Thermoregulation of Shigella and Escherichia coli EIEC pathogenicity. A temperature-dependent structural transition of DNA modulates accessibility of virF promoter to transcriptional repressor H-NS

Abstract

M.Falconi and B.Colonna contributed equally to this workThe expression of plasmid-borne virF of Shigella encoding a transcriptional regulator of the AraC family, is required to initiate a cascade of events resulting in activation of several operons encoding invasion functions. H-NS, one of the main nucleoid-associated proteins, controls the temperature-dependent expression of the virulence genes by repressing the in vivo transcription of virF only below a critical temperature (~32°C). This temperature-dependent transcriptional regulation has been reproduced in vitro and the targets of H-NS on the virF promoter were identified as two sites centred around -250 and -1 separated by an intrinsic DNA curvature. H-NS bound cooperatively to these two sites below 32°C, but not at 37°C. DNA supercoiling within the virF promoter region did not influence H-NS binding but was necessary for the H-NS-mediated transcriptional repression. Electrophoretic analysis between 4 and 60°C showed that the virF promoter fragment, comprising the two H-NS sites, undergoes a specific and temperature-dependent conformational transition at~32°C. Our results suggest that this modification of the DNA target may modulate a cooperative interaction between H-NS molecules bound at two distant sites in the virF promoter region and thus represents the physical basis for the H-NS-dependent thermoregulation of virulence gene expression.

A Biophysical Approach to Transcription Factor Binding Site Discovery

“…For both H-NS and LRP, we find significant peaks overlapping (and just downstream in case of H-NS) of the 70 -binding site. This observation is consistent with the known examples of regulatory H-NS sites: in Figure 7A, we show regions found in several foot- (Lucht et al 1994;Falconi et al 1998;Soutourina et al 1999). In addition, we observe that if we condition the distribution by restricting to only strong 70 promoters, the peak near zero offset disappears, as shown in Figure 7B.…”

“…Note the significant peaks at relative positions near ‫4מ‬ and 8, which correspond to direct overlap, and at positions 25, 35, and 52, where H-NS is downstream of the promoter. Horizontal bars correspond to footprinted H-NS sites from Falconi et al (1998), Lucht et al (1994, and Soutourina et al (1999). (B) Same as A but excluding weak candidate RNAP-70 promoters (i.e., sites with high | E/µ| "score").…”

Thermoregulated expression of virulence genes in enteropathogenic Escherichia coli

“…H-NS is involved in the thermoregulation of virF (Falconi et al, 1998), the first positive activator of a multi-step regulatory cascade leading to expression of the type III secretion system and other virulence effectors (Dorman & Porter, 1998). It was proposed that the physical basis for the thermoregulated expression of virF resides in a temperature-dependent structural modification of the virF promoter, resulting from a temperature-sensitive curved DNA structure (Falconi et al, 1998). H-NS also mediates repression of the virB gene downstream in the Shigella regulatory cascade (Tobe et al, 1993).…”

Anti-silencing: overcoming H-NS-mediated repression of transcription in Gram-negative enteric bacteria

“…In those cases where the matter has been investigated, the AraC-like protein has been found both to antagonize H-NS repression and to exert a positive influence (albeit a modest one) on The virB regulatory virulence gene in Shigella flexneri and enteroinvasive E. coli Falconi et al (1998); Porter & Dorman (2002) promoter function in the absence of the repressor (Jordi et al, 1992;Murphree et al, 1997). Those AraC-like proteins that derepress and activate thermally responsive virulence genes do not appear to bind chemical ligands; they respond instead to a physical signal (temperature).…”