Effect of <i>fis</i> and <i>typA</i> deletion in UTI89 on <i>sslE</i> expression determined via western blot and qRT-PCR analysis

<p>(A) Western blot analysis of SslE using (i) whole-cell lysates and (iii) supernatant fractions prepared from UTI89, UTI89<i>fis</i> and UTI89<i>fis</i>(pFis). (ii) Western blot loading control for whole cell lysate samples using an OmpA antibody. (B) Western blot analysis of SslE using whole-cell lysates and supernatant fractions prepared from UTI89, UTI89<i>typA</i> and UTI89<i>typA</i>(pTypA). (ii) Western blot loading control for whole cell lysate samples using an OmpA antibody. (C) Relative fold-difference of <i>sslE</i> transcript levels of UTI89, UTI89<i>fis</i> and UTI89<i>fis</i>(pFis); mRNA levels were calculated relative to the level of UTI89 <i>sslE</i> mRNA. (D) Relative fold-difference of <i>sslE</i> transcript levels of UTI89, UTI89<i>typA</i> and UTI89<i>typA</i>(pTypA); mRNA levels were calculated relative to the level of UTI89 <i>sslE</i> mRNA. The relative <i>sslE</i> mRNA levels were consistent with the data observed from the western blot analysis. The data was obtained from three independent experiments; error bars indicate standard deviation. Statistical analysis was performed using an unpaired, two-tailed t-test.</p

Effect of <i>fis</i> and <i>hns</i> double deletion on <i>sslE</i> expression in UTI89 determined via western blot and qRT-PCR analysis.

<p>(A) Western blot analysis of SslE using (i) whole-cell lysates and (iii) supernatant fractions prepared from UTI89, UTI89<i>hns</i>, UTI89<i>fis</i> and UTI89<i>fis hns</i>. (ii) Western blot loading control for whole cell lysate samples using an OmpA antibody. The overall level of SslE was reduced in UTI89<i>fis hns</i> compared to wild-type UTI89. (B) Relative fold-difference of <i>sslE</i> transcript levels of UTI89, UTI89<i>hns</i>, UTI89<i>fis</i> and UTI89<i>fis hns</i>. All mRNA levels were calculated relative to the level of UTI89 <i>sslE</i> mRNA. The relative <i>sslE</i> mRNA levels were consistent with the data observed from the western blot analysis. The data was obtained from three independent experiments; error bars indicate standard deviation.</p

Effect of <i>fis</i> deletion on <i>sslE</i> expression in strains IHE3034, 536 and EC958 determined via western blot and qRT-PCR analysis.

<p>Western blot analysis of SslE using preparations from (A) IHE3034, (B) 536 and (C) EC958; each with their respective <i>fis</i> mutant and complemented strains. In each analysis, (i) whole-cell lysates and (iii) supernatant fractions were examined. In addition, (ii) a control for whole cell lysate samples was performed using an OmpA antibody. (D) Relative fold-difference of <i>sslE</i> transcript levels of IHE3034, 536 and EC958, and their respective <i>fis</i> mutant and complemented strains. All mRNA levels were calculated relative to the level of IHE3034 <i>sslE</i> mRNA. The relative <i>sslE</i> mRNA levels were consistent with the data observed from the western blot analysis. The data was obtained from three independent experiments; error bars indicate standard deviation. Statistical analysis was performed using an unpaired, two-tailed t-test.</p

Motility phenotypes of EC958 hyper-motility mutants.

<p>(A) The diameter of the swimming zone from EC958 (wild-type) and mutant strains was measured after 7 hours of incubation at 37°C on 0.25% LB agar. The results are based on three replicates; error bars indicate standard deviations. (B) Comparative motility of the same wild-type and mutant strains in 0.25% LB agar. The results from a single experiment, representative of three independent experiments, are shown.</p

Motility of the (A) EC958mprA, (B) EC958hemK, (C) EC958yjeA mutants and their respective parent and complemented strains.

