Regulatory cross-talk between adhesin operons in Escherichia coli: inhibition of type 1 fimbriae expression by the PapB protein - PubMed (original) (raw)

Regulatory cross-talk between adhesin operons in Escherichia coli: inhibition of type 1 fimbriae expression by the PapB protein

Y Xia et al. EMBO J. 2000.

Abstract

Pathogenic Escherichia coli often carry determinants for several different adhesins. We show a direct communication between two adhesin gene clusters in uropathogenic E.coli: type 1 fimbriae (fim) and pyelonephritis-associated pili (pap). A regulator of pap, PapB, is a key factor in this cross-talk. FimB recombinase turns on type 1 fimbrial expression, and PapB inhibited phase transition by FimB in both off-to-on and on-to-off directions. On-to-off switching requiring FimE was increased by PapB. By analysis of FimB- and FimE-LacZ translational fusions it was concluded that the increase in on-to-off transition rates was via an increase in FimE expression. Inhibition of FimB-promoted switching was via a different mechanism: PapB inhibited FimB-promoted in vitro recombination, indicating that FimB activity was blocked at the fim switch. In vitro analyses showed that PapB bound to several DNA regions of the type 1 fimbrial operon, including the fim switch region. These data show that Pap expression turns off type 1 fimbriae expression in the same cell. Such cross-talk between adhesin gene clusters may bring about appropriate expression at the single cell level.

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Figures

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Fig. 1. Immunofluorescence staining of E.coli J96 cells. Bacteria grown in LB at 37°C were prepared for microscopic analysis as described in Materials and methods. (A) Phase-contrast micrograph of bacterial cells. (B) Anti-Pap–lissamine–rhodamine staining. (C) Anti-type-1–fluorescein isothiocyanate staining.

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Fig. 2. Effect of papB on the on-to-off switching of strain AAEC198A (fimB+fimE+fimA–lacZYA) in defined rich medium at 37°C. The data from two separate experiments with the strain carrying the PapB-producing plasmid pHMG88 (closed symbols) or the vector plasmid pACYC184 (open symbols) are shown. Model rates of 0.9 (▴) and 0.45 (▵) per cell generation are shown by the dotted lines. The frequencies determined from these data are shown in Table III.

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Fig. 3. Effect of purified His-PapB on the in vitro recombination in bacterial extracts containing either FimB or FimE. Recombination assays were set up as described in Materials and methods. Lane 1: control extract (pET11 in NEC26) plus pMM36 (switch starts in the on orientation). Lane 2: FimE extract (pIB382 in NEC26) plus pMM36. Lane 3: FimE extract with 4 μM His-PapB plus pMM36. Lane 4: FimE extract with 8 μM His-PapB plus pMM36. Lane 5: FimE extract with 12 μM His-PapB plus pMM36. Lane 6: FimE extract with 16 μM His-PapB plus pMM36. Lane 7: 1 kb marker. Lane 8: FimB extract (pIB378 in NEC26) plus pMM36. Lane 9: FimB extract with 4 μM His-PapB plus pMM36. Lane 10: FimB extract with 8 μM His-PapB plus pMM36. Lane 11: FimB extract with 12 μM His-PapB plus pMM36. Lane 12: FimB extract with 16 μM His-PapB plus pMM36. Lane 13: control extract (pET11 in NEC26) plus pJL2 (switch in the off orientation). Lane 14: FimB extract plus pJL2. Lane 15: FimB extract with 4 μM His-PapB plus pJL2. Lane 16: FimB extract with 8 μM His-PapB plus pJL2. Lane 17: FimB extract with 12 μM His-PapB plus pJL2. Lane 18: FimB extract with 16 μM His-PapB plus pJL2.

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Fig. 4. PapB binding to the fim regulatory DNA region. (A) Outline of the fimB, fimE, fimA regions and positions of the promoters (P). The relative positions and lengths of different PCR fragments are also indicated. (B and C) Gel mobility shift assays of His-PapB binding to DNA fragments (PCR1–6). The assays were carried out as described in Materials and methods.

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Fig. 5. Schematic illustration of the cross-talk mediated by PapB between the pap and fim gene clusters.

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