<i>E. coli</i> strains and plasmids used in this study.

a<p>Cb^r: carbenicillin resistant, Cm^r: chloramphenicol resistant, Km^r: kanamycin resistant, Spec^r; spectinomycin resistant.</p

Calculated binding rates of pili and bacteria at different pH.

<p>Panels A and B show the binding to galabiose-BSA-coated surface (black bars) and BSA-coated surface (gray bars) for pili and bacteria respectively. Panels C and D show the specific binding rates of pili and bacteria respectively, calculated as difference between binding to galabiose-BSA-coated surfaces and BSA-coated surfaces obtained in SPR experiments.</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

Analysis of OMV preparations.

<p>(A) The OMVs were isolated from the supernatant of a 80-ml bacterial liquid culture incubated 14 hours under shaking conditions. Finally, each OMV preparation was resuspended with 200 μl of 20 mM Tris-Cl (pH 8.0). Five μl of each OMV sample per well was run on 12% PAGE and subjected to silver staining. Total protein amounts (mg) of OMVs from a 80-ml bacterial culture of each strain were determined by Bradford assay and are presented below each lane. Lanes; 1, BW25113; 2, RN102; 3, BW25113/pNTR-SD; 4, RN102/pNTR-SD; 5, RN102/pNT3(<i>hldE</i>). (B and C) AFM images of OMVs prepared from BW25113 and RN102 on 1 μm² surfaces were shown.</p

Loss of outer membrane integrity in strain RN102.

<p>Bacterial samples were collected from 48-hour-cultured biofilms for TEM analysis. TEM images of the bacterial cells and the cell appendages are shown for strains: (A) BW25113, (B) RN102, (C) BW25113/pNTR-SD, (D) RN102/pNTR-SD, and (E) RN102/pNT3(<i>hldE</i>). The outer membranes are indicated by arrows. Representative electron-microphotographs of each strain are shown. A 500-nm-long bar is shown in the lower left corner of each eclectron-micrograph. (F) Western blot analysis of supernatants from BW25113 and RN102. Supernatants were harvested by centrifugation from bacterial liquid culture grown for 48 hours under static conditions. Results of Western blot using anti-Crp, anti-DsbA, anti-OmpC, and anti-OmpA antisera are shown. (G) Supernatants from bacterial liquid cultures of BW25113 or RN102 grown for 48 hours under static conditions were serially diluted with TE. The diluted samples were used as template DNA for PCR using <i>E. coli atoS</i> gene-specific primer pairs. Lanes: 1, without dilution; 2, 10⁻¹ dilution; 3, 10⁻² dilution; 4, 10⁻³ dilution; 5, 10⁻⁴ dilution; 6, 10⁻⁵ dilution; 7, 10⁻⁶ dilution.</p

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

Autoaggregation phenotype by LPS mutants.

<p>Each strain standardized at OD₆₀₀ = 1.0 in PBS was used for autoaggregation assay. The value at OD₆₀₀ after an18-hour incubation is shown as the mean ± SD of results from three independent experiments. Statistical analysis was performed using ANOVA. *<i>P</i><0.05, against autoaggregation level of strain BW25113.</p

AFM micrographs of HB101/pPAP5 cells expressing P pili.

<p>AFM micrographs of HB101/pPAP5 cells expressing P pili.</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

Generalized LPS structures in a series of <i>E. coli</i> LPS mutants used in this study.

<p>A silver-stained polyacrylamide gel after SDS-PAGE is shown in the upper panel. In the lower panel, the drawing of LPS structures especially highlights the core OS portion of LPS. Structures of the major glycoforms of core OS are based on a structural analysis of a K-12 strain, W3100 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051241#pone.0051241-MullerLoennies1" target="_blank">[59]</a> and a <i>waaP</i> mutant of strain R1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051241#pone.0051241-Yethon1" target="_blank">[60]</a>. The LPS core OS composition of the <i>hldE</i> strain (RN102) is the same as that of the <i>waaC</i> strain (RN101), probably because the lack of HldE protein results in arrest of ADP-Hep biosynthesis. It has been reported that in the <i>E. coli</i> R1 strain, inactivation of the <i>waaP</i> gene results not only in the loss of all phosphate groups on HepI and HepII, but also loss of HepIII <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051241#pone.0051241-Yethon1" target="_blank">[60]</a>. We could not detect any difference in size of LPS between RN107 and BW25113 or RN105, although the loss of Gal (shown by an asterisk) was expected theoretically. Therefore, the LPS structure of RN107 was described based on the information of the SDS-PAGE gel in this study. A more precise analysis is required to fully understand the LPS structure of RN107. Each sugar or amino sugar of core OS is shown by a black (Hep), blue (Glc), red (Gal), green (Kdo), or gray (GlcN) circle. Phosphate groups (Phosphate or pyrophosphorylethanolamine) modified on sugar are shown by yellow boxes (P/PPEtN). Hep: L-<i>glycero</i>-D-<i>manno</i>-heptose, Kdo: 3-deoxy-D-<i>manno</i>-oct-2-ulosonic acid, GlcN: <i>N</i>-acetylglucosamine.</p