<p>Left panels, rate of motility expressed as the mean diameter of the swimming zone per hour for each strain (+/-standard deviation; n = 3). Right panels, representative motility plates comparing the swimming phenotype of wild-type, mutant and complemented strains.</p

Promoter region of <i>sslE</i> in UTI89 and analysis of the effect of putative Fis-binding sequences on <i>sslE</i> promoter activity

<p>(A) Promoter region of <i>sslE</i> in UTI89. The transcription start site as determined via 5'RACE is indicated as +1. Putative -10 and -35 promoter sites and the ATG translational start site are indicated accordingly. The region cloned into the promoter-less <i>lacZ</i> reporter plasmid pQF50 is indicated by arrows showing the binding positions of the primers used to amplify the fragment. Putative Fis-binding sites are indicated in bold and overlined. Fis-binding region 1 (F1) consists of one Fis consensus binding sequence. Fis-binding region 2 (F2) consists of three over-lapping consensus sequences. The nucleotide changes introduced to disrupt the Fis-binding sites in the constructs are indicated by an arrow depicting the nucleotide change. (B) β-galactosidase activity represented as Miller units measured for the various <i>sslE</i> promoter-<i>lacZ</i> reporter plasmid constructs in UTI89<i>lacI-Z</i> (wild-type) and UTI89<i>lacI-Z fis</i>:<i>kan</i> (<i>fis</i> mutant) strains. Plasmid pQF50-<i>sslE</i> containing the intact <i>sslE</i> promoter resulted in approximately double the β-galactosidase activity in the wild-type compared to the <i>fis</i> mutant (p < 0.05; t-test). Plasmid pQF50-<i>sslE</i>M1 containing mutations in F1 resulted in similar levels of β-galactosidase activity to pQF50-<i>sslE</i> in both strains. Plasmids pQF50-<i>sslE</i>M2 and pQF50-<i>sslE</i>M3, which were disrupted in F2 and F1/F2, respectively, resulted in a similarly reduced β-galactosidase activity in both the wild-type and the <i>fis</i> mutant strains (not significant; ns), indicating that F2 is important for Fis activation of the <i>sslE</i> promoter.</p

Fitness of CFT073 capsule (<i>kpsD</i>) and O antigen (<i>waaL</i>) mutants during <i>in vivo</i> competition with CFT073 WT and corresponding complemented (<i>kpsD</i>^C, <i>waaL</i>^C) strains.

<p>C57B/L6 mice were infected transurethally with a 1∶1 mixture of CFT073<i>kpsD</i> and CFT073 WT or CFT073<i>kpsD</i>^C; CFT073<i>waaL</i> and CFT073 WT or CFT073<i>waaL</i>^C. (A) Each marker represents LOG₁₀ total CFU recovered from each mouse per 0.1 g of bladder tissue. Lines connect data points for the same mouse, and horizontal bars represent median values. (B) Each marker represents the LOG₁₀ competitive index calculated for each individual mouse; competitive indices are the ratio of the mutant 0.1 g of bladder tissue to that of WT or complemented strain. Dashed lines represent hypothetical competitive index of 1 (LOG₁₀1 = 0), which indicates no difference in fitness between the two strains. Horizontal bars represent group medians, and each competition group had 6 to 8 mice. All mutants were significantly outcompeted by CFT073 WT or their respective complemented strains for bladder colonization (<i>P</i><0.05). The O6 antigen was significantly more important than the K2 capsular antigen for bladder colonization (<i>P<</i>0.05).</p

Posouzení nosných konstrukcí při regeneraci a rekonstrukci staveb

Import 20/04/2006Prezenční výpůjčkaVŠB - Technická univerzita Ostrava. Fakulta hornicko-geologická. Institut bezpečnostního inženýrství (547

Genes identified by TraDIS in the motility screen.

<p>Genes identified by TraDIS in the motility screen.</p

Western blot and qRT-PCR analysis to examine <i>sslE</i> expression in ExPEC strains.

<p>(A) Western blot analysis of SslE from (i) whole-cell lysates and (iii) supernatant fractions prepared from UTI89, UTI89<i>sslE</i>, IHE3034, IHE3034<i>sslE</i>, 536, EC958 and MG1655. SslE has a predicted size of approximately 167 kDa. Two major cross-reacting bands of this size were detected (indicated by arrows), possibly representing full-length and processed SslE. No cross-reacting bands were detected in samples prepared from UTI89<i>sslE</i> and IHE3034<i>sslE</i>, demonstrating the specificity of antibody. (ii) Loading control for whole cell lysate samples. The same samples used above were examined by western blot using an OmpA antibody. Similar levels of OmpA were detected in all samples, indicating equivalent loading of total protein. (B) Relative fold-difference of <i>sslE</i> transcript levels of ExPEC strains UTI89, IHE3034, 536, EC958 as determined by qRT-PCR. All mRNA levels were calculated relative to the level of UTI89 <i>sslE</i> mRNA. The relative <i>sslE</i> mRNA levels were consistent with the data observed from the western blot analysis. The data was obtained from three independent experiments; error bars indicate standard deviation.</p