PMC

EIA analyses of strain NU149 grown in a pH 5.5 or 7 environment mixed with plain Sepharose beads, mannose-coated Sepharose beads, or mannose-coated Sepharose beads with 2% free mannose (wt/vol) added. Columns represent NU149 grown in pH 5.5 LB mixed with mannose-coated Sepharose beads (black column), plain Sepharose beads (white column), or mannose-coated Sepharose beads plus 2% free D-mannose (gray column) as well as NU149 grown in pH 7 LB mixed with mannose-coated Sepharose beads (left striped column), plain Sepharose beads (white dots column), or mannose-coated Sepharose beads plus 2% free D-mannose (right striped column). Optical densities at 492 (O.D.₄₉₂) were determined. The data represents the means ± standard deviations from at least three separate experiments. ∗ equals P < 0.05 and ∗∗ equals P < 0.0001.

Effects of fimB transcription in strain NU149/pWS145-38 containing a fimB-lux transcriptional fusion grown in a pH 5.5 or 7 environment mixed with plain Sepharose beads, mannose-coated Sepharose beads, of mannose-coated Sepharose beads with 2% free mannose (wt/vol) added. Columns represent NU149 grown in pH 5.5 LB mixed with mannose-coated Sepharose beads (black column), plain Sepharose beads (white column), or mannose-coated Sepharose beads plus 2% free D-mannose (gray column) as well as NU149 grown in pH 7 LB mixed with mannose-coated Sepharose beads (left striped column), plain Sepharose beads (white dots column), or mannose-coated Sepharose beads plus 2% free D-mannose (right striped column). The RLU/CFU were calculated by using a luminometer to measure luminescence, subtracting out the background, and then dividing by viable counts. The data represents that the means ± standard deviations are indicated from at least three separate runs. ∗ equals P < 0.05 and ∗∗ equals P < 0.0001.

Quantitative determination of mRNA regulation by LD-RT-PCR analysis of cDNAs of strain NU149 cells mixed with plain Sepharose beads (−) or mannose-coated Sepharose beads (+) for 10, 25, 60, or 120 min. The FimB1/FimB2, FimE1/FimE2, and EcFtsZ1/EcFtsZ2 primer pairs were used to amplify serially twofold diluted cDNAs and targeted fimB (379 bp product), fimE (392 bp product), and ftsZ (302 bp product) transcripts, respectively. All PCR products were electrophoresed on 1.5% agarose gels. The following dilutions of cDNAs were used: undiluted (lane 1), 1/2 (lane 2), 1/4 (lane 3), 1/8 (lane 4), 1/16 (lane 5), 1/32 (lane 6), 1/64 (lane 7), 1/128 (lane 8), and 1/256 (lane 9). The data represent at least three separate runs.

Determination of the fimS invertible element orientation in strain NU149 mixed with plain Sepharose beads or mannose-coated Sepharose beads in pH 5.5 or pH 7.0 media by PCR analysis. The PCR analysis was performed with chromosomal DNA isolated from the NU149 cells using the INV and FIMA primers to amplify Phase-ON-oriented DNA (450 bp product), FIME and INV primers to amplify Phase-OFF-oriented DNA (750 bp product), and EcFtsZ1 and EcFtsZ2 primers to amplify the ftsZ gene (302 bp product). The products were standardized against the ftsZ product using ImageQuant software and the corrected values for both orientations were standardized to the respective 0 h time point. The lanes were loaded as follows: MW = molecular weight standard; lane 1, NU149 at time 0 h; lane 2, NU149 time 24 h, mannose-coated at pH 7.0; lane 3, NU149 time 24 h, plain at 24 h; lane 4, NU149 time 24 h, mannose-coated at pH 5.5; lane 5, NU149 time 24 h, plain at pH 5.5. All PCR products were electrophoresed on 1.5% agarose gels.

Quantitative determination of the fimS invertible element orientation of E. coli cells with a plasmid that has the FimH protein represented as wild type (WT), Null, Q133K, or N46A mixed with mannose-coated Sepharose beads (+) for 0 h, 4 h, or 24 h. The PCR analysis was performed with twofold dilutions of chromosomal DNA isolated from the E. coli cells using the INV and FIMA primers to amplify Phase-ON-oriented DNA (450 bp product) or FIME and INV primers to amplify Phase-OFF-oriented DNA (750 bp product). All PCR products were electrophoresed on 1.5% agarose gels. The following dilutions of DNA were used: undiluted (lane 1), 1/2 (lane 2), 1/4 (lane 3), 1/8 (lane 4), 1/16 (lane 5), 1/32 (lane 6), 1/64 (lane 7), 1/128 (lane 8), 1/256 (lane 9), and 1/512 (lane 10). The data represent at least three separate runs.