<i>P. mirabilis hfq</i> locus and Hfq protein.

<p>(<b>A</b>) Genomic organization of the <i>hfq</i> locus in <i>P. mirabilis</i>. The nucleotide numbers of the locus in the genome are listed on the top. (<b>B</b>) Alignment of Hfq proteins <i>of E. coli</i>, <i>P. mirabilis</i>, <i>P. aeruginosa</i>, <i>Salmonella</i> Typhimurium, <i>V. cholerae</i> and <i>Y. enterocolitica</i> using the NASTAR- MegAlign program. The highly conserved Sm1 and Sm2 motifs are indicated.</p

<i>P. mirabilis hfq</i> was required for motility and production of flagellin, flagella and hemolysin.

<p>(<b>A</b>) Swimming motility of the wild-type and hfq mutant. Aliquots (5 µl) of overnight culture were inoculated centrally into the LB swimming plates. The plates were incubated at 37°C. The representative picture was taken at 16 h after inoculation. (<b>B</b>) Swarming motility of the wild-type and hfq mutant. Aliquots (5 µl) of overnight culture were inoculated centrally onto the LB swarming plates. The plates were incubated at 37°C and the migration distance was measured hourly after inoculation. (<b>C</b>) The SDS-PAGE profile of flagellin of wild-type and <i>hfq</i> mutant at 3 h and 5 h after inoculation on swarming plates. The band of flagellin is indicated by an arrow. (<b>D</b>) TEM pictures of wild-type and <i>hfq</i> mutant cells. Bacterial cultures were applied onto a grid, cells were stained with 1% PTA and pictures were taken. Flagella are indicated by arrows. (<b>E</b>) Hemolysin activities of wild-type and <i>hfq</i> mutant during the swarming migration process. The representative result from three independent experiments is shown in <b>A</b>, <b>C</b> and <b>D</b>. In <b>B</b> and <b>E</b>, the data represent the average of three independent experiments with standard deviation. wt, wild-type; hfq, <i>hfq</i> mutant.</p

<i>P. mirabilis hfq</i> was required for the RpoS level and RpoS-expressing plasmid fails to rescue <i>hfq</i> mutant from the H₂O₂ killing in the stationary phase.

<p>(<b>A</b>) The level of <i>rpoS</i> mRNA in wild-type, <i>hfq</i> mutant and Hfq-complemented strain. The amounts of <i>rpoS</i> mRNA from overnight cultures of wild-type, <i>hfq</i> mutant and Hfq-complemented strain were quantified by real-time RT-PCR. The value of the wild-type was set at 100%. (<b>B</b>) The survival of wild-type, <i>hfq</i> mutant, <i>rpoS</i> mutant, RpoS-complemented strain and <i>hfq</i> mutant with the RpoS-expressing plasmid after exposure to H₂O₂. The H₂O₂ survival was determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085626#pone-0085626-g003" target="_blank">Fig. 3A</a> except that overnight cultures was used instead of cultures of the exponential phase. All the data represent the average of three independent experiments with standard deviation. wt, wild-type; hfq, <i>hfq</i> mutant; hfqc, Hfq-complemented strain; ropS, <i>rpoS</i> mutant; rpoSc, RpoS-complemented strain; hfq-pRpoS, <i>hfq</i> mutant with the RpoS- expressing plasmid.</p

Bacterial strains and plasmids used in this study.

<p>Bacterial strains and plasmids used in this study.</p

MICs of gentamicin (Gm), streptomycin (Sm), spectinomycin (Spe), ampicillin (Amp), ciprofloxacin (Cip), tetracycline (Tc), chloramphenicol (Cm) and polymyxin B (PB) for wild-type <i>P. mirabilis</i> (wt) and <i>hfq</i> mutant (hfq).

<p>MICs of gentamicin (Gm), streptomycin (Sm), spectinomycin (Spe), ampicillin (Amp), ciprofloxacin (Cip), tetracycline (Tc), chloramphenicol (Cm) and polymyxin B (PB) for wild-type <i>P. mirabilis</i> (wt) and <i>hfq</i> mutant (hfq).</p

Primers used in this study.

<p>Primers used in this study.</p

The loss of <i>P. mirabilis hfq</i> affected the OMP profile and RpoE expression.

<p>(<b>A</b>) The SDS-PAGE profile of OMPs from overnight cultures of wild-type and <i>hfq</i> mutant. The representative result from three independent experiments is shown. 1, PMI1017; 2, OmpF; 3, OmpA. (<b>B</b>) The <i>rpoE</i> promoter activities of wild-type, <i>hfq</i> mutant and Hfq-complemented strain. The activity of promoter was determined by the reporter assay in the <i>rpoE-xylE</i> reporter plasmid- transformed wild-type, <i>hfq</i> mutant and Hfq-complemented strain after incubation in LB broth for 4, 5 and 6 h. The data represent the average of three independent experiments with standard deviation. wt, wild-type; hfq, <i>hfq</i> mutant; hfqca, Hfq-complemented strain; M, marker.</p

<i>P. mirabilis hfq</i> was required for colonization of the urinary tract, infection of the burned wound, induction of cytokine expression and survival in macrophage.

