mrI in P. mirabilis. To our knowledge, this is the first report demonstrating that a natural compound, HQ17-2, can reduce virulence factor expression and enhance efficacy of PB in P. mirabilis. This study not only uncovers new biological effects of HQ17-2 but also provides evidence for the potential of HQ17-2 in clinical applications. Materials and Methods Bacterial Strains, Plasmids, Reagents and Growth Conditions The bacterial strains and plasmids used in this study are listed in Swarming Assay The swarming migration assays was performed as described previously. Briefly, an overnight bacterial culture was inoculated centrally onto the surface of dry LB swarming plates containing 2% agar with or without HQ17-2, which were then incubated at 37uC. The swarming migration distance was measured by following swarm fronts of the bacterial cells at 1 h intervals. Measurement of Hemolysin Activity An overnight LB culture of the bacteria was inoculated onto the surface of dry LB swarming plates with or without HQ17-2, which were then incubated at 37uC. Hemolysin activity was determined 2 h after inoculation and hourly thereafter. Preparation of cells for hemolysin assay and measurement of hemolysin activity were performed as described previously. Cell Invasion Assay The cell invasion assay was performed as described by Jiang et al.. An overnight P. mirabilis culture was diluted 100-fold and incubated for 3 h before the cell invasion assay was performed. In this study, HQ17-2 was included in the bacterial suspension during the 1.5 h infection period to investigate the effect of HQ17-2 on the cell invasion ability of P. mirabilis. Construction of P. mirabilis rcsB Mutant For construction of the rcsB mutant, the primer pair, RcsB1/ RcsB2, was used to amplify the central region of rcsB. The PCR product was cloned into pGEM-T Easy vector to form pGEM-rcsBc. The pGEM-rcsBc was then digested with XbaI, and ligated with 2 Hydroquinone Modulates Virulence, Drug Sensitivity Strain or plasmid Proteus mirabilis N2 dA10 dA10c dIp dIpc rcsB rcsBc rcsBov CI1,11 Plasmids pGEM-T Easy pUT/mini-Tn5 pACYC184-rppA-xylE pACYC184-pmrI-xylE pACYC184-flhDC-xylE doi:10.1371/journal.pone.0045563.t001 Genotype or relevant phenotype Source or reference Wild-type; Tcr N2 derivative; rppA-knockout mutant; PBs Kmr dA10 containing pGEM-T Easy-rppA; rppA-complemented strain; Ampr N2 derivative; pmrI knockout mutant; PBs Kmr dIp containing pACYC184-pmrI; pmrI-complemented strain; Cmr N2 derivative; rcsB-knockout mutant; Kmr rcsB containing pACYC184-rcsB; rcsB-complemented strain; Cm r This study This study This study Clinical isolates rcsB-overexpressed N2; rcsB cloned into the high-copy pGEM-T Easy with intact rcsB oriented behind lac promoter; Ampr Highly resistant to PB High-copy TA cloning vector; Ampr A suicide plasmid requiring Pir protein for replication and containing mini-Tn5 cassette containing Kmr gene pACYC184 containing rppA promoter sequence and xylE coding region; Cmr pACYC184 containing pmrI promoter sequence and xylE coding region; Cm r Promega This study This study pACYC184 containing flhDC promoter sequence and xylE coding region; Cmr the Km-resistant cassette to form pGEM-rcsBc/Km. The rcsBc/Kmcontaining fragment was cut with SalI-SphI and ligated into Debio1347 site SalISphI 
digested pUT vector to form pUT-rcscBc/Km. For inactivation of rcsB gene by homologous recombination, pUT-rcsBc/Km was transferred from E. coli S17-1 to P. mirabilis N2 by conjugation. Transconjugants w