent on codon usage.

<p>Different pBCSJC001 derivative plasmids were constructed where the leucine codon in the fourth position of the i-tag (TTA) was replaced by the alternative leucine codons (TTG, CTT, CTA, CTG), and/or the glutamate codon in the fifth position of the i-tag (GAA) was replaced by the alternative glutamate codon (GAG). 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 (strain BCSMH030 used as a negative control), CTGGAG (frequency of 0.0002, strain BCSJC031), CTAGAG (frequency of 0.0002, strain BCSJC029), CTCGAG (frequency of 0.0003, strain BCSJC028), TTAGAG (frequency of 0.0004, strain BCSJC026), CTTGAG (frequency of 0.0004, strain BCSJC030), CTGGAA (frequency of 0.0005, strain BCSJC025), CTAGAA (frequency codon usage of 0.0006, strain BCSJC024), TTGGAG (frequency of 0.0006, strain BCSJC027), CTCGAA (frequency of 0.0007, strain BCSJC006), TTAGAA (frequency of 0.0010, strain BCSJC001), CTTGAA (frequency of 0.0011, strain BCSJC023) and TTGGAA (frequency of 0.0015, strain BCSJC022). At least 100 cells of each strain were quantified. Representative images of each strain are shown. Scale bar: 1 µm.</p

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

Bacterial strains and plasmids.

<p>Bacterial strains and plasmids.</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

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

<p>(<b>A</b>) 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) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113796#pone.0113796-Sabelnikov1" target="_blank">[18]</a><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113796#pone.0113796-Lacks3" target="_blank">[19]</a>. (<b>B</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). (<b>C</b>) 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 <i>S. pneumoniae</i> 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. (<b>D</b>) 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 <i>S. pneumoniae</i> 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.</p

Genes encoding the l-rhamnose biosynthesis pathway are distributed in listeriae and other bacterial species.

<p>Comparison of the genomic organization of the l-rhamnose pathway genes in the genus <i>Listeria</i> and other bacteria. The corresponding species and strains are indicated on the left (<i>Lmo</i>, <i>Listeria monocytogenes</i>; <i>Lin</i>, <i>Listeria innocua</i>; <i>Lse</i>, <i>Listeria seeligeri</i>; <i>Liv</i>, <i>Listeria ivanovii</i>; <i>Lwe</i>, <i>Listeria welshimeri</i>; <i>Smu</i>, <i>Streptococcus mutans</i>; <i>Mtu</i>, <i>Mycobacterium tuberculosis</i>; <i>Sen</i>, <i>Salmonella enterica</i> serovar Typhimurium; <i>Sfl</i>, <i>Shigella flexneri</i>; <i>Pae</i>, <i>Pseudomonas aeruginosa</i>) and listerial serotypes are indicated on the right. Genes are represented by boxed arrows and their names are provided for strain EGD-e. Operons are underlined by dashed arrows and homologs of the <i>rml</i> genes are shown with identical colors. Numbered gaps indicate the genetic distance (Mb, mega base pairs) between <i>rml</i> genes located far apart in the chromosome. Bacterial genomic sequences were obtained from NCBI database and chromosomal alignments assembled using Microbial Genomic context Viewer and Adobe Illustrator.</p

L-Rhamnosylation of <i>Listeria monocytogenes</i> Wall Teichoic Acids Promotes Resistance to Antimicrobial Peptides by Delaying Interaction with the Membrane

