DNA supercoiling, orientation of the fimS element, and quantification of fis, hns, hupB, ihfA, and lrp mRNA levels in AIEC LF82 associated with intestinal epithelial cells. (A) The wild-type strain harboring pUC18 reporter was used to infect Intestine-407 cells for 3 h or was grown in MEM or conditioned MEM. Plasmid DNA was separated by electrophoresis on 1% agarose gel containing 2.5 μg/ml chloroquine, and topoisomers were detected by hybridization with DIG-labeled pUC18 probe. (B) Determination of the orientation of the fim operon invertible element in wild-type AIEC LF82 strain grown in MEM or in conditioned MEM for 3 h or after a 3-h infection period of Intestine-407 cells. A 450-bp product revealed ON orientation, and the 750-bp product revealed the OFF orientation of the invertible element. (C) Determination of the amount of the FimA subunit by Western blotting using antibodies raised against type 1 pili was performed. As a control, Western blot analysis was performed using antibodies raised against the α-subunit of RNA polymerase. (D) mRNA levels of fis, hns, hupB, ihfA, and lrp genes encoding histone-like proteins in AIEC LF82 bacteria after a 3-h infection period of Intestine-407 cells relative to levels in AIEC LF82 bacteria grown for 3 h in MEM cell culture medium using real-time RT-PCR. As controls, 16S rRNA levels were measured. Only experiments showing the same levels of 16S rRNA for each sample were taken into account. Data represent means of at least three separate experiments; bars indicate and standard errors of the means (SEM). Lanes 1, wild-type AIEC LF82 strain grown in MEM; lanes 2, AIEC LF82 after a 3-h infection period of Intestine-407 cells; lanes 3, AIEC LF82 grown in conditioned MEM for 3 h.

Type 1 pilus expression and adhesion ability of LF82 wild-type strain and LF82 fis::Km mutant with induced YhjH and Fis expression. (A) Transmission electron microscopy (TEM) examination of bacteria stained using immunogold labeling with antibodies raised against purified type 1 pili (black arrow). Flagella (gray arrow) were observed only on the surface of wild-type bacteria. The black scale bar represents 500 nm. (B) In LF82 ΔfliA and LF82 fis::Km mutants harboring the pBADyhjH construct, expression of YhjH protein was induced using 0.1% arabinose. Intestine-407 cell-associated bacteria were quantified after centrifugation and a 3-h infection period. The mean number of cell-associated LF82 bacteria was 7.2 × 10⁶ ± 3.9 × 10⁵ CFU. Results are expressed as cell-associated (adherent plus intracellular) bacteria relative to those obtained for strain LF82, taken as 100%. Each value is the mean ± SEM of at least four separate experiments. The expression of functional type 1 pili was evaluated by determination of the yeast aggregation. (C) Determination of the amount of FimA subunit by Western blotting using antibodies raised against type 1 pili. (D) Overexpression of Fis was induced in LF82 fis::Km and the wild-type strain harboring pBADfis construct with 0.1% arabinose. Intestine-407 cell-associated bacteria were quantified after centrifugation and a 3-h infection period. The mean number of cell-associated LF82 bacteria was 7.8 × 10⁶ ± 7.8 × 10⁵ CFU. *, P < 0.05; **, P < 0.01.

Identification of specific FBSs in the fim operon. (A) Sequence logo generated from 65 Fis-defined FBSs and their complements displays the relative functional contribution of bases within the 15-bp core sequence of the Fis-binding site. Each column (position) in a motif can be characterized by the amount of information it contains (measured in bits). Highly conserved positions in the motif have high information content; positions where all letters are equally likely have low information content. (B) Location of putative identified FBS in regulatory regions of fim operon.

Validation of putative FBS by EMSA. Binding of purified Fis to the 75-bp labeled DNA fragments and with an excess of unlabeled DNA bearing the fimE promoter (A), intergenic nanC-fimB region (B), or an unrelated DNA sequence (C). The DNA fragments were incubated with increasing concentrations of Fis or with 0.045 nM Fis in the presence of 1,000-fold excess of nonspecific competitor poly(dI-dC). The protein was incubated with the DNA for 15 min at room temperature, and samples were analyzed on a native 12% polyacrylamide gel.

DNase I footprinting using an automated capillary sequencer in the fim regulatory regions. Electropherograms showed a protection pattern of the fimE (left panel) and nanC-fimB (right panel) promoters after digestion with DNase I following incubation in the presence (bottom panel) or absence (top panel) of Fis (0.45 nM). Corresponding DNA sequences are given below the electropherograms and focus on the region corresponding to the in silico identified FBS (blue peaks). Red peaks represent the DNA ladder.

Regulation of fimE, fimB, and nanC expression by Fis in the absence or presence of sialic acid. (A) Fold variation of nanC, fimB, and fimE mRNA levels in the LF82 fis::Km mutant relative to those in the wild-type LF82 strain using real-time RT-PCR. (B) Determination of the orientation of the fimS element in absence (+) or in presence (−) of 0.1% sialic acid in wild-type strain LF82 or in the LF82 fis::Km mutant (see the legend to Fig. ). Determination of the amount of FimA subunit by Western blotting using antibodies raised against type 1 pili. (D) Fold variation of nanC and fimB mRNA levels in LF82 fis::Km mutant and wild-type strain LF82 in the presence of 0.1% sialic acid relative to levels in the absence of sialic acid. As controls, 16S rRNA levels were measured. Only experiments showing the same levels of 16S rRNA for each sample were taken into account. Data represent means of at least three separate experiments; bars indicate SEM. *, P < 0.05; **, P < 0.01.

Model of regulation of type 1 pilus expression in AIEC strain LF82 grown in cell culture medium (MEM) or in contact with intestinal epithelial cells. (A) In MEM, AIEC LF82 bacteria expressed high levels of Fis, leading to a repression of nanC and activation of fimE expression. As a consequence, there is equilibrium between the expression of the two recombinases FimE and FimB, and the population of AIEC LF82 bacteria in MEM is equally distributed among the ON and the OFF phases. (B) AIEC LF82 bacteria in contact with intestinal epithelial cells exhibited decreased Fis expression, leading to Fis-dependent decreased activation of fimE, and nullified inhibition of nanC, encoding NanC, the N-acetylneuraminic acid (Neu₅Ac) outer membrane channel, which allows entry of this amino sugar into bacteria. As a consequence, in addition to decreased levels of the recombinase FimE, FimB expression is decreased. Consequently, AIEC LF82 bacteria associated with intestinal epithelial cells are highly piliated due to the predominant ON phase of the fim promoter. OM, outer membrane.