OmpR regulation of the uropathogenic Escherichia coli fimB gene in an acidic/high osmolality environment

Abstract

Uropathogenic Escherichia coli (UPEC) causes more than 90 % of all human urinary tract infections through type 1 piliated UPEC cells binding to bladder epithelial cells. The FimB and FimE site-specific recombinases orient the fimS element containing the fimA structural gene promoter. Regulation of fimB and fimE depends on environmental pH and osmolality. The EnvZ/OmpR two-component system affects osmoregulation in E. coli. To ascertain if OmpR directly regulated the fimB gene promoters, gel mobility shift and DNase I footprinting experiments were performed using OmpR or phosphorylated OmpR (OmpR-P) mixed with the fimB promoter regions of UPEC strain NU149. Both OmpR-P and OmpR bound weakly to one fimB promoter. Because there was weak binding to one fimB promoter, strain NU149 was grown in different pH and osmolality environments, and total RNAs were extracted from each population and converted to cDNAs. Quantitative reverse-transcriptase PCR showed no differences in ompR transcription among the different growth conditions. Conversely, Western blots showed a significant increase in OmpR protein in UPEC cells grown in a combined low pH/high osmolality environment versus a neutral pH/high osmolality environment. In a high osmolality environment, the ompR mutant expressed more fimB transcripts and Phase-ON positioning of the fimS element as well as higher type 1 pili levels than wild-type cells. Together these results suggest that OmpR may be post-transcriptionally regulated in UPEC cells growing in a low pH/high osmolality environment, which regulates fimB in UPEC.

Fig. 1.

Gel mobility shift assays that use OmpR protein. (a) A gel shift with the 445 bp ³²P-labelled fimB promoter region and OmpR. (b) A gel shift with the 445 bp ³²P-labelled fimB promoter region and OmpR-P. (c) A gel shift with the 445 bp ³²P-labelled fimB promoter region and D55A OmpR. (d) A gel shift with the 340 bp ³²P-labelled fimE promoter region. Lanes were loaded as follows: (1) DNA alone, (2) DNA plus 0.5 µM OmpR, (3) DNA plus 1 µM OmpR, (4) DNA plus 2 µM OmpR, (5) DNA plus 5 µM Omp and (6) DNA plus 8 µM OmpR.

Fig. 2.

DNase I footprinting analysis of unphosphorylated (OmpR) and phosphorylated OmpR (OmpR-P) at the fimB promoter region 2. The concentration (nM) of OmpR and OmpR-P used in each lane is indicated at the bottom. The lanes with ‘0’ indicate the cleavage pattern by DNase I in the absence of OmpR/OmpR-P. The coordinates indicate positions relative to the fimB promoter 2 site. Regions of protection are indicated by black bars for OmpR (+2 to −18) and OmpR-P (+2 to −22) and a hypersensitive site is indicated by an arrow.

Fig. 3.

Quantitative determination of ompR transcript and OmpR expression levels in exponentially grown UPEC NU149 cells in different environments. (a) qRT-PCR analysis of UPEC NU149 cells grown to mid-exponential phase in pH 5.5, pH 5.5 plus 800 mOsm NaCl (5.5+), pH 7.0 and pH 7.0 plus 800 mOsm NaCl (7.0+) LB. The fold change in ompR transcript levels that were corrected using ftsZ are shown as the mean±sd from three separate runs. (b) Western blot analysis of OmpR protein expression. One hundred nanograms of purified OmpR protein or 10 µg of total protein isolated from NU149 grown in pH 5.5, 5.5+ (pH 5.5 LB with 800 mOsm NaCl), 7.0 and 7.0+ (pH 7.0 LB with 800 mOsm NaCl) LB. (c) One hundred nanograms of purified OmpR protein or 10 µg of total protein isolated from NU149 grown in pH 7.0 LB with (+) or without (−) IPTG. The blots were probed with absorbed polyclonal rabbit anti-OmpR antibody.

Fig. 4.

Quantitative determination of fimB transcript expression by qRT-PCR. UPEC NU149 (solid black bars) and NU149 OmpR1 (open white bars) cells were grown to mid-exponential phase in pH 5.5, pH 5.5 plus 800 mOsm NaCl (5.5+), pH 7.0 and 7.0 plus 800 mOsm NaCl (7.0+) LB. The fold change in fimB transcript levels that were corrected using ftsZ are shown as the mean±sd from two separate runs.

Fig. 5.

Determination of the fimS invertible element orientation by PCR on chromosomal DNAs isolated from NU149, NU149 OmpR1 and NU149 OmpR1/pFR29* cells grown in pH 5.5 or 7.0 LB with or without added NaCl (+, added NaCl). Cells were harvested during mid-exponential phase following aerobic growth with agitation at 250 r.p.m. at 37 °C in pH 7.0 LB media with either no added NaCl (low salt) or 800 mOsm added NaCl (high salt) or in pH 5.5 LB medium with either no added NaCl (low salt) or 800 mM NaCl (high salt). Multiplex PCRs were set up with INV and FIMA primers to amplify Phase-ON-oriented DNA (ON, 450 bp product) (Schwan et al., 1992), FIME and INV primers to amplify Phase-OFF-oriented DNA (OFF, 750 bp product) (Schwan et al., 1992), and EcFtsZ 1 and 2 primers to amplify the ftsZ gene (302 bp product) (Schwan et al., 2007). Each multiplex was run at least three separate times. WT, wild-type NU149; R, ompR strain; R+, ompR strain complemented with pFR29*. The lanes were loaded onto a 1.5 % agarose gel as follows: lane 1, NU149 (pH 7.0, low salt); lane 2, NU149 OmpR1 (pH 7.0, low salt); lane 3, NU149 OmpR1/pFR29* (pH 7.0, low salt); lane 4, NU149 (pH 7.0, high salt); lane 5, NU149 OmpR1 (pH 7.0, high salt); lane 6, NU149 OmpR1/pFR29* (pH 7.0, high salt); lane 7, NU149 (pH 5.5, low salt); lane 8, NU149 OmpR1 (pH 5.5, low salt); lane 9, NU149 OmpR1/pFR29* (pH 5.5, low salt); lane 10, NU149 (pH 5.5, high salt); lane 11, NU149 OmpR1 (pH 5.5, high salt); and lane 12, NU149 OmpR1/pFR29* (pH 5.5, high salt). 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).

Fig. 6.

Electron micrographs of NU149 OmpR1 (a) and NU149 (b) cells grown in pH 7.0 LB with 800 mOsm NaCl. Magnification, ca. ×100 000.