Confirmation of NO sensitivity.

<p>Individual Tn mutants were exposed to 1.25 mM DEA/NO or culture medium alone along with the parental strain 5A18NP1. Genomic equivalents were quantified following a three-day outgrowth period, and an outgrowth ratio was determined for each strain as the ratio of genomic equivalents in the treated sample compared to the untreated sample. See <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006225#ppat.1006225.s009" target="_blank">S5 Table</a> for Tn clone numbers. *, P < 0.01 compared to 5A18NP1; §, P < 0.05 compared to 5A18NP1 by 1-way ANOVA followed by Dunnett’s test. N.S., not significant.</p

Putative <i>B</i>. <i>burgdorferi</i> genes involved in RNS and ROS resistance.

<p>Putative <i>B</i>. <i>burgdorferi</i> genes involved in RNS and ROS resistance.</p

Frequency ratios of individual Tn mutants with insertions in putative ROS and RNS resistance genes.

<p>We prioritized genes for further study if they had overall frequency ratios <0.33 in both replicates after exposure to DEA/NO (A), TBHP (B), or H₂O₂ (C). The frequency ratios of individual Tn mutants with insertions in these genes are shown for both replicate 1 (black circles) and replicate 2 (gray circles). The median is indicated with a bar. Genes that were selected for further analysis are shown in blue.</p

Confirmation of ROS sensitivity.

<p>Individual Tn mutants, complemented mutants, and the parental strain were exposed to H₂O₂, TBHP, or culture medium alone. Bacterial cell density was quantified by dark-field microscopy following a three-day outgrowth period. An outgrowth ratio was determined for each strain as the ratio of cell numbers in the treated sample compared to the untreated sample. Results for 125 μM H₂O₂ (A) or 7.5 mM TBHP (B) are shown. *, P < 0.01 compared to 5A18NP1; §, P < 0.05 compared to 5A18NP1 by one-way ANOVA followed by Dunnett’s test. See <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006225#ppat.1006225.s009" target="_blank">S5 Table</a> for Tn clone numbers and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006225#ppat.1006225.s010" target="_blank">S6 Table</a> for a description of the complemented Tn mutants (Tn::<i>bb0017</i>-comp, DM104; Tn::<i>bb0164</i>-comp, JH511).</p

Infectivity of 39 Tn mutants in C57BL/6 and <i>gp91</i>^phox-/- mice.

<p>Three groups of six C57BL/6 and three groups of five or six <i>gp91</i>^{<i>phox</i>-/-} mice were infected with a mini Tn library containing 39 Tn mutants at a dose of 1×10⁵ bacteria. Pools of organs were collected from each group of mice and cultured in BSK-II medium to expand the <i>B</i>. <i>burgdorferi</i> population. The frequency of each Tn mutant in the three C57BL/6 organ pools and the three <i>gp91</i>^{<i>phox</i>-/-} organ pools was determined using Tn-seq. The boundaries of each box indicate the minimum and maximum values, with the median indicated by a bar. §, mutants are missing plasmids important for murine infection. T09TC420 (Tn::<i>bb0017</i>-2) is missing lp28-1. T07TC421 (Tn::<i>uvrD</i>-1) is missing lp36. Thus, the effects of the Tn insertion in these two mutants cannot be discerned. The arrow indicates the limit of detection of the assay. *, the Tn::<i>pncA</i> mutant included in the mini-library was subsequently found to contain a second Tn insertion in the <i>hk1</i> gene. Hk1 is not required for murine infection [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006225#ppat.1006225.ref047" target="_blank">47</a>].</p

Mn levels are decreased in the Tn::<i>bb0164</i> mutant compared to the parental strain.

<p>Cellular levels of Mn (A) or Zn (B) were determined using ICP-MS in the 5A18NP1 parental strain as well as in several Tn mutants and their respective complemented strains. Mn levels were also measured in the <i>bmtA</i> mutant and its parental 297 strain [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006225#ppat.1006225.ref022" target="_blank">22</a>]. All strains were grown to early stationary phase in BSK-II medium. Data represent the mean ± standard deviation of three independent experiments. *, P<0.0001 by one-way ANOVA followed by Tukey’s test. Quantitative reverse transcriptase PCR (RT-qPCR) was used to measure expression levels of <i>bmtA</i> (C) and <i>bb0164</i> (D) in the 5A18NP1, Tn::<i>bb0164</i>, 297, and <i>bmtA</i> strains. *, P<0.001 by one-way ANOVA followed by Tukey’s test. N.S., not significant. N.D., not detected. See <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006225#ppat.1006225.s010" target="_blank">S6 Table</a> for a description of the complemented Tn mutants (Tn::<i>bb0017</i>-comp, DM104; Tn::<i>bb0164</i>-comp, JH511).</p