<p>(<b>A</b>) Colonization of wild-type, <i>hfq</i> mutant or the Hfq-complemented strain in the mouse bladder and kidneys. Bacterial loads were determined in the bladder and kidneys on day 3 and day 6 after transurethral inoculation with suspension containing equal amount of bacteria (10⁷ CFU). Horizontal bars indicate median values for each group. Filled and open circles and triangles represent wild-type, <i>hfq</i> mutant and Hfq-complemented strain retrieved from five C57BL/6 mice, respectively. *, Significant difference by Student's t-test analysis (*p<0.01). (<b>B</b>) Bacterial loads in the burned wounds of rats infected with <i>P. mirabilis</i> wild-type, <i>hfq</i> mutant or the Hfq-complemented strain. Bacterial loads were determined from the wounds of three Wistar rats after inoculation with suspension containing equal amount of bacteria (2×10⁶ CFU/wound). The data represent the average of three rats with standard deviation. (<b>C</b>) <i>hfq</i> mutation induced expression of IL-8 and MIF. IL-8 and MIF mRNA levels of NTUB1 cells after challenging with the wild-type, <i>hfq</i> mutant or the Hfq-complemented strain were measured by real-time RT-PCR. The value of NTUB1 cells without challenging (nil) was set at 100%. (<b>D</b>) Survival of the wild-type, <i>hfq</i> mutant or the Hfq-complemented strain in macrophage. RAW264.7 macrophage cells were infected with bacteria for 30 min at an MOI of 10 and intracellular survival of the bacteria was determined by the streptomycin protection assay at 0, 2 and 4 h. The value obtained with the wild-type cells at 0 h after streptomycin treatment was set at 100%. The data represent the average of three independent experiments with standard deviation in <b>C</b> and <b>D</b>. wt, wild-type; hfq, <i>hfq</i> mutant; hfqc, Hfq-complemented strain.</p

10′(<em>Z</em>),13′(<em>E</em>)-Heptadecadienylhydroquinone Inhibits Swarming and Virulence Factors and Increases Polymyxin B Susceptibility in <em>Proteus mirabilis</em>

<div><p>In this study, we demonstrated that 10′(<em>Z</em>), 13′(<em>E</em>)-heptadecadienylhydroquinone (HQ17-2), isolated from the lacquer tree, could decrease swarming motility and hemolysin activity but increase polymyxin B (PB) susceptibilityof <em>Proteus mirabilis</em> which is intrinsically highly-resistant to PB. The increased PB susceptibility induced by HQ17-2 was also observed in clinical isolates and biofilm-grown cells. HQ17-2 could inhibit swarming in the wild-type and <em>rppA</em> mutant but not in the <em>rcsB</em> mutant, indicating that HQ17-2 inhibits swarming through the RcsB-dependent pathway, a two-component signaling pathway negatively regulating swarming and virulence factor expression. The inhibition of hemolysin activity by HQ17-2 is also mediated through the RcsB-dependent pathway, because HQ17-2 could not inhibit hemolysin activity in the <em>rcsB</em> mutant. Moreover, the finding that HQ17-2 inhibits the expression of <em>flhDC</em> gene in the wild-type and <em>rcsB-</em>complemented strain but not in the <em>rcsB</em> mutant supports the notion. By contrast, HQ17-2 could increase PB susceptibility in the wild-type and <em>rcsB</em> mutant but not in the <em>rppA</em> mutant, indicating that HQ17-2 increases PB susceptibility through the RppA-dependent pathway, a signaling pathway positively regulating PB resistance. In addition, HQ17-2 could inhibit the promoter activities of <em>rppA</em> and <em>pmrI</em>, a gene positively regulated by RppA and involved in PB resistance, in the wild-type but not in the <em>rppA</em> mutant. The inhibition of <em>rppA</em> and <em>pmrI</em> expression caused lipopolysaccharide purified from HQ17-2-treated cells to have higher affinity for PB. Altogether, this study uncovers new biological effects of HQ17-2 and provides evidence for the potential of HQ17-2 in clinical applications.</p> </div

RcsB regulates <i>flhDC</i> expression and HQ17-2 inhibits <i>flhDC</i> expression in the wild-type and the <i>rcsB-</i>complemented <i>P. mirabilis</i> but not in the <i>rcsB</i> mutant.

<p>(<b>A</b>) and (<b>B</b>) The expression of <i>flhDC</i> in the wild type <i>P. mirabilis</i> (N2), the <i>rcsB</i> mutant (rcsB) and the <i>rcsB-</i>overexpressed strain (rcsBov). In <b>A</b>, the mRNA amount of <i>flhDC</i> was quantified by real-time RT-PCR at 4 h after inoculation. In <b>B</b>, the activities of XylE in the <i>flhDC</i>-<i>xylE</i> reporter plasmid-transformed N2, rcsB, and rcsBov strains were determined at 4 h after inoculation by the reporter assay. (<b>C</b>) The expression of <i>flhDC</i> mRNAs in wild-type <i>P. mirabilis</i>, the <i>rcsB</i> mutant and the <i>rcsB</i>-complemented strain in the presence or absence of 72.6 µM HQ17-2. The mRNA amount of <i>flhDC</i> was quantified by real-time RT-PCR after incubation with HQ17-2 for 4 h. In <b>A</b> and <b>B</b>, the value obtained for the wild-type was defined as 100%, and other values were expressed relative to this value. *, a significant difference was observed (P<0.01) in comparing to the wild-type. In <b>C</b>, the value obtained for the wild-type cells in the absence of HQ17-2 was defined as 100%. All data represent the averages of three independent experiments with standard deviations. A significant difference was observed by Student’s <i>t-</i>test analysis (*, P<0.01).</p