IscR controls iron-dependent biofilm formation in Escherichia coli by regulating type I fimbria expression

Abstract

Biofilm formation is a complex developmental process regulated by multiple environmental signals. In addition to other nutrients, the transition metal iron can also regulate biofilm formation. Iron-dependent regulation of biofilm formation varies by bacterial species, and the exact regulatory pathways that control iron-dependent biofilm formation are often unknown or only partially characterized. To address this gap in our knowledge, we examined the role of iron availability in regulating biofilm formation in Escherichia coli. The results indicate that biofilm formation is repressed under low-iron conditions in E. coli. Furthermore, a key iron regulator, IscR, controls biofilm formation in response to changes in cellular Fe-S homeostasis. IscR regulates the FimE recombinase to control expression of type I fimbriae in E. coli. We propose that iron-dependent regulation of FimE via IscR leads to decreased surface attachment and biofilm dispersal under iron-limiting conditions.

FIG. 1.

Inhibition of biofilm formation by the iron chelator dipyridyl. (A) Wild-type strain was grown in increasing amounts of 2,2′-dipyridyl. (B) Wild-type strain was incubated in LB or LB with 200 μM 2,2-dipyryidyl. Increasing concentrations of ferric chloride or magnesium chloride were added to the dipyridyl-containing samples. Strains were grown at 25°C for 24 h in LB. Final planktonic growth was recorded at 600 nm (light gray bars). After washing, staining with crystal violet, and solubilization, final biofilm formation was recorded at 570 nm (dark gray bars). The average of triplicate experiments is shown.

FIG. 2.

Dispersion of mature biofilm by the iron chelator dipyridyl. Wild-type strain was grown at 25°C for 24 h in LB. Planktonic cells were removed by washing with sterile media and fresh LB (dark gray bars) or fresh LB with 200 μM 2,2′-dipyridyl (light gray bars) was added (time = 0). At various time points, wells from each condition were washed, stained with crystal violet, and solubilized. Final biofilm formation was recorded at 570 nm. The average of triplicate experiments is shown.

FIG. 3.

Role of IscR in regulating biofilm formation. Strains indicated were grown at 25°C for 24 h in LB. Final planktonic growth was recorded at 600 nm (light gray bars). After washing, staining with crystal violet, and solubilization, final biofilm formation was recorded at 570 nm (dark gray bars). The average of triplicate experiments is shown. For panel C, 0.05% l-arabinose was also added to all strains at the time of inoculation. piscR expresses wild-type IscR and piscR-CTM expresses the C92A-C98A-C104A triple cysteine mutant of IscR. pFWO2 is the empty parent control plasmid.

FIG. 4.

Type I fimbriae are required for the ΔiscR biofilm phenotype. Strains indicated were grown at 25°C for 24 h in the LB. Final planktonic growth was recorded at 600 nm (light gray bars). After washing, staining with crystal violet, and solubilization, final biofilm formation was recorded at 570 nm (dark gray bars). The average of triplicate experiments is shown.

FIG. 5.

Measurement of fimA ON/OFF status in wild-type and ΔiscR strains. (A) Diagram showing the PCR fragment used to determine whether fimS (the fimA promoter) is in the ON or OFF state. Relative orientation of the HinfI restriction site and the fimA promoter are shown. Different fragments generated by HinfI digestion in both ON and OFF state are labeled. (B) Gel showing the relative abundance of ON and OFF fimS fragments in wild-type and ΔiscR strains. Quantification of ON fimS is shown below the gel (see Materials and Methods).

FIG. 6.

Role of FimE in the ΔiscR biofilm phenotype. Wild-type or ΔiscR strains containing the (A) Φ(fimB-lacZ) or (B) Φ(fimE-lacZ) transcriptional fusions were grown in LB until mid-exponential phase or overnight to stationary phase. The β-galactosidase activity was measured and the Miller units were calculated as described previously. The average of triplicate experiments is shown. (C) The strains indicated were grown at 25°C for 24 h in LB. Final biofilm formation was recorded at 570 nm (dark gray bars). The average of triplicate experiments is shown. pRI-fimE constitutively expresses the FimE recombinase. pRI is the empty parent control plasmid.

FIG. 7.

EMSA analysis of apoIscR binding to the fimB-fimE intergenic region. Unlabeled oligonucleotides were added to compete with the labeled fimB-fimE intergenic region PCR fragment (bottom). The PfimE oligonucleotide corresponds to the proposed IscR binding site in the fimB-fimE intergenic region (top). The PsufA oligonucleotide corresponds to the previously mapped binding site for IscR in the sufA promoter. The nonspecific oligonucleotide is an unrelated sequence of the same length. All oligonucleotide sequences are listed in Table S1 in the supplemental material. Each oligonucleotide was added to the same three final concentrations (200, 600, or 1,000 nM), while apoIscR is present at 500 nM in all samples.

FIG. 8.

Proposed model for fimE regulation by IscR under iron and iron-sulfur replete or iron and iron-sulfur limiting conditions. Induction of the FimE recombinase by apoIscR leads to inversion of the fimS region to the OFF position and decreases expression of the fimAICDFGH locus. Decreased expression of type I fimbriae diminishes biofilm formation under iron and iron-sulfur limiting conditions.