Elevated levels of flagellin were present on the bacterial surface of some mutants with improved swimming and swarming motility.

<p>Flagellins were sheared from bacterial surface, analyzed by Western blotting with antibodies to FliC and FljB and blots were quantified by densitometry using Quantity One software. Surface expression of flagellins was normalized to the level on the surface of the wild type strain. Samples for each mutant were prepared in three independent experiments.</p

Confirmation of hypermotility phenotypes.

<p>Stationary cultures normalized by OD₆₀₀ were spotted on swimming or swarming agar along with wild type strain, ATCC14028s. Rough strain, ATCC 14028r, was used as a negative control on swarming agar. After 3.5 and 5 hours of incubation at 37°C on swimming or swarming plates, respectively, the diameter of each bacterial growth area was measured in three independent experiments, with each experiment performed in triplicate.</p

Pathway clustering of mutants with altered motility.

<p>*Based on COGs (Clusters of Orthologous Groups of protein).</p><p>**Mutants with previously known motility phenotype are shown in bold.</p><p>***Mutants in fimbrial genes are underlined.</p><p>Pathway clustering of mutants with altered motility.</p

Distribution of motility phenotypes in single gene knock out collection.

<p>Distribution of motility phenotypes in single gene knock out collection.</p

Histopathological changes in the cecum of infected chicks at day 3 post-infection.

<p>Groups of 3 White Leghorn chicks were inoculated intragastrically by gavage with 10⁹ CFU of the wild type <i>S</i>. Typhimurium 14028<b> </b>s strain, the ΔT6SS_SPI-6 mutant strain or the Δ<i>clpV</i> mutant strain. At day 3 post-infection the chicks were sacrificed and the ceca were excised, fixed, stained with hematoxylin and eosin, and analyzed for histopathological lesions. Representative images of stained sections (400X) and scores for histopathological lesions in the cecum of infected chicks are shown (-, no changes; +, mild; ++, strong; +++, severe). White arrows indicate heterophil infiltration.</p

Motility scores of single deletion mutants of <i>Salmonella</i> Typhimurium ATCC14028 on swimming (A) and swarming (B) agar.

<p>Stationary phase cultures were transferred to motility agar, and motility was scored after 3.5 and 5 hours of incubation at 37°C on swimming or swarming plates, respectively. Motility was scored on the scale from 0 to 10 with wild type motility equal to 5. Data are presented as average swimming or swarming score from experiments with triplicate samples, performed on three independent occasions. Dots located outside of shaded area indicate scores greater than 5.25 and lower than 1.25 to define mutants with increased or reduced (<25 % of wild type) motility.</p

Mutants with enhanced motility.

<p>* - Diameter of swimming and swarming rings were measured and compared to wild type. Results are shown as the mean of six independent experiments.</p><p>** - Bold indicates statistical significance, <i>p</i><0.05.</p><p>Mutants with enhanced motility.</p

Primers used in this study.

a<p>Italics indicate the region that anneals to the 5′ or 3′ end of the antibiotic resistance cassette used for the mutagenesis. Underline indicates <i>XbaI</i> restriction sites used for cloning an internal region of homology to T6SS of SPI-6 into R995 plasmid.</p

Distribution of <i>S.</i> Typhimurium 14028 s and SPI-6 T6SS mutants in the gastrointestinal tract and internal organs of orally infected chickens.

<p>Four-day-old White Leghorn chicks were infected by gavage with 10⁹ CFU of either the wild-type <i>S</i>. Typhimurium 14028<b> </b>s, the ΔT6SS_SPI-6 mutant or the Δ<i>clpV</i> mutant strains. After 1, 3 and 9 days post-infection, the chicks were humanely euthanized and the ileum, cecum, liver and spleen were aseptically removed. Tissues were homogenized and viable bacterial counts were determined. Data are mean values of log₁₀ CFU/g of tissue, from five animals at each time point.</p

<i>In vivo</i> competition between the ΔT6SS_SPI-6 mutants complemented <i>in trans</i> with T6SS_SPI-6 or T6SS_SPI-19 and the wild type <i>S</i>. Typhimurium 14028 s.

<p>Fifteen four-day-old White Leghorn chicks were orally infected with 10⁹ CFU of a mixture at a 1∶1 ratio of strains WT/R995, ΔT6SS_SPI-6/R995+SPI-6 and ΔT6SS_SPI-6/R995+SPI-19. At 1, 3 and 9 days post-infection groups of five chicks were sacrificed and the organs were excised, homogenized, and serially diluted for determination of bacterial loads. Bars represent the geometric mean of the log converted ratio of the mutant CFU to the wild type CFU normalized to the equivalent ratio in the inoculum. Error bars denote standard error. Statistical significance was determined using a two-tailed Student’s <i>t</i> test, and asterisks indicate statistically significant differences between normalized output ratios (*<i>P</i><0.05). ^‡Indicate statistically significant differences between normalized output ratios and the equivalent ratio in the inoculum (^‡<i>P</i><0.05).</p