IscR does not modulate the temporal induction of nuclease colicins.

<p>A) Measured β-galactosidase activities of strain BW25113, carrying the <i>lac</i> expression vector pRW50 containing various nuclease colicin promoter fragments. Each value represents the mean ± SD of at least three independent measurements, the arrow indicates the time of addition of a sub-lethal concentration of nalidixic acid (37 μM) and the dashed lines represent optical density (OD₆₀₀). B) The binding of purified LexA protein to a P³² end-labelled <i>cea8</i> fragment was investigated using DNase I footprint analysis. The location of the two LexA binding sites, LexA₁ and LexA₂ is indicated by orange boxes and the -10 and -35 promoter elements by blue boxes. The final concentration of LexA protein used in reactions was 12.5, 25, 50 and 100 nM (lanes 2–5). C) The binding of purified LexA and RNA polymerase (RNAP) to a P³² end-labelled <i>cea8</i> fragment was investigated using EMSA analysis. DNA fragments were first incubated for 15 min with various concentrations of LexA (100, 200 or 400 nM) followed by the addition of 100 nM RNAP with further incubation for 15 min at 37°C. The location of free DNA, the LexA/DNA and RNAP/DNA complexes is indicated. D) The binding of purified IscR protein to P³² end-labelled colicin K (<i>cka</i>) or <i>cea8</i> promoter fragments was assayed using EMSA analysis. The concentration of IscR used was 0.4, 0.8, 1.6 and 3.2 μM. The location of free DNA and the IscR/DNA complex is marked. E) Assays of colicin production in BW25113 and its Δ<i>iscR</i> derivative cells, carrying various colicin-encoding plasmids. Equal amounts of cells were collected at hourly time points after the addition of nalidixic acid (0 h) and crude cell extracts were placed into wells in agar plates overlaid with soft agar, harbouring the <i>E</i>. <i>coli</i> K-12 indicator strain DH5α pBR322. Experiments were performed in duplicate and representative growth curves are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005354#pgen.1005354.s002" target="_blank">S2 Fig</a>.</p

Binding of AsnC protein to <i>cea8</i> is altered by L-asparagine.

<p>A) Organisation of the colicin E8 promoter region. The figure shows the DNA sequence of the <i>cea8</i> promoter from position -93 to +100. The -35 and -10 core promoter elements and the predicted start of transcription (+1) are shown in bold type and the start of translation (ATG) is underlined. The location of the two LexA binding sites (LexA₁ and LexA₂) is shown by orange boxes. The AsnC-induced hypersensitive sites observed by DNase I footprinting are starred. B) The binding of purified AsnC protein to a P³² end-labelled <i>cea8</i> fragment in the presence and absence of L-asparagine (± L-Asn) was investigated using EMSA analysis. The concentration of AsnC in lanes 2–7 and 9–14 was 0.5, 1.05, 2.1, 4.2, 8.4 and 12.6 μM, respectively. The location of free DNA, the position of the wells and the various AsnC/DNA complexes is indicated. C) An <i>in vitro</i> DNase I footprint experiment analysing the binding of purified AsnC to the <i>cea8</i> promoter. The concentration of AsnC in lanes 2–6 and 8–12 was 0.5, 1.0, 2.1, 4.2, and 6.3 μM, respectively. The AsnC-induced hypersensitive sites are starred (also see panel A) and the location of the different protection patterns observed due to L-asparagine is indicated by red boxes.</p

LexA and AsnC can bind to <i>cea8</i> promoter region simultaneously.

