Conventional immunofluorescence microscopy showing localization of SecA in the non-capsulated mutant Δ<i>cpsE</i> compared to the parental WT NEM316.

<p>(A) Immunofluorescence microscopy of bacteria harvested in mid-exponential phase and visualized with fluorescent vancomycin (green) or plus rabbit anti-SecA pAb (red). Note that SecA is more concentrated in the constricting septa and its neighboring region in a pattern very similar to that reported for the non-capsulated strain of <i>S. pneumoniae </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065832#pone.0065832-Tsui1" target="_blank">[31]</a>. (B) Immunofluorescence of bacteria harvested in mid-exponential phase and visualized with rabbit pAb against Bsp and PilB.</p

Uncoupling transcription and translation in the <i>pil1</i> operon.

<p>Quantitative RT-PCR were performed on RNAs extracted from UCN34 WT and Pil1+_var23 variant grown without or with chloramphenicol (8 µg/mL) to induce ribosome stalling in leader peptide mRNA. The expression levels were normalized using 16S rRNA as an internal standard and are indicated as the n-fold change with respect to untreated WT strain UCN34, expressed as means and standard deviations of at least three separate experiments. The gene <i>tanA</i> was used as a reference gene whose transcription is not affected by addition of chloramphenicol. Asterisks represent P values (*<i>P</i><0.05) evaluated using a Student <i>t</i> test. The location in the <i>pil1</i> operon of the four primer pairs used is indicated at the bottom.</p

Model of pilus regulation by an attenuation-like mechanism.

<p>(A) <i>S. gallolyticus</i> UCN34 WT displays a heterogeneous <i>pil1</i> expression and consists of two main subpopulations, a majority of low piliated cells (Pil1_low, 70%) and a minority of hyper piliated cells (Pil1_high, 30%). The Pil1_low cells, characterized by a basal expression of <i>pil1</i>, possess a regulatory leader peptide-encoding gene with 22 GCAGA repeats that ends 39 bp upstream the hairpin transcription terminator. In-frame addition/deletion of GCAGA did not modify the distance between the leader peptide stop codon and the terminator. In this case, most transcripts initiated at P<i>pil1</i> promoter end at this premature terminator and the observed low expression of <i>pil1</i> probably occurs by readthrough transcription. The Pil1_high cells, characterized by a strong expression of <i>pil1</i>, displayed out-of-frame addition/deletion of repeats resulting in the synthesis of longer regulatory peptides whose stop codon are located within or dowstream of the hairpin terminator. Translation of these long regulatory leader peptides enhances <i>pil1</i> genes transcription by preventing the formation of the transcription terminator. (B) Flow cytometry analysis of UCN34 WT and isogenic mutant derivatives (Δ<i>term</i>, ATG*, and 3 STOPs) labeled with anti-PilB pAb. The WT and mutant profiles are depicted by gray area and black lines, respectively. Note that, as expected, the UCN34 and “back to the WT” profiles, depicted by dotted lines, are almost entirely superimposable. (C) Immunolabeling screening of the Pil1+_var23 and Δ<i>term</i> strains with anti-PilB pAb. As opposed to the Pil1+_var23 variant, the Δ<i>term</i> mutant is homogeneous and only generates Pil1+ colonies. This is the consequence of the deletion of the transcription terminator that blocks this strain in the Pil1_high configuration. (D) Quantitative RT-PCR were performed on RNAs extracted from WT UCN34 and mutant derivatives using pilB primers. The expression levels were normalized using 16S rRNA as an internal standard and are indicated as the n-fold change with respect to WT UCN34, expressed as means and standard deviations of at least three independent experiments with four technical replicates. Asterisks represent P values (** <i>P</i><0.05) evaluated using a Student <i>t</i> test.</p

Expression in GBS of Bsp recombinant proteins with structurally unrelated signal peptides.

