7 research outputs found

    Surface distribution of Bsp in <i>S.</i><i>agalactiae</i> NEM316 and derivatives.

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    <p>Bacteria were harvested in exponential phase (OD<sub>600</sub> = 0.3) and labeled with DAPI (blue) plus rabbit anti-Bsp pAb (green). The bacterial strains studied were: NEM316 (WT) (positive control); NEM316Δ<i>bsp</i>/pTCV-erm without insert (negative control); NEM316Δ<i>bsp</i>/pTCV-<i>erm</i> expressing recombinant Bsp with the signal peptide of CspA, Alp2, PilB, or Gbs0791. Data are representative of three independent experiments.</p

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

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    <p>Bacteria grown overnight in 10 ml of TH (OD<sub>600</sub>≈2) were diluted to get an initial OD<sub>600</sub> of 0.05 (1/40 dilution) and grown at 37°C until OD<sub>600</sub> reached 0.5 and re-diluted again in TH (1/10) until reaching an OD<sub>600</sub> of 0.5 and diluted again (1/10) before final collection at mid-exponential phase (OD<sub>600</sub> 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

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

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    <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 (OD<sub>600</sub> 0.3) or stationary (OD<sub>600</sub> 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

    Distribution of SecA at the surface of <i>S.</i><i>agalactiae</i> (GBS NEM316) and <i>S. pyogenes</i> (GAS M18).

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    <p>(A) Western blot image showing that the polyclonal antibody directed against <i>E. coli</i> SecA recognizes a band of approximately 90 kDa in both GAS M18 and GBS NEM316 strains. (B) Conventional immunofluorescence microscopy showing the differential distribution of SecA at the surface of GBS NEM316 versus GAS strain M18 collected in exponential and stationary phase of growth. Heterogeneity of SecA distribution was quantified by eye following analysis of randomly selected fields.</p

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

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    <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

    Surface localization of SrtA and unrelated cell wall-anchored proteins in <i>S.</i><i>agalactiae</i> NEM316.

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    <p>(A, B) Bacteria harvested in exponential phase (OD<sub>600</sub> = 0.3) were labeled with (A) DAPI (blue) or guinea pig anti-SrtA pAb (green) and (B) with rabbit pAb against PilB, Gbs0791, Alp2, or CspA (red).</p

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

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    <p>(A, B) IFM images of bacteria harvested in exponential phase (OD<sub>600</sub> = 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
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