Transposon mutagenesis identified OmpR and LptD as regulators of T6SS-4 expression.

<p>(A) Location of the transposon in the three strains (<i>Tn</i>23, <i>Tn</i>31 and <i>Tn</i>52) displaying lower T6SS-4 expression isolated in the random screen. (B) β-galactosidase activities (upper panel, in Miller units) and fluorescence levels (lower panel, in arbitrary units) of <i>Y. pseudotuberculosis</i> RL31748-4 (no fusion), <i>Y.p.</i> RL31758-41 (WT, carrying the promoter-<i>lacZ</i> fusion at the locus and the promoter-<i>gfp</i> fusion at the <i>ara</i> locus) and of the transposon strains carrying the pBAD24 empty vector (−) or pBAD-ompR (+). <i>p</i>-values obtained using paired Student’s <i>t-</i>test analyses are indicated (***, <i>p</i>≤0.0001).</p

T6SS-4 thermoregulation is OmpR-independent.

<p>β-galactosidase activities (upper panel, in Miller units) and fluorescence levels (lower panel, in arbitrary units) of the T6SS-4 promoter fusions at 28°C and 37°C in the wild-type (A) and <i>ompR1</i> transposon (B) strains. Identical results were obtained for the <i>ompR2</i> transposon strain (data not shown). <i>p</i>-values obtained using paired Student’s <i>t-</i>test analyses are indicated (***, <i>p</i>≤0.0001).</p

OmpR does not regulate the other <i>Y. pseudotuberculosis</i> T6SS loci.

<p>β-galactosidase activities (upper panel, in Miller units) and fluorescence levels (lower panel, in arbitrary units) of <i>Y. pseudotuberculosis</i> with <i>lacZ</i> and <i>gfp</i> fusions to T6SS-1, T6SS-2, T6SS-3-rev, T6SS-3-fwd, T6SS-4, T6SS-5 and <i>vgrG</i> (IP31758_0696) putative promoters, and carrying the pBAD24 empty vector (−) or pBAD-ompR (+). <i>p</i>-values obtained using paired Student’s <i>t-</i>test analyses are indicated (NS, non significant [<i>p</i>>0.05]; ***, <i>p</i>≤0.0001).</p

T6SS-4 gene expression is responsive to osmotic and cell envelope stresses through an OmpR-dependent mechanism.

<p>β-galactosidase activities (upper panel, in Miller units) and fluorescence levels (lower panel, in arbitrary units) of the T6SS-4 promoter in the WT or transposon <i>ompR1</i> strain carrying the pBAD24 empty vector (−) or pBAD-ompR (+) after exposure – or not – to 0.6 M Sucrose or to sodium deoxycholate (DOC) for 60 min. Identical results were obtained for the <i>ompR2</i> transposon strain (data not shown). <i>p</i>-values obtained using paired Student’s <i>t-</i>test analyses are indicated (NS [non significant], <i>p</i>>0.05; ***, <i>p</i>≤0.0001).</p

OmpR binds to the promoter region of T6SS-4.

<p>(A) Intergenic sequence upstream the first gene of the T6SS-4 operon. The TTG putative initiation codon is underlined, as the ATG initiation codon of the divergent gene upstream T6SS-4. The framed sequences in bold letters correspond to putative OmpR binding sites identified by <i>in silico</i> analyses using Virtual Footprint. A third OmpR binding site was experimentally identified upstream this intergenic region <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066615#pone.0066615-Zhang2" target="_blank">[63]</a>. (B) Electrophoretic mobility shift assays of the <i>Y. pseudotuberculosis ompF</i> (upper panel) or T6SS-4 (lower panel) promoters using phosphorylated purified OmpR protein (lane 1, no protein; lane 2, 10 nM; lane 3, 20 nM; lane 4, 40 nM; lane 5, 60 nM; lane 6, 80 nM). Lanes 7 and 8: competition experiments with unlabelled T6SS-4 (upper panel) or <i>ompF</i> (lower panel) promoter PCR fragments at a promoter:competitor 1∶4 (lane 7) or 1∶20 (lane 8) ratio, in presence of 80 nM phosphorylated purified OmpR protein. Controls include incubation with the purified ferric uptake regulator Fur (lane 9, 80 nM) or incubation of the OmpR-independent enteroaggregative <i>E. coli sci-1</i> promoter PCR fragment (P<i>sci1</i>) with phosphorylated purified OmpR (lane 10, 80 nM). The positions of the free probes and of the shift fragments (*) are indicated.</p

The α-helix located between residues 104 and 130 of TssB1 is required for T6SS function and interaction with TssC1.

<p>(A) Hcp release assay. Hcp_FLAG release was assessed by separating whole cells (C) and culture supernatant (SN) fractions from 2×10⁹ wild-type (WT), Δ<i>tssB1</i> (Δ<i>tssB</i>) cells or Δ<i>tssB1</i> cells producing TssB1 (<i>tssB⁺</i>) or TssB1 deleted of the α-helix 104-130 (<i>tssB</i>Δ<i>^hel</i>). Proteins were separated by 12.5%-acrylamide SDS-PAGE and Hcp was immunodetected using anti-FLAG monoclonal antibody (lower panel). The periplasmic TolB protein (immunodetected using an anti-TolB polyclonal antibodies, upper panel) was used as a marker to verify that no lysis occured. (B) Bacterial two-hybrid assay. BTH101 reporter cells carrying pairs of plasmids producing the indicated T6SS proteins (B, TssB1; BΔhel, TssB1 deleted of the α-helix; C1, TssC1) fused to the T18 or T25 domain of the <i>Bordetella</i> adenylate cyclase were spotted on X-Gal indicator LB agar plates.</p

The hydrophobic motif of the TssB1 α-helix is required for T6SS function and interaction with TssC1.

