Hfq impact on bladder cell infection.

<p>PD07i bladder cells cultured in 24-well plates were infected with UPEC strains cultured overnight at 37°C in LB medium. Mean CFU counts of triplicates from three independent experiments were plotted and standard deviation calculated. UTI89Δ<i>hfq</i> showed a statistically significant reduction in adhesion as well as invasion (*<i>p-value</i> <0.05).</p

Model illustrating PapR-mediated modulation of P-fimbriae phase variation.

<p>The <i>pap</i> gene cluster encodes two regulatory proteins, PapI and PapB, that work in concert with other global regulators such as LRP, H-NS and Dam methylase to control P-fimbrial phase variation between the OFF and ON states. LRP mediated transcriptional activation of PapR sRNA results in the degradation of <i>papI</i> mRNA. An absence of functional PapI results in a failure to switch from an OFF to an ON phase, and a failure therein to express P-fimbriae on the surface.</p

LRP mediated transcriptional activation of PapR.

<p>(A) PapR transcriptional start site was mapped using primer extension reaction loaded in the lane marked 1 alongside Sanger sequencing reactions for the four nucleotides represented in lanes GCAT. (B) Northern blotting was used to detect PapR levels in UTI89/pNDM220 (lane 1), UTI89Δ<i>lrp</i>/pNDM220 (lane 3) and UTI89Δ<i>hfq</i>/pNDM220 (lane 5) with the respective complemented strains UTI89Δ<i>lrp</i>/pSKlrp (lane 2) and UTI89Δ<i>hfq</i>/pJMJ220 (lane 4), all cultured in LB medium. 5S RNA was used as the internal loading control. (C) Illustration of the genomic context of PapR in the UTI89 genome, drawn to scale. LRP was found to positively regulate <i>papR</i> transcription.</p

Characterization of PapR function.

<p>(A) Heme- and yeast cell agglutination assays performed with UTI89/pNDM220, UTI89Δ<i>papR</i>/pNDM220 and UTI89Δ<i>papR</i>/pSK1 with and without the addition of α-D-mannose. Scale bars set at 50 μm. (B) PD07i bladder cells and IMCD3 kidney collecting duct cells cultured in 24-well plates were infected with UTI89/pNDM220, UTI89Δ<i>papR</i>/pNDM220, UTI89Δ<i>papR</i>/pSK1 and UTI89Δ<i>hfq</i>. Strains were either left untreated (-) or treated (+) with 3% α-D-mannose and used for infection. Bacterial adhesion was assessed by calculating mean CFU counts from three independent experiments. Statistical significance was calculated using Students t-test (*<i>p-value</i> <0.05). (C) Flow cytometry of UTI89/pNDM220, UTI89Δ<i>papR</i>/pNDM220 and UTI89Δ<i>papR</i>/pSK1 grown overnight in LB medium and immunolabelled with α-PapA and α-Fim antibodies was used to detect the extent of P- and type-1 fimbriated cells respectively. Mean fluorescence from three independent experiments was plotted along with the standard deviations. Statistical significance was calculated using Students t-test (*<i>p-value</i> <0.05).</p

Novel sRNAs detected in UTI89.

<p>(A) Northern blots showing transcript levels for PapR and C271. Lanes 1 and 2 represent RNA from culture reference and infection respectively. (B) Graphical output of normalized sequence reads mapping to PapR and C271 in culture reference and infection visualized using Integrated Genome Viewer (Broad Institute). The table below includes normalized total read counts within the PapR and C271 probes.</p

The structure of LeoA shows it to be related to prokaryotic and eukaryotic dynamin-like proteins.

<p>A: Comparison of different dynamin structures to the structure of LeoA, unequivocally showing it to belong to the dynamin family of proteins. B: Although the trunk structures superimpose poorly, their overall architecture and topology is conserved (scheme on the right). C: Stereo plot superposition of BDLP1 (PDB 2J68, GDP-bound form) and LeoA nucleotide-binding pockets. In this study GTP binding or hydrolysis by LeoA was not observed. The structure of the LeoA GTPase domain shows a distorted geometry, especially around loop and helices 225–248, although this sort of deviation is not uncommon for nucleotide-free structures of genuine nucleotide-binding proteins. BDLP1 is in blue, LeoA in orange, with the GDP from the BDLP1 structure in grey. The GTPase domains of BDLP1 and LeoA (residues 54–281 and 69–324, respectively) were aligned using Cα atoms only, with a resulting RMSD of 3.1 Å. D: LeoA reveals a novel conformation for the dynamin family. Superposition of BDLP1-apo (2J69), BDLP1-GMPPNP (2W6D) and human dynamin 1 (3ZVR) crystal structures is shown, using the LeoA (4AUR) GTPase domain as a reference. The attachment angle of the trunks to the GTPase domains is very different, and this presumably leads to different polymer assembly on lipid membranes and consequently different functional mechanisms.</p

Crystallographic data.

<p>SeMet data values for peak wavelength, only.</p>1<p>Values in parentheses refer to the highest recorded resolution shell.</p>2<p>Anomalous correlation coefficient between half sets (SCALA) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107211#pone.0107211-Winn1" target="_blank">[19]</a>.</p>3<p>5% of reflections were randomly selected before refinement.</p>4<p>Percentage of residues in the Ramachandran plot (PROCHECK) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107211#pone.0107211-Winn1" target="_blank">[19]</a>.</p><p>Crystallographic data.</p

LeoA is part of a conserved putative operon.

