Both TssM1 size variants are required for activity of the CTS1 T6SS.

<p>(A) <i>C. rodentium</i> WT cells or <i>ΔtssM1</i> cells expressing WT <i>tssM1</i> (WT; producing both forms of TssM1: TssM1-FL and TssM1[1–807]) or TssM1 variants bearing mutations in the poly-A tract (AAG; producing only the TssM1[1–807] variant) or mutations in the poly-A tract and a deletion of the last nucleotide (AAGΔA; producing only the TssM1-FL variant). (B) <i>C. rodentium</i> cells producing the AAGΔA and AAG variants from the chromosome and producing the other variant from the pBAD18 vector. Hcp release assay (top panels). The extracellular proteins were isolated by separating whole cells (C) and the supernatant fraction (S). Proteins were visualized by Coomassie blue staining after 15%-acrylamide SDS-PAGE. Molecular mass markers are indicated on the left. Growth competition assay (bottom panels). Growth competition assay was monitored by mixing fluorescently labeled GFP⁺<i>E. coli</i> W3110 prey cells with the indicated strains of <i>C. rodentium</i> as predators. Mixes were spotted onto LB agar supplemented with IPTG (500 µM) and arabinose (2%), and incubated for 14 h at 30°C. The relative fluorescence (upper graph) is expressed in arbitrary units (A.U.) and is the mean of fluorescence levels obtained from three independent experiments (each measured in triplicate). Fluorescent images of the competition assays (obtained with a LI-COR Odyssey imager) are shown below the upper graph.</p

The TssM1 full-length variant is produced through slippage on a poly-A tract.

<p>(A) Representation of the nucleotide sequence of <i>C. rodentium tssM1</i> and of the mutagenized variants used in panel (B). The poly-A tract is shown in red, the mutagenized nucleotides are shown in green. (B) Western-blot analyses of <i>C. rodentium</i> cells producing the indicated TssM1 protein variant bearing a FLAG-epitope tag at the N-terminus and a 6×His tag upstream the stop codon resulting from +1 frameshifting. Crude extracts from 10⁹ cells were subjected to 10%-acrylamide SDS-PAGE and immunodetection using the anti-FLAG and anti-5His monoclonal antibodies. The positions of the TssM1-FL and -[1–807] variants are indicated on the right, with the schematic representation in the middle. The relative intensities of these two TssM1 variants are indicated (as %). Molecular weight markers (in kDa) are indicated on the left. (C) Representation of the nucleotide sequence of <i>C. rodentium tssM1</i> constructs used for fluorescence studies shown in panel (D). The poly-A tract is shown in red, the mutagenized nucleotides are shown in green and additional nucleotides are shown in blue. (D) Fluorescence levels (in arbitrary units, A.U.) of cells producing the different TssM1 variants shown in panel (C).</p

Both TssM1 size variants are required for activity of the CTS1 T6SS.

<p>(A) <i>C. rodentium</i> WT cells or <i>ΔtssM1</i> cells expressing WT <i>tssM1</i> (WT; producing both forms of TssM1: TssM1-FL and TssM1[1–807]) or TssM1 variants bearing mutations in the poly-A tract (AAG; producing only the TssM1[1–807] variant) or mutations in the poly-A tract and a deletion of the last nucleotide (AAGΔA; producing only the TssM1-FL variant). (B) <i>C. rodentium</i> cells producing the AAGΔA and AAG variants from the chromosome and producing the other variant from the pBAD18 vector. Hcp release assay (top panels). The extracellular proteins were isolated by separating whole cells (C) and the supernatant fraction (S). Proteins were visualized by Coomassie blue staining after 15%-acrylamide SDS-PAGE. Molecular mass markers are indicated on the left. Growth competition assay (bottom panels). Growth competition assay was monitored by mixing fluorescently labeled GFP⁺<i>E. coli</i> W3110 prey cells with the indicated strains of <i>C. rodentium</i> as predators. Mixes were spotted onto LB agar supplemented with IPTG (500 µM) and arabinose (2%), and incubated for 14 h at 30°C. The relative fluorescence (upper graph) is expressed in arbitrary units (A.U.) and is the mean of fluorescence levels obtained from three independent experiments (each measured in triplicate). Fluorescent images of the competition assays (obtained with a LI-COR Odyssey imager) are shown below the upper graph.</p

Transcriptional slippage is conserved in <i>Y. pseudotuberculosis</i>.