<p>Amino-acid sequence (A) and helical wheel projection (B) of the TssB1 α-helix (from residue Pro-103 to residue Leu-131). The different motifs described in this study are highlighted in different colours: blue, N-terminal hydrophobic; red, polar/charged; green, leucine-rich. The residues of these motifs mutagenized in this study are underlined (A) or striped (B). Top-view (C) and side-view (D) projections of the TssB1 α-helix. The targeted residues are colored as in panel (A). Top- and side-views have been modelled using PyMOL v0.99. (E) Hcp release assay. Hcp_FLAG release was assessed by separating whole cells (C) and culture supernatant (SN) fractions from 2×10⁹ Δ<i>tssB1</i> cells producing TssB1 (<i>tssB⁺</i>) or TssB1 bearing substitutions within the hydrophobic (<i>tssB^VIL</i>), the polar/charged (<i>tssB^RR</i>) or the leucin-rich (<i>tssB^LL</i>) motif. Proteins were separated by 12.5%-acrylamide SDS-PAGE and Hcp and TolB were immunodetected using anti-FLAG monoclonal (lower panel) and anti-TolB polyclonal (upper panel) antibodies. (F) Bacterial two-hybrid assay. BTH101 reporter cells producing the T25 domain of the <i>Bordetella</i> adenylate cyclase fused to TssC1 (T25-C) and the T18 domain fused to TssB1 (T18-B) or TssB1 variants bearing substitutions within the hydrophobic (T18-B^VIL), the polar/charged (T18-B^RR) or the leucin-rich (T18-B^LL) motif were spotted on X-Gal indicator LB agar plates.</p

Mutagenesis study of the hydrophobic motif of the TssB1 α-helix.

<p>(A) Hcp release assay. Hcp_FLAG release was assessed by separating whole cells (C) and culture supernatant (SN) fractions from 2×10⁹ Δ<i>tssB1</i> cells producing TssB1 (<i>tssB⁺</i>) or TssB1 bearing double or single substitutions within the hydrophobic motif (<i>tssB^VI</i>, V106W-I110W; <i>tssB^IL</i>, I110W-L117W; <i>tssB^VL</i>, V106W-L117W; <i>tssB^V</i>, V106W; <i>tssB^I</i>, I110W; <i>tssB^L</i>, L117W). Proteins were separated by 12.5%-acrylamide SDS-PAGE and Hcp and TolB were immunodetected using anti-FLAG monoclonal (lower panel) and anti-TolB polyclonal (upper panel) antibodies. (B) Bacterial two-hybrid assay. BTH101 reporter cells producing the T25 domain of the <i>Bordetella</i> adenylate cyclase fused to TssC1 (T25-C) and the T18 domain fused to TssB1 (T18-B) or TssB1 variants bearing substitutions within the hydrophobic motif (T18-B^VI, V106W-I110W; T18-B^IL, I110W-L117W; T18-B^VL, V106W-L117W; T18-B^V, V106W; T18-B^I, I110W; T18-B^L, L117W) were spotted on X-Gal indicator LB agar plates.</p

The hydrophobic motif of the TssB1 α-helix is required for T6SS sheath assembly.

<p>Time-lapse fluorescence microscopy recordings showing sheath dynamics in Δ<i>tssBC1</i> (Δ<i>tssB1</i>-Δ<i>tssC1</i>) or Δ<i>tssB1</i> cells producing TssB1-sfGFP (TssB1) (A) or TssB1-sfGFP bearing the indicated substitutions (B). Individual images were taken every 30 sec. Assembly and contraction events are indicated by the white open triangles. Scale bars are 2 µm.</p

An ABC Transporter with Two Periplasmic Binding Proteins Involved in Iron Acquisition in <i>Pseudomonas aeruginosa</i>

Pyoverdine I is the main siderophore secreted by <i>Pseudomonas aeruginosa</i> PAO1 to obtain access to iron. After extracellular iron chelation, pyoverdine-Fe uptake into the bacteria involves a specific outer-membrane transporter, FpvA. Iron is then released in the periplasm by a mechanism involving no siderophore modification but probably iron reduction. The proteins involved in this dissociation step are currently unknown. The pyoverdine locus contains the <i>fpvCDEF</i> operon, which contains four genes. These genes encode an ABC transporter of unknown function with the distinguishing characteristic of encompassing two periplasmic binding proteins, FpvC and FpvF, associated with the ATPase, FpvE, and the permease, FpvD. Deletion of these four genes partially inhibited cytoplasmic uptake of ⁵⁵Fe in the presence of pyoverdine and markedly slowed down the <i>in vivo</i> kinetics of iron release from the siderophore. This transporter is therefore involved in iron acquisition by pyoverdine in <i>P. aeruginosa</i>. Sequence alignments clearly showed that FpvC and FpvF belong to two different subgroups of periplasmic binding proteins. FpvC appears to be a metal-binding protein, whereas FpvF has homology with ferrisiderophore binding proteins. <i>In vivo</i> cross-linking assays and incubation of purified FpvC and FpvF proteins showed formation of complexes between both proteins. These complexes were able to bind <i>in vitro</i> PVDI-Fe, PVDI-Ga, or apo PVDI. This is the first example of an ABC transporter involved in iron acquisition <i>via</i> siderophores, with two periplasmic binding proteins interacting with the ferrisiderophore. The possible roles of FpvCDEF in iron uptake by the PVDI pathway are discussed