Osmolarity and pH growth conditions regulate fim gene transcription and type 1 pilus expression in uropathogenic Escherichia coli

Abstract

A comparative study was performed to determine the effects of pH, osmolarity, and human urine on the transcription of several fim genes, as well as the overall expression of type 1 pili. Several fim-lacZYA fusions were constructed on single-copy plasmids to test a range of pHs and a range of osmolarities. Growth in acidic medium slightly reduced expression from all of the fim promoters (fimA, fimB, and fimE). Increased osmolarity in neutral-pH medium repressed fimA and fimB transcription by approximately 50% when 400 mM NaCl was used and nearly threefold when 800 mM NaCl was used, whereas fimE transcription rose slightly as the osmolarity increased. This effect was more pronounced in high-osmolarity acidic media; fimB and fimA expression decreased fivefold in growth media containing 800 mM NaCl compared to expression in growth media without added NaCl. Moreover, fimE expression doubled under the same high-osmolarity conditions compared to expression in a low-osmolarity acidic environment. When a fimB-lacZ or fimE-lacZ fusion was inserted into the chromosome of strain AAEC189, fimE expression changed slightly as the osmolarity increased, but fimB expression decreased by 50% in a low-pH high-osmolarity environment. When strain AAEC189 with either a plasmid-borne fimB-lacZ fusion or a plasmid-borne fimE-lacZ fusion was grown in human urine, similar changes in the levels of fimB and fimE expression were observed. Limiting-dilution reverse transcription-PCR confirmed that these changes in fim expression occurred in clinical isolates of uropathogenic Escherichia coli grown in media with different pHs and different osmolarities. Furthermore, the invertible switch region in uropathogenic strain NU149 shifted from favoring the phase-on position in a neutral-pH low-osmolarity environment to favoring the phase-off position in a low-pH high-osmolarity environment. Results obtained with an ompR mutant strain demonstrated that fimB expression was derepressed and that OmpR may neutralize repression by an acid response regulator of fimE expression in a low-pH environment. In addition, H-NS was verified to be important in regulation of fimB, but it had only a slight effect on fimE under the specific pH and osmotic growth conditions tested. Enzyme immunoassays with anti-type 1 pilus antibody and hemagglutination assays showed that fewer type 1 pili were detected with cells in a low-pH high-osmolarity environment. Together, these observations demonstrate that a combination of low pH and high osmolarity regulates the transcription of fim genes, which favors a shift in the invertible element to the phase-off orientation and a loss of type 1 pilus expression. Taken together, our data suggest that the environmental cues that we tested may regulate expression of type 1 pili in specific in vivo niches, such as murine kidneys and possibly human kidneys.

FIG. 1.

Effects of pH and osmolarity on fimA, fimB, and fimE expression as determined with lacZYA transcriptional fusions in strain AAEC189. The β-galactosidase (β-Gal) activity (expressed in Miller units) was determined after 20 min; means ± standard deviations are indicated. Osmolarity effects were tested by using NaCl as the osmolyte. (A) Different pHs; (B) different osmolarities in pH 7.0 media; (C) different osmlarities in pH 5.5 media.

FIG. 2.

Effects of growth in human urine on fimA, fimB, and fimE expression as determined by using lacZYA transcriptional fusions in strain AAEC189. (A) Different percentages of human urine mixed with LB medium; (B) different pHs of human urine, adjusted with sodium phosphate buffer. The β-galactosidase (β-Gal) activity is expressed in Miller units; means ± standard deviations are indicated.

FIG. 3.

Quantitative determination of mRNA regulation by LD-RT-PCR analysis of cDNAs of strain NU149 cells grown in pH 7.0 LB medium with low osmolarity, in pH 5.5 LB medium with low osmolarity, and in pH 5.5 LB medium with high osmolarity. 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 folowing dilutions of cDNAs were used: undiluted (lanes 1), 1/2 (lanes 2), 1/4 (lanes 3), 1/8 (lanes 4), 1/16 (lanes 5), 1/32 (lanes 6), 1/64 (lanes 7), 1/128 (lanes 8), and 1/256 (lanes 9).

FIG. 4.

Determination of the invertible element orientation in strain NU149 grown under different environmental conditions by PCR analysis. The PCR analysis was performed with chromosomal DNA isolated from NU149 cells grown in pH 7.0 LB medium with no added NaCl (low osmolarity), in pH 5.5 LB medium with no added NaCl (low osmolarity), and in pH 5.5 LB medium with 400 mM NaCl (high osmolarity), using the INV and FIMA primers to amplify phase-on-oriented DNA (450-bp product) and the FIME and INV primers to amplify phase-off-oriented DNA (750-bp product). The following dilutions were used for the PCR: undiluted (lanes 1), 1/2 (lanes2), 1/4 (lanes 3), 1/8 (lanes 4), 1/16 (lanes 5), 1/32 (lanes 6), 1/64 (lanes 7), 1/128 (lanes 8), and 1/256 (lanes 9). All PCR products were electrophoresed on 1.5% agarose gels.

FIG. 5.

EIA analyses of strains NU149 and J96 grown in media with different osmolarities. Optical densities at 492 nm (O.D.₄₉₂) were determined, and means ± standard deviations are indicated. a, agar grown; None, no NaCl added. (A) Bacteria grown in pH 7.0 media; (B) bacteria grown in pH 5.5 media.