RfaH suppresses small RNA MicA inhibition of fimB expression in Escherichia coli K-12

Abstract

The phase variation (reversible on-off switching) of the type 1 fimbrial adhesin of Escherichia coli involves a DNA inversion catalyzed by FimB (switching in either direction) or FimE (on-to-off switching). Here, we demonstrate that RfaH activates expression of a FimB-LacZ protein fusion while having a modest inhibitory effect on a comparable fimB-lacZ operon construct and on a FimE-LacZ protein fusion, indicating that RfaH selectively controls fimB expression at the posttranscriptional level. Further work demonstrates that loss of RfaH enables small RNA (sRNA) MicA inhibition of fimB expression even in the absence of exogenous inducing stress. This effect is explained by induction of σ(E), and hence MicA, in the absence of RfaH. Additional work confirms that the procaine-dependent induction of micA requires OmpR, as reported previously (A. Coornaert et al., Mol. Microbiol. 76:467-479, 2010, doi:10.1111/j.1365-2958.2010.07115.x), but also demonstrates that RfaH inhibition of fimB transcription is enhanced by procaine independently of OmpR. While the effect of procaine on fimB transcription is shown to be independent of RcsB, it was found to require SlyA, another known regulator of fimB transcription. These results demonstrate a complex role for RfaH as a regulator of fimB expression.

Fig 1

The effects of ΔrfaH and ΔslyA mutations on the β-galactosidase produced by FimB-LacZ translational (tl) and fimB-lacZ transcriptional (tc) fusions. The wild-type (wt) and mutant strains indicated were grown and processed as described in Materials and Methods.

Fig 2

The effects of ΔrfaH on FimB off-to-on recombination per cell per generation. The bacteria were grown and processed as described in Materials and Methods. The values shown are the means of at least five measurements.

Fig 3

The organization of the fimB promoter and 5′ UTR. The extents of deletion mutations Δ1 to Δ3 are indicated by solid lines. Also indicated are the positions of the MicA target sequence in the fimB mRNA (26), the predicted fimB Shine-Dalgarno sequence (SD), and the ops site-like element OLE. The fimB promoter −35 and −10 regions (shaded rectangles), transcriptional start site and direction (arrow), and previously characterized SlyA binding sites O_SA1 and O_SA2 (34) are also shown. The start of the fimB ORF is indicated by the labeled box. Sp (SphI) and EO (EcoO109I) correspond to the restriction endonuclease sites used in this study. The ClaI site used lies within the fimB ORF further downstream than the region included in the diagram. The scale of the diagram (100 bp) is indicated by an additional horizontal line. The parallel diagonal lines denote that O_SA1 and O_SA2 lie further upstream of the fimB promoter than indicated by the linear scale of the diagram.

Fig 4

The effects of Δ1 to Δ3 mutations on the β-galactosidase produced by a FimB-LacZ fusion. The wild-type (wt) and mutant strains indicated were grown and processed as described in Materials and Methods.

Fig 5

The effects of micA, rfaH, and micA rfaH double mutations on the β-galactosidase produced by a FimB-LacZ fusion. The wild-type (wt) and mutant strains indicated were grown and processed as described in Materials and Methods, except that the growth medium used contained 1 mM nicotinic acid to allow growth of the rseA mutants which contain a linked nadB::Tn10 mutation.

Fig 6

The effects of rfaH and rseA on the β-galactosidase produced by a micA-lacZ fusion. The wild-type (wt) and mutant strains indicated were grown and processed as described in Materials and Methods, except that the growth medium used contained 1 mM nicotinic acid to allow growth of the rseA mutants which contain a linked nadB::Tn10 mutation.

Fig 7

The effect of rfaH and rseA on the β-galactosidase produced by a rpoHP3::lacZ fusion. The strains indicated were grown and processed as described in Materials and Methods, except that the growth medium used contained 1 mM nicotinic acid to allow growth of the rseA mutants which contain a linked nadB::Tn10 mutation.

Fig 8

The effects of micA, rfaH, and micA rfaH double mutations on the β-galactosidase produced by a FimB-LacZ fusion in the presence and absence of procaine. Procaine was included at the concentrations (0 to 20 mM) specified. The wild-type (wt) and mutant strains indicated were grown and processed as described in Materials and Methods.

Fig 9

The effects of procaine on FimB off-to-on recombination per cell per generation. Procaine was included at the concentrations (0 to 20 mM) specified. The wild-type strain was grown and processed as described in Materials and Methods. The values shown are the means of at least five measurements.

Fig 10

The effects of rfaH and slyA mutations on the β-galactosidase produced by a fimB-lacZ transcriptional fusion in the presence and absence of procaine. Procaine was included at the concentrations (0 to 20 mM) specified. The wild-type (wt) and mutant strains indicated were grown and processed as described in Materials and Methods.

Fig 11

Model for the control of fimB expression and type 1 fimbriation by RfaH and MicA. (a) Misfolded outer membrane proteins (MOMP) activate protease DegS to cleave RseA, releasing σ^E to activate micA transcription (37, 46). (b) Procaine (Pc) activates EnvZ/OmpR to also induce σ^E and hence micA expression (39). (c) Mutation of rfaH also leads to changes in LPS core biosynthesis that cause misfolding of outer membrane proteins and hence induction of σ^E and hence micA expression. (d) Procaine activates an alternative envelope stress pathway (Sx-Rx). (e) RfaH is postulated to inhibit fimB transcription indirectly by activation expression of Sx and/or Rx. (f and g) SlyA enhances fimB expression directly (34) (f) but is also postulated (g) to enhance expression of an unknown factor (Fx) that prevents Sx-Rx signaling in the absence of procaine. (h) FimB catalyzes off-to-on inversion of fimS to enhance expression of type 1 fimbriae (T1F). (i) Type 1 fimbriae stimulate inflammation by enhancing LPS-activated host signaling pathways (10–12). Stimulatory and inhibitory interactions are indicated by arrows and diamonds, respectively. Dotted lines represent speculative pathways. OM, outer membrane; IM, inner membrane.