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As a follow-up to the LD-RT-PCR results, fimB expression was also monitored using strain NU149/pWS145-38 cells grown in pH 5.5 and pH 7.0 LB were mixed with plain Sepharose beads, mannose-coated Sepharose beads, and mannose-coated Sepharose beads with 2% free mannose added.
Binding of type 1 pili to mannose receptors appeared to shift transcription to favor fimB over fimE.
Changes in the levels of fimB and fimE transcripts combined with alterations in the invertible element suggested that the type 1 pilus expression was conceivably altered after type 1 pilus binding to mannose.
Several mutants were examined (including a cpxR mutant strain) to try to elucidate which gene product may be regulating the fim genes following binding to mannose receptors, but no gene was linked directly with the regulatory changes affecting fimB or fimE (data not shown).
In this study, we have shown that FimH mutants associated with the mannose-binding pocket [29] affected the FimH ligand binding to mannose residues and subsequent changes in fimB and fimE transcription within the UPEC cells.
The binding of FimH to its tethered mannose receptor causes transcriptional activation of the fimB gene that leads to increased type 1 pili expression.
However, only one study has examined fimB expression in UPEC growing in murine bladders, but this study was limited to a 48 h period and did not examine fim recombinase gene transcription in infected murine kidneys [35].
In order to address whether there is temporal regulation of fim genes in UPEC cells growing in murine urinary tracts, we constructed fimA, fimB, and fimE-lux transcriptional fusions and moved these fusions into a UPEC strain.
The pP5-48 plasmid DNA containing the fimB promoters [28] was extracted as described above, leading to creation of the pWS141-2, pWS144-27, and ultimately the pWS145-38 plasmid.
At 8 hpi, fimB expression was 0.419 RLU/CFU (Figure 3(c)).
Unlike the fimA and fimB transcription results in UPEC infected murine bladders, fimE transcription was much lower than either fimA or fimB transcription.
The fim-lux fusion results demonstrated a temporal regulation of fimA, fimB, and fimE within a UPEC strain growing in murine urinary tracts.