<p>(A) <i>Bam</i>HI-<i>Not</i>I PCR fragments carrying the ribosome binding site (RBS) and the signal peptides (SP) of 5 SecA-dependent substrates (Bsp, Alp2, Gbs0791, PilB, and CspA) were fused in frame with a <i>Not</i>I-<i>Pst</i>I PCR fragment coding the Bsp protein devoid of its signal peptide (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065832#pone.0065832.s004" target="_blank">Table S1</a>). The resulting <i>Bam</i>HI-<i>Pst</i>I fragments were cloned downstream the constitutive P<i>tetM</i> promoter from the low-copy-number pTCV<i>-erm</i>. The SP and Bsp sequences are indicated in upper-bold italic characters and upper-bold characters, respectively. The boxed RP motif in all proteins corresponded to the translation of the two internal codons of the <i>Not</i>I restriction site (CGGCCG). All but one SP were predicted with SignalP 4.1 (<a href="http://www.cbs.dtu.dk/services/SignalP/" target="_blank">www.cbs.dtu.dk/services/SignalP/</a>) whereas the remaining (Alp2) was predicted with PrediSi (<a href="http://www.predisi.de" target="_blank">www.predisi.de</a>). The AA residues in the SP thought to direct localized secretion at the bacterial surface are indicated in red characters. Arrowheads indicate the predicted site of cleavage of the various SP. (B) Analysis of surface display of Bsp recombinant proteins in a Δ<i>bsp</i> mutant strain by immunoblotting. Whole bacterial cells harvested in exponential (OD₆₀₀ 0.3) or stationary (OD₆₀₀ 1.2) phases were washed, resuspended in phosphate buffer saline to similar density and spotted on nitrocellulose. Membranes were hybridized with specific anti-Bsp antibodies or with anti-GBS pAb (loading control). (C) Western blotting analysis of culture supernatants. Proteins were separated on 4–12% gradient Tris-acetate Criterion XT SDS-PAGE gel, then transferred onto a nitrocellulose membrane, and detected by immunoblotting with specific anti-Bsp and anti-CAMP antibodies. In (B) and (C), the Δ<i>bsp</i> mutant strain harboring pTCV-<i>erm</i> (negative control) or pTCV-<i>erm</i> directing synthesis of recombinant Bsp proteins associated with Alp2, Gbs0791, PilB, and CspA signal peptides were used.</p

Uptake of <i>S. gallolyticus</i> UCN34 WT, Δ<i>pil1</i>, and Δ<i>term</i> (Pil1+_locked) strains by human THP-1 macrophages.

<p>(A) Indirect immunofluorescence microscopy showing phagocytosis of Δ<i>pil1</i> or Δ<i>term</i> (Pil1+_locked) mutants by THP-1 macrophages in the presence of anti-Pil1 antibody (opsonophagocytic assay). Actin is depicted in grey-purple, Hoeschst 33342 in blue, intracellular bacteria in green, and extracellular bacteria in yellow (green+red). (B) Percentage of phagocytosis by THP-1 human macrophages after 1 h of infection with WT, Δ<i>pil1</i>, and Δ<i>term</i> (Pil1+_locked) strains, with or without an anti-Pil1 antibody. Values are representative of at least 4 independant experiments. (C) Flow-cytometry analysis of Pil1 expression performed on the UCN34 WT from the inoculum (grey) or from intracellular UCN34 bacteria recovered from THP-1 macrophages in an opsonophagocytic assay with an anti-PilB pAb (black lines are 3 replicates from one experiment).</p

Isolation, phenotypic analyses and sequencing of Pil1+ variants.

<p>(A) Detection of Pil1+ variants by colony blot using anti-PilB pAb. Isolated bacteria on a TH agar plate (left) and corresponding replicate hybridized with anti-PilB pAb (right). Arrowheads indicate the few variants displaying a Pil1+ phenotype. (B) Serial analysis of Pil1 expression in WT and Pil1+_var populations. Individual colonies of WT UCN34 strain isolated on TH agar plates were grown in 96 well plates, transferred to a Nylon membrane, and probed with anti-PilB. Analysis of one Pil1+ variant revealed that it subsequently generates a majority of Pil1+ colonies (90–95%) and a minority of Pil1_low cells (5–10%) thus displaying phenotypic pilus heterogeneity as the WT UCN34. (C–F) Phenotypic comparison of WT UCN34 and Pil1+_var23 variant. Scanning immunogold electron microscopy (C) and flow cytometry (D) analyses of bacteria labeled with anti-PilB pAb. Western blot analysis (E) of cell wall protein extracts revealed with anti-PilB pAb. Quantitative RT-PCR analysis (F) of RNAs extracted from exponentially growing <i>S. gallolyticus</i> cells. The expression levels were normalized using 16S rRNA as an internal standard and are indicated as the n-fold change with respect to the WT strain UCN34, expressed as means and standard deviations of at least three independent experiments with four technical replicates. Asterisks represent P values (*<i>P</i><0.05) evaluated using a Student <i>t</i> test. The location in the <i>pil1</i> operon of the four primer pairs used is indicated at the bottom. (G) Sequence and flow cytometry analyses of WT UCN34 and Pil1+_var strains. Frameshift addition/deletion GCAGA repeats in the leader peptide encoding gene did not modify the Pil1 expression profile of UCN34 WT whereas out-of-frame addition were always associated with a Pil1+ phenotype.</p

Heterogeneous expression of Pil1 pilus at the <i>Streptococcus gallolyticus</i> UCN34 surface.