<p>A) The panel shows an EMSA analysing the binding of purified LexA and AsnC protein to a P³² end-labelled <i>cea8</i> fragment in the presence of L-asparagine (+ L-Asn). The concentration of AsnC in lanes 2–7 and 9–14 was 0.5, 1.05, 2.1, 4.2, 8.4 and 12.6 μM, respectively. LexA protein was present in reactions at concentration of 400 nM. The location of free DNA, the position of the wells and the various protein/DNA complexes is marked. B) The binding of purified LexA and AsnC proteins to the <i>cea8</i> promoter fragment was studied by DNase I footprinting in the presence and absence of L-asparagine (± L-Asn). AsnC was included at concentrations of 1.0, 2.1 and 4.2 μM and LexA at a concentration of 400 nM. The prominent hypersensitive bands, corresponding to positions +1 and -16, that are produced by the concurrent binding of AsnC and LexA, are starred. Red boxes indicate the position of AsnC interactions, which are affected by L-Asn. The location of the two LexA binding sites, LexA₁ and LexA₂, is indicated by orange boxes and the -10 and -35 promoter elements by blue boxes. C) The panel shows an extended run of the footprint analysis from panel B, focusing on positions -80 to -60 upstream of the transcription start site. Labels are identical to those described above. Panels D-F) show models of the different nucleoprotein complexes formed at the <i>cea8</i> promoter by the binding of LexA and AsnC.</p

The <i>cae8</i> promoter is sensitive to the amino acid L-asparagine.

<p>The figure shows measured β-galactosidase activities from wild-type BW25113 cells, carrying the <i>cea8</i> promoter region subcloned into pRW50. Cells were grown in M9 minimal medium containing 0.5 mM NH₄Cl until an OD₆₀₀ of ~0.2, when the culture was split in to two and grown further in the presence of either 10 mM NH₄Cl or 20 mM L-asparagine. The arrow indicates the time of addition of nalidixic acid and the dashed lines represent OD₆₀₀. Each value is the average of duplicate experiments and the standard deviation is shown.</p

AsnC modulates the temporal induction of different DNA- and RNA-degrading colicins.

<p>Expression of the (A) <i>cea2</i>::<i>lacZ</i> and the (B) <i>cea6</i>::<i>lacZ</i> fusions in strain BW25113 (wt) and the Δ<i>iscR</i> and Δ<i>asnC</i> mutants. For panels A and B each value represents the mean ± SD of at least two independent measurements, the arrow indicates the time of the addition of a sub-lethal concentration of nalidixic acid (37 μM) and the dashed lines represent OD₆₀₀. C) Growth curves of BW25113 (wt) and Δ<i>asnC</i> cells harbouring naturally occurring plasmids encoding either the DNA degrading colicin E2, the tRNA cleaving colicin E5 or the rRNA cleaving colicin E6. The arrow indicates the time of addition of nalidixic acid. Experiments were performed in duplicate and representative growth curves are shown. D) Assays of colicin production in BW25113 and Δ<i>asnC</i> cells, carrying various colicin-encoding plasmids. Equal amounts of cells were collected at hourly time points after the addition of nalidixic acid (0 h) and crude cell extracts were applied into wells in agar plates, overlaid with soft agar harbouring the colicin sensitive strain DH5α pBR322. The experiments were performed in duplicate. E) AsnC protein has little effect on the expression of the pore-forming colicin K. The activity of the <i>cka</i> promoter was measured in strain BW25113 (wt) and its Δ<i>iscR</i> or Δ<i>asnC</i> mutant derivatives. Each value represents the mean ± SD of four independent measurements, the arrow indicates the time of addition of a sub-lethal concentration of nalidixic acid (37 μM) and the dashed lines represent the OD₆₀₀.</p

Reporter OMVs capture rapid kinetics of vesicle uptake by host cells in real time.