<p>A: overview of the <i>tia</i> locus in <i>E. coli</i> ETEC H10407; modified from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107211#pone.0107211-Fleckenstein1" target="_blank">[13]</a>. An annotated version of the locus with probable promoter and RBS sites is shown in Figure S1 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107211#pone.0107211.s001" target="_blank">File S1</a>. B: Surface plot of the LeoA monomer coloured by domains: yellow, GTPase domain; red, neck domain; blue, trunk domain; green, putative paddle region. Similarly coloured hydropathy plots (TMpred) provide transmembrane prediction for LeoA and LeoB, respectively. C: <i>Orf2</i> and <i>orf3</i> of the <i>tia</i> locus align well against <i>orf4</i>, which encodes LeoA and the alignment spans the entire length of LeoA. It seems that LeoA is encoded in tandem with another in sequence related <i>orf</i> that is split into two chains. <i>Orf2</i> and <i>orf3</i> have been renamed here <i>leoC</i> and <i>leoB</i>, respectively. Sequences aligned: LeoA (E. coli ETEC H10407), WP_011717023.1 (<i>Shewanella sp. ANA-3</i>), WP_007214706.1 (<i>Bacteroides cellulosilyticus</i>), WP_001006159.1 (<i>Helicobacter pylori</i>), WP_001006151.1 (<i>Helicobacter pylori</i>), WP_014535968.1 (<i>Helicobacter pylori</i>), WP_000787447.1 (<i>Helicobacter pylori</i>), WP_001006093.1 (<i>Helicobacter pylori</i>), WP_000787451.1 (<i>Helicobacter pylori</i>), WP_005966123.1 (<i>Fusobacterium periodonticum</i>). D: Tandem genes for bacterial DLPs are common. Previously reported were IniA and IniC, and DynA, which is a fusion of two DLP genes <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107211#pone.0107211-Alland1" target="_blank">[37]</a>. <i>Nostoc</i> BDLP1 occurs in tandem with BDLP2 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107211#pone.0107211-Brmann1" target="_blank">[6]</a>. YjdA does not seem to follow this pattern. LeoABC shows splitting of the first gene into two as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107211#pone-0107211-g001" target="_blank">Figure 1B and C</a>. Dimensions are approximate. A large non-coding region (525 bp) between BDLP1 and BDLP2 is indicated. E: Purified His₆-tagged LeoA protein, over-expressed in <i>E. coli</i> and purified by metal affinity and size exclusion chromatography.</p

The crystal structure of LeoA.

<p>A: Schematic sequence alignment showing the similarity of LeoA to <i>Nostoc</i> BDLP1 and eukaryotic mitofusin Fzo1. In order to make the ClustalW alignment more stable, the 10 top hits from BLAST searches, with each of the three sequences, were included for each family. Conservation stretches across all three families, with all major domains of known function and fold being conserved, leading to the conclusion that LeoA is a <i>bona fide</i> bacterial dynamin-like protein. B: The 2.7 Å crystal structure of LeoA from <i>E. coli</i> ETEC H10407 shows an elongated molecule with the conserved GTPase domain followed by the trunk and tip regions. The conformation of the trunk relative to the GTPase domain, mediated by Gly274, is novel and reveals a ‘flattened’ conformation reminiscent of that observed in the DLP human guanylate-binding protein 1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107211#pone.0107211-Prakash1" target="_blank">[32]</a>. The putative paddle region at the trunk tip is dominated by hydrophobic residues in conserved positions known to be critical for lipid binding in <i>Nostoc</i> BDLP1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107211#pone.0107211-Low3" target="_blank">[9]</a>. A rainbow colour scheme is used from the N (blue) to the C (red) terminus. C: Close-up of neck and GTPase domains, rotated approximately by 180° with respect to main part of panel B.</p

LeoA is localised in the periplasm and the <i>leoAB</i> genes enhance vesicle-based protein export.

<p>A: Using a polyclonal LeoA antibody, the sub-cellular localisation of LeoA in WT <i>E. coli</i> ETEC H10407 was investigated. The protein at endogenous expression levels is easily detected and this signal disappears in a <i>leoA</i> KO strain, demonstrating specificity. A classical fractionation experiment with sub-cellular marker proteins clearly shows LeoA to be localised in the periplasm and possibly in the inner membrane. Figure S5 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107211#pone.0107211.s001" target="_blank">File S1</a>, top shows the same experiment with a 3xFLAG-tagged version of LeoA. B: Immunofluorescence using the <i>leoA</i>-3xFLAG fusion strain shows a punctate pattern, which is specific to the presence of the fusion (C, right). See Figure S2 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107211#pone.0107211.s001" target="_blank">File S1</a> for quantification of preferential polar and midcell localisation. D: Quantified Western blots demonstrating the influence of Leo proteins on protein secretion into the culture supernatant, presumably via vesicles. Both OmpA and a twin arginine-exported GFP reporter construct tend to accumulate in H10407Δ<i>leoAB</i> whole-cell lysates (left). GroEL served as an internal loading control (Figure S6 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107211#pone.0107211.s001" target="_blank">File S1</a> shows the original blots and quantification data from two biological replicates). Right: conversely, OmpA and Tat-GFP levels are reduced by about 50% in culture supernatants (vesicle fractions) from the H10407Δ<i>leoAB</i> mutant strain. Supplying extra LeoA protein from a plasmid reverses the effect of the <i>leoA</i> deletion (last column).</p