<p>(A–B) Scanning immunogold electron microscopy and immunofluorescence of <i>S. gallolyticus</i> UCN34 strain and <i>L. lactis</i> NZ9000 pOri23Ω<i>pil1</i> strain expressing <i>pil1</i>. Pili were revealed with an anti-PilB polyclonal antibody (pAb) coupled to 10 nm gold beads (A) or the DyLight 488 rabbit secondary antibody (B). (C) Flow cytometry analysis of <i>S. gallolyticus</i> UCN34 cell populations labeled with anti-PilB pAb and the DyLight 488 rabbit secondary antibody. Results are represented as dot plots (left) or as a graph (right). The two groups (left) or peaks (right) in black correspond to the heterogeneous UCN34 WT strain constituted of a weakly piliated subpopulation (Pil1_low cells) and of a hyper piliated subpopulation (Pil1_high cells) representing 67% and 28% of the total population respectively. The negative population in gray corresponds to the isogenic Δ<i>pil1</i> mutant (Pil1_neg).</p

Distribution of SecA in <i>S.</i><i>agalactiae</i> NEM316.

<p>Bacteria grown overnight in 10 ml of TH (OD₆₀₀≈2) were diluted to get an initial OD₆₀₀ of 0.05 (1/40 dilution) and grown at 37°C until OD₆₀₀ reached 0.5 and re-diluted again in TH (1/10) until reaching an OD₆₀₀ of 0.5 and diluted again (1/10) before final collection at mid-exponential phase (OD₆₀₀ of 0.3) to get a homogenous population of exponentially growing cells. Bacteria were pretreated with lysozyme (1 mg/mL final concentration) for 15 min at 37°C and then permeabilized with PBS-Triton X-100 (0.4%) for 5 min at RT, washed twice with PBS and then fixed with PBS containing 3% paraformaldehyde for 15 min at RT. (A) Differential interference contrast (DIC) and immunofluorescence microscopy (IFM) of bacteria harvested in mid-exponential phase and visualized with rabbit anti-SecA pAb (red) or fluorescent vancomycin (green) plus rabbit anti-SecA pAb (red). White arrows in the last panel indicate potential constriction septa. Image representative of at least 800 GBS chains analyzed (B) Deconvolution images of sequential z-sections (0.3 µm) of NEM316 cells labeled with rabbit anti-SecA pAb presented as maximum intensity projections.</p

Subcellular localization of Bsp and CAMP factor in <i>S.</i><i>agalactiae</i> NEM316.

<p>(A, B) IFM images of bacteria harvested in exponential phase (OD₆₀₀ = 0.3) and labeled with specific antibodies directed against Bsp (A) or CAMP factor (B) revealed with AlexaFluor 594- fluorescent secondary antibody (red). Outline of the cells was visualized by DIC and active zone of peptidoglycan synthesis with fluorescent vancomycin (green). (C) Signal peptides of Bsp and CAMP proteins. The amino acids constituting the YSIRK motif are highlighted in red. Schematic localization of vancomycin, Bsp, and CAMP factor at non-constricting septa.</p

Heterogeneous expression of Pil1 depends on its 5′ upstream region.

<p>(A–D) Flow cytometry profiles of the WT UCN34 strain, the isogenic Δ<i>pil1</i> mutant, and the Δ<i>pil1</i> complemented with pTCV<i>erm</i>-P<i>tet</i>-<i>pil1</i> or pTCV<i>erm</i>-P<i>pil1</i>-<i>pil1</i>. (A) The WT strain displays two cell subpopulations, a majority of Pil1_low (66%) and a minority of Pil1_high (27%). (B) The Δ<i>pil1</i> mutant that does not express the Pil1 pilus. (C) The Δ<i>pil1</i> mutant complemented with the plasmid pTCV<i>erm</i>-P<i>tet</i>-<i>pil1</i>, P<i>tet</i> being a constitutive promoter, displays a single population of highly piliated cells. (D) The Δ<i>pil1</i> mutant complemented with the plasmid pTCV<i>erm</i>-P<i>pil1</i>-<i>pil1</i>, P<i>pil1</i> being the entire <i>gallo2180-pilA</i> intergenic region, displays two subpopulations. (C–D) Immunofluorescence staining with anti-PilB pAb (green) confirms that only the P<i>pil1</i> promoter and downstream sequences restored the heterogeneous expression of the Pil1 pilus (Pil1_low, 76%; Pil1_high, 16%). Bacteria were stained with DAPI (blue).</p