Phenotypical analysis of the Δ<i>mfd</i> mutant in mutagenic conditions.

<p>(A) <i>B</i>. <i>cereus</i> wild type and Δ<i>mfd</i> mutant strains were inoculated in LB medium at a starting optical density (OD) of 0.07 and grown at 25°C with agitation. The OD was measured every hour at 600 nm. This graph represents representative growth curves out of at least five independent experiments. (B) <i>B</i>. <i>cereus</i> wild type and Δ<i>mfd</i> mutant strains were grown at 37°C under agitation until mid exponential growth phase. Serial dilutions were plated on agar plates containing 50 ng/mL mitomycin C. Plates were incubated ON at 37°C and bacterial survival was assessed by observing the growth zone. Images correspond to a representative example out of at least 3 independent experiments done in duplicates. (C) <i>B</i>. <i>cereus</i> wild type and Δ<i>mfd</i> mutant strains were grown at 37°C under agitation until mid exponential growth phase. Serial dilutions were plated on agar plates and exposed to UV light for 0 to 15 seconds at 5J/m². Plates were incubated ON at 37°C and bacterial survival was assessed by observing the growth zone. Images correspond to a representative example out of at least 3 independent experiments done in duplicates. (D) <i>B</i>. <i>cereus</i> wild type and Δ<i>mfd</i> mutant strains were grown in LB medium until entry into stationary growth phase. Culture supernatant was filtered and added to HeLa cells. Cytotoxicity was measured by the trypan blue method after 2 h of incubation. Results are means of three independent experiments. (E) Bacterial strains were grown in LB medium at 37°C under agitation or without agitation (semi anaerobiosis). This graph represents representative growth curves out of at least three independent experiments. (F) Bacterial strains were grown in LB medium at 37°C under agitation, then diluted with the pH adjusted to 5, 6 or 7. Cfu were calculated after 24 h of growth by plating serial dilutions on LB agar plates. Results are means of at least three independent experiments. (G) <i>B</i>. <i>cereus</i> wild type and Δ<i>mfd</i> mutant strains were cultured in LB medium for 24 h in the presence of the anti microbial peptide cecropin A. Cfu were calculated by plating serial dilutions on LB agar plates. Results are means of three independent experiments.</p

Role of Mfd in <i>in vivo</i> survival following NO stress.

<p>C57/Bl6/Sev 129 mice (Wt Mice) and NOS2-/- mice (iNOS-KO mice) were inoculated intranasally with <i>B</i>. <i>cereus</i> wild type and Δ<i>mfd</i> mutant bacteria (5.10⁶/mice). Wild type and <i>Δmfd</i> mutant strains were recovered from the lung (A) and from the brain (B) following mice death. The cfu recovered post infection was calculated by plating the bacteria on LB agar plates. For each mouse, the same symbol is used for lung (A) and brain (B) values. Cfu is shown for 5 wild type mice infected with Bc407, 4 wild type mice infected with Δ<i>mfd</i>, 3 KO mice infected with Bc407 and 3 KO mice infected with Δ<i>mfd</i>.</p

In the context of NO stress, we show that <i>mfd</i> and <i>recBC</i>/<i>addAB</i> deletions are epistatic.

<p>As RecBC participates in the repair of Double Strand Breaks (DSB), this indicates that Mfd also participates in DSB repair. It is unlikely that Mfd prevents DSBs because, in that case, RecBC would be essential for DNA repair in a <i>mfd</i> mutant. By contrast, we hypothesized that following the DSBs induced by NO exposure, RecBC has not directly access to Double strand ends (DSEs). Thus, Mfd would act first by removing the RNAP blocked on DNA lesions. Then the RecBCD complex can be recruited to repairs the DSB. RecBCD unwinds the DNA helix and degrades single strands. When RecBDC encounters a Chi site on the DNA, the degradation of the 5’ terminus is enhanced leaving a 3’ overhang and leads to the formation of an ssDNA. RecA binds to ssDNA and promotes repair by recombination with a homologous molecule of DNA [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163321#pone.0163321.ref050" target="_blank">50</a>].</p

Mfd confers resistance to NO stress.