<p>(A) Representation of the nucleotide sequence of <i>Y. pseudotuberculosis tssM3</i>. The poly-A tract is shown in red. The codons and the amino-acids resulting from translation of the WT sequence, as well as those produced from +1 or +2 frameshifting are indicated with the size of the TssM3 variants. (B) Western-blot analyses of <i>Y. pseudotuberculosis</i> cells producing the TssM3 WT protein or a derivative mutated in the poly-A tract (AAG) bearing a FLAG-epitope tag at the N-terminus. Crude extracts from 10⁹ cells were subjected to 10%-acrylamide SDS-PAGE and immunodetection using the anti-FLAG monoclonal antibody. The positions of the TssM3-FL and -[1–802] variants are indicated on the right using the schematic representation. The relative intensities of these two variants are indicated (as %). Molecular weight markers (in kDa) are indicated on the left. (C) Representation of the nucleotide sequence of <i>Y. pseudotuberculosis tssM3</i> constructs used for fluorescence studies shown in panel (D). The poly-A tract is shown in red and additional nucleotides are shown in blue. (D) Fluorescence levels (in arbitrary units, A.U.) of cells producing the different TssM1 variants shown in panel (C).</p

The <i>C. rodentium tssM1</i> gene encode two size variants.

<p>(A) Representation of the nucleotide sequence of <i>C. rodentium tssM1</i>. The 11-nt poly-A tract is shown in red. The codons and the amino-acids resulting from translation of the WT sequence, as well as those produced from +1 or +2 frameshifting are indicated (stops identified by the asterisks) with the size of the corresponding TssM1 variants. (B) Schematic representation of the putative localizations and topologies of the TssM1[1–807] (produced from the WT sequence), TssM1-FL (produced from +1 frameshifting) and TssM1[1–802] (produced from +2 frameshifting) proteins. The locations of the N-terminal FLAG and C-terminal 6×His epitope tags are indicated by red and blue balls respectively (IM, inner membrane; OM, outer membrane). (C) Western-blot analyses of <i>C. rodentium</i> cells producing (WT) or not (vector) the TssM1 protein bearing a FLAG-epitope tag at the N-terminus and a 6×His tag upstream the stop codon resulting from +1 frameshifting. Crude extracts from 10⁹ cells were subjected to 10%-acrylamide SDS-PAGE and immunodetection using the anti-FLAG and anti-5His monoclonal antibodies. The positions of the TssM1-FL and -[1–807] variants are indicated on the right, with the schematic representation in the middle. The relative intensities of these two TssM1 variants are indicated (as %). Molecular weight markers (in kDa) are indicated on the left.</p

The ratio between the two TssM1 variants is critical for CTS1 T6SS function.

<p>Hcp release assays. <i>C. rodentium</i> Δ<i>tssM1</i> cells expressing WT <i>tssM1</i> (WT; producing both forms of TssM1: TssM1-FL and TssM1[1–807]) or TssM1 variants bearing mutations in the poly-A tract (AAG; producing only the TssM1[1–807] variant) or mutations in the poly-A tract and a deletion of the last nucleotide (AAG<i>Δ</i>A; producing only the TssM1-FL variant) from the pASK-IBA37+ vector (A) or producing the indicated variants from the pBAD18 and pASK-IBA37+ vectors (B). Extracellular proteins were isolated by separating whole cells (C) and the supernatant fraction (S) from cultures induced with arabinose and anhydrotetracyclin. TssM1 variants were immunodetected using the anti-FLAG monoclonal antibody (upper panels). The relative intensities of the short and long TssM1 variants are indicated (as %). Total proteins were visualized by Coomassie blue staining (lower panels). Molecular mass markers are indicated on the left.</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

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