Temporal Regulation of fim Genes in Uropathogenic Escherichia coli during Infection of the Murine Urinary Tract

Figure 4

Determination of the fimS invertible element orientation by PCR on chromosomal DNA isolated from NU149 grown in LB as well as NU149 infected murine bladder and kidney homogenates spanning a five-day infection period. Two random different UPEC infected murine bladder and kidney homogenates were screened for each time point. Multiplex PCRs were set up with INV and FIMA primers to amplify Phase-ON-oriented DNA (ON, 450 bp product) [28], FIME and INV primers to amplify Phase-OFF-oriented DNA (OFF, 750 bp product) [41], and EcFtsZ 1 and 2 primers to amplify the ftsZ gene (302 bp product) [45]. Each multiplex was run at least three separate times. The lanes were loaded onto a 1.5 % agarose gel as follows: lane 1, NU149 inoculum; lane 2, NU149 infected bladder mouse 1 day 0.33; lane 3, NU149 infected bladder mouse 2 day 0.33; lane 4, NU149 infected kidney mouse 1 day 0.33; lane 5, NU149 infected kidney mouse 2 day 0.33; lane 6, NU149 infected bladder mouse 1 day 1; lane 7, NU149 infected bladder mouse 2 day 1; lane 8, NU149 infected kidney mouse 1 day 1; lane 9, NU149 infected kidney mouse 2 day 1; lane 10, NU149 infected bladder mouse 1 day 3; lane 11, NU149 infected bladder mouse 2 day 3; lane 12, NU149 infected kidney mouse 1 day 3; lane 13, NU149 infected kidney mouse 2 day 3; lane 14, NU149 infected bladder mouse 1 day 5; lane 15, NU149 infected bladder mouse 2 day 5; lane 16, NU149 infected kidney mouse 1 day 5; lane 17, NU149 infected kidney mouse 2 day 5. For each lane, the intensities of the OFF and ON states were quantified using ImageQuant software (Molecular Dynamics) and corrected to the intensity of the ftsZ band. The corrected values for both states were standardized to the respective wild-type band (lane 1).