<p>(A) Mice peritoneal macrophages isolated from wild type (BM wt) and NOS2-/- (BM KO) mice were infected with <i>B</i>. <i>cereus</i> wild type and Δ<i>mfd</i> mutant strains. At the indicated time points, bacterial survival was calculated by plating and normalized to the cfu obtained at t0. Results represent mean values of at least 3 independent experiments done in triplicates. (B) Bacteria were exposed directly to chemically generated NO (1.5 mM sodium nitrite) in a cell free system for the indicated times. The bacteria were then harvested and plated on agar plates to evaluate bacterial survival. Results represent mean values of at least 3 independent experiments done in triplicates.</p

The Bacterial Mfd Protein Prevents DNA Damage Induced by the Host Nitrogen Immune Response in a NER-Independent but RecBC-Dependent Pathway - Fig 4

<p><b>(A) AddAB but not UvrA is involved to prevent NO stress</b>. <i>B</i>. <i>cereus</i> wild type and mutant strains were exposed to 1.5 mM NO for 1 h in a cell-free system. Bacteria were harvested and plated on agar plates to evaluate bacterial survival. Cfu counts were normalized to initial cfu. The results reported are mean values of at least three independent experiments. P values are calculated using the Student test. <b>(B) Survival of <i>B</i>. <i>cereus</i> mutant strains with Raw cells.</b> Raw cells were infected with <i>B</i>. <i>cereus</i> wild type and mutant strains at a multiplicity of infection of 10 at 37°C. After 20 h of incubation, bacteria were recovered by scraping and counted by plating serial dilutions on agar plates. Alternatively, the NO inhibitor NMMLA was added to the cells at 1 mM.</p

NO IC 50 on <i>B</i>. <i>cereus</i> mutants.

<p>NO IC 50 on <i>B</i>. <i>cereus</i> mutants.</p

HlyII induces transient membrane permeability.

<p>Macrophages were incubated with increasing concentrations of HlyII (0 to 0.5 µg/mL) for 2 h, and membrane permeability was assessed by trypan blue dye exclusion (black scale). A representative image is shown for non-treated cells (B), and cells incubated with HlyII at 0.2 µg/mL (C) and 0.5 µg/mL (D). Alternatively, macrophages were incubated with HlyII (0 to 0.5 µg/mL) for 2 h, and washed to remove the toxin. The macrophages were then allowed to recover in fresh medium supplemented with FBS for 24 h. After recovering, membrane permeability was assessed by trypan blue dye exclusion (grey scale). A representative image is shown of recovered cells after incubation with HlyII at 0.2 µg/mL and 24 h recovery (E).</p

Cells have normal metabolic activity following incubation with HlyII.

<p>Macrophages were incubated with increasing concentrations of HlyII (0 to 0.5 µg/mL) for the times indicated. Cellular metabolic activity was evaluated by measuring absorbance at 490 nm, which is proportional to NADH- or NADPH-dependent reduction of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (MTS) for up to 4 h after incubation with the toxin. The values were normalized to those for untreated cells (no toxin).</p

Model of the role and expression of <i>hlyII</i> during infection.

<p>A) As long as iron and glucose are abundant in the bacterial environment, the bacteria will be able to use these resources for growth. Glucose will enter the bacteria as glucose 6P (blue rectangles) and will bind HlyIIR (orange plain cross). Iron (purple circles) will bind Fur (red ovals). These bindings will promote HlyIIR and Fur repressor activities, leading to HlyIIR- and Fur-based transcriptional repression on <i>hlyII</i> gene expression. B) During host infection, bacteria find themselves in an environment, which is low in glucose and iron. Levels of these nutrients are further lowered during bacterial proliferation. The decrease in the concentration of glucose during bacterial proliferation will lead to an inhibition of HlyIIR activity, thus allowing <i>hlyII</i> expression. The decrease in the concentration of iron, partially due to its sequestration by immune cells, will lead to an inhibition of Fur activity, thus allowing <i>hlyII</i> expression. Thus, when glucose and iron are getting scarce, <i>hlyII</i> expression is activated. HlyII will then be released in the environment and induce macrophage and erythrocyte lysis. The dead cells will release their intracellular content, allowing access to metabolites that are essential for bacterial growth.</p

HlyIIR negatively regulates <i>hlyII</i> expression in <i>B. cereus</i>.

<p>(A) Expression levels of the target <i>hlyII</i> gene relative to the endogenous standard 16S RNA were measured by RT-qPCR throughout bacterial growth in the wild type Bt407 (circle) and the Bt407Δ<i>hlyIIR</i> (square) strains. Data are expressed as the ratio of <i>hlyII</i> mRNA normalized to 16S RNA. Values are means of two independent experiments. (B) The specific β-galactosidase activity (Miller unit) of strains Bt407 and Bt407 Δ<i>hlyIIR</i> harboring the transcriptional pHT-P<i>hlyII</i>’<i>Z</i> fusion were measured from bacteria grown in LB medium at 37°C from 2 h before the culture entry into stationary phase (t−2) to 4 h after (t4). Results represent mean values of at least three independent experiments. (C) The specific β-galactosidase activity (Miller unit) of strain Bt407 harboring the pHT-P<i>hlyIIR</i>’<i>Z</i> fusion was measured from bacteria grown in LB medium at 37°C from 2 h before the culture entered into stationary phase (t−2) to 4 h after (t4). Results represent mean values of at least three independent experiments.</p