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

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

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

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

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

Activity of <i>S. aureus</i> nuclease in different <i>B. subtilis</i> backgrounds.

<p>Growth (OD_{600 nm}) and nuclease relative activity in culture supernatants (scale 1–10) are presented for three different <i>B. subtilis</i> backgrounds. Filled diamonds, growth; open squares, nuclease relative activity. Time is time after the beginning of nuclease induction by addition of 2 mM IPTG. Mean values of three independent experiments are presented. Error bars indicate the SEM.</p

Schematic representation of the high-throughput cloning strategy.

<p><b>A. </b><i>B. subtilis</i> Gateway expression vector pDG148-GW with an inducible P<i>spac</i> promoter. lacI, lac repressor gene; AmpR, kanR, phlR, CmR, genes providing resistance to ampicilin, kanamycin, phleomycin, or chloramphenicol, respectively; ccdB, gene coding for the cytotoxic CcdB protein. <b>B.</b> Full-length coding sequences (CDS) are amplified by nested PCR using a set of CDS-specific (primary) primers and a set of universal (secondary) primers. The resulting PCR product contains the CDS preceded by a synthetic SD sequence (brackets) and a <i>Sap</i>I restriction site (underlined). <i>att</i>B1.1 and <i>att</i>B2.1 sites for site-specific recombination are indicated by braces; start and stop codons of the CDS, in italic-bold.</p

Immune modulation effect of secreted <i>E. coli</i> flagellin in different <i>B. subtilis</i> backgrounds.

<p>Bars indicate the relative activity of a luciferase reporter gene under the control of an NF-κB dependent promoter in an intestinal epithelial cell line (HT-29), <i>in vitro</i>. Bacterial backgrounds of which the culture supernatant was tested are indicated on the abscissa: grey bars, supernatants of bacterial cultures in which flagellin (if present) had been induced with IPTG; open bars, supernatants of bacterial cultures in which flagellin had not been induced. -, HT-29 cells only; T1, TNF-α (1 ng/ml). Mean values of three independent experiments are presented. Error bars indicate the SEM. F-BS, presence (+) or absence (−) of <i>B. subtilis</i> flagellin gene; P, presence or absence of multiple <i>B. subtilis</i> protease genes; pDG, presence or absence of pDG148 (empty vector); pF-EC, presence or absence of pDGfliC-SEC encoding a secreted form of <i>E. coli</i> flagellin.</p

Properties of <i>B. subtilis wt</i> and mutant strains.

<p><b>A.</b> Effect of <i>B. subtilis</i> strains on NF-κB activation. Bars indicate the relative activity of a luciferase reporter gene under the control of an NF-κB dependent promoter in an intestinal epithelial cell line (HT-29), <i>in vitro</i>. Bacterial strains are indicated on the abscissa. b, live bacteria from exponentially growing cultures; s, culture supernatant; -, HT-29 cells only; T1, TNF-α (1 ng/ml). Mean values of three independent experiments are presented. Error bars indicate the SEM. F-BS, presence (+) or absence (−) of <i>B. subtilis</i> flagellin gene; P, presence or absence of multiple <i>B. subtilis</i> protease genes; EM, presence or absence of extracellular matrix genes (<i>eps</i> and <i>tas</i>A). <b>B. </b><i>B. subtilis</i> phenotypes as viewed by phase-contrast microscopy. Exponentially growing bacteria are shown, 100X magnification. <b>C.</b> Effect of <i>B. subtilis</i> strains on IL8 secretion. Bars indicate the concentration of secreted IL8 after incubation of HT-29 cells with the bacteria or bacterial culture supernatants indicated on the abscissa. Abbreviations as in Fig. 2A. The same bacterial cultures were used for the NF-κB activation assay shown in Fig. 2A and the IL8 secretion assay shown in Fig. 2C.</p