<div><p><i>Listeria monocytogenes</i> is an opportunistic Gram-positive bacterial pathogen responsible for listeriosis, a human foodborne disease. Its cell wall is densely decorated with wall teichoic acids (WTAs), a class of anionic glycopolymers that play key roles in bacterial physiology, including protection against the activity of antimicrobial peptides (AMPs). In other Gram-positive pathogens, WTA modification by amine-containing groups such as D-alanine was largely correlated with resistance to AMPs. However, in <i>L</i>. <i>monocytogenes</i>, where WTA modification is achieved solely <i>via</i> glycosylation, WTA-associated mechanisms of AMP resistance were unknown. Here, we show that the L-rhamnosylation of <i>L</i>. <i>monocytogenes</i> WTAs relies not only on the <i>rmlACBD</i> locus, which encodes the biosynthetic pathway for L-rhamnose, but also on <i>rmlT</i> encoding a putative rhamnosyltransferase. We demonstrate that this WTA tailoring mechanism promotes resistance to AMPs, unveiling a novel link between WTA glycosylation and bacterial resistance to host defense peptides. Using <i>in vitro</i> binding assays, fluorescence-based techniques and electron microscopy, we show that the presence of L-rhamnosylated WTAs at the surface of <i>L</i>. <i>monocytogenes</i> delays the crossing of the cell wall by AMPs and postpones their contact with the listerial membrane. We propose that WTA L-rhamnosylation promotes <i>L</i>. <i>monocytogenes</i> survival by decreasing the cell wall permeability to AMPs, thus hindering their access and detrimental interaction with the plasma membrane. Strikingly, we reveal a key contribution of WTA L-rhamnosylation for <i>L</i>. <i>monocytogenes</i> virulence in a mouse model of infection.</p></div

WTA l-rhamnosylation delays AMP interaction with the <i>Lm</i> plasma membrane.

<p>(<b>A</b>) Depolarization rate of <i>Lm</i> strains in response to CRAMP. Mid-exponential-phase bacteria pre-stained (15 min) with 30 μM DiOC₂(3) were challenged with 50 μg/ml CRAMP and changes in the membrane potential, expressed as the ratio of CRAMP-treated versus untreated samples, were monitored during 30 min. Data represent the mean±SD of three independent experiments. (<b>B</b>) SYTOX Green uptake kinetics of <i>Lm</i> strains in response to CRAMP-mediated membrane permeabilization. Exponential-phase bacteria were incubated (37°C) with PBS (white symbols) or 50 μg/ml CRAMP (black symbols), in the presence of 1 μM SYTOX Green, and the increase in green fluorescence emission was recorded over time. (<b>C and D</b>) Transmission electron microscopy analysis of the subcellular distribution of CRAMP in immunogold-labeled sections of mid-exponential-phase wild type and Δ<i>rmlACBD Lm</i> strains treated with 50 μg/ml CRAMP (15 min, 37°C). (C) Representative images of contrasted sections of <i>Lm</i> cells showing CRAMP-specific gold labeling (10-nm black dots). Scale bar: 0.2 μm. (D) Quantification of the subcellular partition of CRAMP labeling in wild type and Δ<i>rmlACBD Lm</i> strains, for two independent assays. The percentages of cell envelope- and cytoplasm-associated gold dots per bacterium were quantified (at least 90 cells per strain) and the results expressed for each strain as mean±SD. (<b>E and F</b>) Western blot analysis of levels of CRAMP bound to purified cell wall of different <i>Lm</i> strains. Purified cell wall (100 μg) was incubated with CRAMP (5 min), washed and digested overnight with mutanolysin. (E) Supernatants from mutanolysin-treated samples were resolved in 16% Tris-tricine SDS-PAGE and immunoblotted for CRAMP. The <i>Lm</i> cell wall-anchored protein InlA was used as loading control. (<b>F</b>) Quantification of the relative CRAMP levels represented as the mean±SD of four independent blots. *, <i>p</i>≤0.05; **, <i>p</i>≤0.01.</p

A functional <i>rml</i> operon is required for glycosylation of <i>Lm</i> WTAs with l-rhamnose.

<p>(<b>A</b>) Alcian blue-stained 20% polyacrylamide gel containing WTA extracts from logarithmic-phase cultures of different <i>Lm</i> strains. (<b>B–D</b>) HPAEC-PAD analyses of the sugar composition of the (B) WTA, (C) peptidoglycan and (D) cytoplasmic fractions isolated from the indicated <i>Lm</i> strains. Samples were hydrolyzed in 3 M HCl (2 h, 95°C), diluted with water and lyophilized before injection into the HPLC equipment. Standards for ribitol (Rib), l-rhamnose (Rha), glucosamine (GlcN), and muramic acid (Mur) were eluted under identical conditions to allow peak identification.</p