Growth of wild type 4/74 and Δ<i>pspE</i>, Δ<i>glpE</i> and Δ<i>pspE/glpE</i> strains in the presence 0.01% SDS.

<p>Strains were grown in 100 ml flasks containing 20 ml M9 supplemented with 0.01% SDS. The growth of wild type and mutated strains was similar.</p

Survival of wild type 4/74 and Δ<i>pspE</i>, Δ<i>glpE</i> and Δ<i>pspE/glpE</i> strains in the presence of peroxynitrite treatment. Bacteria grown to logarithmic phase (OD_600nm = 0.04±0.01) were treated with 360 µM peroxynitrite for 15 min.

<p>Survival of bacteria was determined by calculating the number of bacteria after 15 min in relation to the number of bacteria at the beginning of the experiment. Results show mean values of at least three independent experiments ± SEM.</p

Growth of wild type and <i>glpE</i> and <i>pspE</i> mutant strains in M9 medium with and without GSNO.

<p>Bacteria grown to stationary phase in M9 were re-grown in M9 medium without supplement and in media with varying concentrations of GSNO. The figure shows results for growth in M9, M9+0.5 mM GSNO and M9+1 mM GSNO. Growth of the wild type (black), Δ<i>glpE</i> (blue), Δ<i>pspE</i> (green) and Δ<i>glpE</i>/Δ<i>pspE</i> (red) strains was monitored for 20 h at 37°C with intermediate shaking of the plate. The data show representative results of two independent replicates. Growth experiments showed similar growth of Δ<i>glpE</i>, Δ<i>pspE</i> and Δ<i>glpE</i>/Δ<i>pspE</i> strains compared to their isogenic wild type strain.</p

Growth of WT (4/74) and <i>pspE</i> and <i>glpE</i> mutated and complemented strains in M9 medium with 10 mM H₂O₂.

<p>Wild type (black), Δ<i>glpE</i> (blue), Δ<i>pspE</i> (green), Δ<i>glpE</i>/Δ<i>pspE</i> (red) and Δ<i>glpE</i>+<i>glpE</i> strains were inoculated to an OD₆₀₀ value of 0.05, H₂O₂ was added and growth was followed for 16 hours. Results shown are representative of two biological repeats.</p

Infection of J774 macrophages with wild type 4/74 and Δ<i>pspE</i>, Δ<i>glpE</i> and Δ<i>pspE/glpE</i> strains.

<p>J774A.1 macrophages were infected with complemented-opsonized bacteria in a MOI of 10 and incubated for 25 min. Extracellular bacteria were killed by treatment with 100 µg ml⁻¹ gentamicin. Bacteria were released from the macrophages 1 h p.i., 4 h p.i. and 24 h p.i. and the number of intracellular bacteria was determined by CFU ml⁻¹ calculations and was expressed relatively to CFU at T1. The results show mean values ± SEM of at least three independent replicates. The small inserts shows results of an internal control experiment conducted with a SPI-2 (<i>ΔssaV</i>) deficient strain showing the expected reduced intracellular survival/replication of the mutated strain.</p

Infection of epithelial INT-407 cells wild type 4/74 and Δ<i>pspE</i>, Δ<i>glpE</i> and Δ<i>pspE/glpE</i> strains.

<p>Monolayers of INT-407 cells were infected with bacteria grown to logarithmic phase in a MOI of 100 and incubated for 15 min. Extracellular bacteria were killed by treatment with 100 µg ml⁻¹ gentamicin. Number of intracellular bacteria was determined by CFU ml⁻¹ calculations and values were adjusted against values for the wild type. The results represent mean values of at least three independent experiments ± SEM. Significant difference (**p<0.01) was determined by one-sample t-test analysis.</p

Oligonucleotide sequences for PCR based amplification and sequencing.

<p>Oligonucleotide sequences for PCR based amplification and sequencing.</p

Bacterial strains and plasmids used in this study.

<p>Bacterial strains and plasmids used in this study.</p

Competitive indices of <i>S</i>. Typhimurium 4/74 wild type bacteria relative to mutant bacteria in the mouse spleen.

<p>C57/BL6 mice were infected i.p. with equal numbers of mutant and wild type bacteria (each 5×10³ CFU). After 4 to 6 days, mice were sacrificed and the spleen was removed. Serial dilutions were spotted on LB agar plates and number of wild type and mutant bacteria in a total of 100 colonies was further determined by selection of the resistance marker. Competitive indices (C.I.) were calculated as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070829#pone.0070829-Jelsbak1" target="_blank">[42]</a>. The results are shown as mean values ± STD based on the number of mice tested as indicated in brackets. Significant differences from 1.0 (**p<0.01) were determined by one-sample t-test analysis.</p

Gene organization and graphic representation of putrescine/spermidine transporters and biosynthesis pathways.

<p>(A) The putrescine and spermidine transporters are localized at three distinct loci; <i>potABCD</i> interrupted by <i>sifA</i>, <i>potFGHI</i>, and <i>potE</i>. The genotype of the transporter mutant (<i>potCD;I;E</i>) is indicated by an asterix above deleted genes. Below is shown a graphic presentation of the transporters with their substrate affinity indicated by p for putrescine and s for spermidine. Expression during infection of cell-cultures is indicated above genes <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036149#pone.0036149-Eriksson1" target="_blank">[36]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036149#pone.0036149-Hautefort1" target="_blank">[37]</a>. (B) The putrescine and spermidine biosynthesis genes are localized at five distinct genetic loci; <i>speA</i>, <i>speB</i>, <i>speC</i>, <i>speDE</i>, and <i>speF</i>. The genotype of the biosynthesis mutant (<i>speB;C;E;F</i>) is indicated by an asterix above deleted genes. Below is shown a graphic presentation of the biosynthesis pathways present in bacteria, reviewed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036149#pone.0036149-Shah1" target="_blank">[67]</a>. SAM: S-adenosylmethionine.</p