The <i>smpB-ssrA</i> Mutation Affects Survival of Mice Infected with <i>Y. pseudotuberculosis</i>

<div><p>(A) Thirty mice, in groups of five mice per strain in three independent experiments, were challenged via the orogastric route with 2 × 10⁹ CFU of wild-type (WT) or ΔBA strain.</p><p>(B) For complementation studies, a total of 16 mice, in groups of four mice per strain in two independent experiments, were infected via the orogastric route with wild-type, ΔBA (p<i>smpBssrA</i>), or ΔBA (pBR322<i>)</i> strains. Mice were monitored for at least 21 d post bacterial infection.</p></div

Wild-Type and ΔBA Strains Display Different Patterns of Tissue Colonization

<p>Groups of seven mice per strain were infected via the orogastric route with 2 × 10⁹ cells and sacrificed on day 4 of infection. Harvested tissues were processed for CFU determination as described in Materials and Methods. Each symbol represents the CFU contained in the indicated tissue sample from one mouse. Data were analyzed by Student <i>t-</i>test in order to determine statistical significance of CFU counts in Peyer's Patches (<i>p</i> < 0.051), MLN (<i>p</i> < 0.271), and spleen (<i>p</i> < 0.044). The <i>smpB-ssrA</i> mutant was found to be able to reach extra-intestinal sites but unable to proliferate efficiently within the MLN and spleen.</p

The ΔBA Mutant Strain Has Reduced Endogenous Levels of Effector Proteins

<p>Cultures of wild-type (WT) or ΔBA strains of <i>Y. pseudotuberculosis</i> were grown in low-calcium medium at 37 °C for 3.5 h. Bacteria were harvested and analyzed for endogenous levels of YopB, YopD, LcrV, LcrH, and YopE proteins by Western blot analysis using Yop-specific polyclonal antibodies.</p

The <i>smpB-ssrA</i> Mutation Affects Levels of Yop Transcripts

<div><p>(A) Relative mRNA levels of the indicated Yops were determined by quantitative real-time PCR as described in Materials and Methods. Each value represents the average of three independent experiments. Standard deviation bars are indicated on each column.</p><p>(B) Quantitative real-time PCR data were independently confirmed by Northern blot analysis of <i>yopB</i> mRNA. Total RNA samples were resolved on a 1% agarose-formaldehyde gel, transferred to nylon membranes, and probed with a biotin-labeled <i>yopB</i> specific probe.</p><p>WT, wild type.</p></div

SsrA-Tagging Variants Complement the ΔBA Mutant to Different Degrees

<div><p>(A) Cultures of wild-type (WT) or the ΔBA mutant strains were grown in low-calcium medium at 37 °C. Bacteria were removed by centrifugation and the secreted proteins were precipitated with TCA, resolved by electrophoresis on a 10%-polyacrylamide Tris-tricine gel, and stained with Coomassie Brilliant Blue.</p><p>(B) To measure motility, equal numbers of bacteria from each strain were inoculated onto 0.25% agar-T medium plates and images were obtained after incubation for 48 h at room temperature.</p><p>MW stds, protein molecular weight standards.</p></div

The ΔBA Mutant Shows Delayed Host Cell Cytotoxicity

<p>HeLa cells (A) were infected at an MOI of 20 with wild-type <i>Y. pseudotuberculosis</i> (B), Δ<i>yopE</i> strain used as a control (C), the ΔBA mutant (D), and the mutant strain complemented with the p<i>smpBssrA</i> plasmid (E) or control plasmid (F). Morphology of the infected HeLa cells was monitored at 30-min intervals under a phase-contrast microscope. Images were captured 2.5-h post infection.</p

Affect of <i>smpB-ssrA</i> Mutation on VirF and YmoA Protein Levels

<p>Cultures of wild-type (WT) or ΔBA strains of <i>Y. pseudotuberculosis</i> were grown in secretion non-permissive (lanes 1 and 2) or permissive medium (lanes 3 and 4) at 37 °C for 3.5 h as described in Materials and Methods. Bacteria were harvested and analyzed for VirF (A) and YmoA (B) proteins by Western blot analysis using specific polyclonal antibodies. Purified YmoA protein was used as a control (lane 5).</p

The ΔBA Mutant Has Impaired Motility

<div><p>(A) An equal number of cells from each bacterial strain were inoculated onto 0.25% agar-T medium plates and incubated for 48 h at room temperature.</p><p>(B) Wild-type and mutant cells were immobilized on grids, negatively stained with phosphotungstic acid, and analyzed using a JEOL JEM-1200EX II transmission electron microscope. Magnification = 12,000×; scale bar = 500 nm.</p><p>WT, wild type.</p></div

SmpB-SsrA Mediated Protein Tagging Activity Is Observed in <i>Y. pseudotuberculosis</i>

<div><p>(A) Schematic representation of the λ-cI-N-trpAt reporter construct encoded on the pKW540 plasmid and anticipated outcomes of protein tagging in wild-type (WT) or ΔBA strains.</p><p>(B) The λ-cI-N-trpAt reporter was induced in wild-type and mutant (ΔBA) strains, and protein samples were analyzed by Western blot using HRP-conjugated anti-H6 serum.</p><p>MW stds, protein molecular weight standards.</p></div