<p>(A) CCF2-AM loaded Hela cells were exposed to OMVs from EHEC carrying ClyA-Bla (red), or vector control (grey) at an MOI of 1000 for 3 hours. Ratio of blue:green fluorescence) over time was plotted as mean ± stdev (n = 3). (B) R_max was determined from data in S2A to visualize speed of uptake and is shown are means ± stdev (n = 3). Significance was determined by analysis of variance, with a Brown Forsythe test to determine equal variance. (**) p≤0.01. (C) Absolute FRET changes after 3 h were determined from data in (A) and plotted as efficiency of OMV uptake. Data shown are means ± stdev (n = 3). Significance was determined by ANOVA, with a Brown Forsythe test to determine equal variance. (**) p≤0.01. (D) CCF2-AM loaded Hela cells were imaged by confocal microscopy and merged blue/green images representative of 15 images (n = 3) are shown. Scale bars, 20 μm. (E) Hela cells incubated with cellmask orange-labelled OMVs (red) for 10 and 60 min and slice views of z-stacks were acquired by confocal microscopy. Scale bars, 10 μm.</p

LPS composition determines major route and kinetics of OMV entry into host cells.

<p>Whilst it is well established that pathogenic species utilize OMVs during infection, the specific adaptations which allow OMVs to contribute to pathogenesis require further exploration. This work has developed a new approach to overcome current methodological limitations and provide consistent data for future studies and allow new insights into the interactions of OMVs with host cells during infection. This method has shown the relevance of LPS composition, in particular the presence of O antigen, in determining the entry route and kinetics of OMVs. Further work in this area may reveal targets for inhibition of these processes, and enable attenuation of infections by preventing the OMV-associated delivery of virulence factors.</p

OMVs lacking O antigen are biased towards clathrin-mediated endocytosis, while OMVs with O antigen can efficiently access host cells via lipid rafts.

<p>Hela cells were either left untreated (control, red), or pre-treated with 5 μg/ml papain (lilac), 1 μg/ml chlorpromazine (pink), 5 mM methyl-β-cyclodextrin (light green) or 1 μg/ml filipin (turquoise), and exposed to ClyA-Bla OMVs isolated from EHEC (A), EAEC (B) or K12 (C) with or without O antigen at an MOI of 1000 for 3 hours. Total FRET changes after 3 hrs were determined from data in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006760#ppat.1006760.s007" target="_blank">S6 Fig</a> and data shown are means ± stdev (n = 3). Significance compared to the control group was determined using ANOVA, with a Brown Forsythe test to determine equal variance. (***) indicates p≤0.001, (**) p≤0.01, (*) p≤0.05, (ns) not significant.</p

Genetically encoded Bla probes are enriched in <i>E</i>. <i>coli</i> OMVs and retain their enzymatic activity.

<p>(A) Expression of genetically encoded Bla probes is induced in bacteria and secreted OMVs are isolated for all subsequent experiments. Entry of OMVs containing Bla probes into host cells can be detected using a continuous FRET assay. (B) Whole cell lysate (WCL), supernatant (sup) and outer membrane vesicles (OMV) fractions isolated from EHEC expressing ClyA-Bla, carrying empty vector, or no vector were separated by SDS-PAGE and expression of ClyA-Bla was detected by Western Blotting and probing with α-Bla antibody. (C) Specific enzyme activity in whole cell lysate, supernatant, OMV or solubilized OMV fractions isolated from EHEC expressing ClyA-Bla, Bla-ClyA, or carrying empty vector (data shown are means ± stdev, n = 3).</p

EHEC OMVs enter host cells more rapidly and efficiently than <i>E</i>. <i>coli</i> K12 OMVs.

<p>(A) CCF2-AM loaded Hela cells were exposed to OMVs from EHEC (red) or <i>E</i>. <i>coli</i> K12 (blue) carrying ClyA-Bla, at an MOI of 1000 for 3 hours. Ratios of blue:green fluorescence over time were plotted as means ± stdev (n = 3). Maximum rates (B) were determined from data in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006760#ppat.1006760.s003" target="_blank">S2 Fig</a> and absolute FRET signal changes after 3 hrs (C) were determined from data in (A) and plotted to visualize overall efficiency of uptake for EHEC (red) and K12 (blue) OMVs. Data shown are means ± stdev (n = 3). Significance was determined by ANOVA, with a Brown Forsythe test to determine equal variance. (***) p≤0.001, (**) p≤0.01.</p