High-affinity DNA binding sites for H-NS provide a molecular basis for selective silencing within proteobacterial genomes

Abstract

The global transcriptional regulator H-NS selectively silences bacterial genes associated with pathogenicity and responses to environmental insults. Although there is ample evidence that H-NS binds preferentially to DNA containing curved regions, we show here that a major basis for this selectivity is the presence of a conserved sequence motif in H-NS target transcriptons. We further show that there is a strong tendency for the H-NS binding sites to be clustered, both within operons and in genes contained in the pathogenicity-associated islands. In accordance with previously published findings, we show that these motifs occur in AT-rich regions of DNA. On the basis of these observations, we propose that H-NS silences extensive regions of the bacterial chromosome by binding first to nucleating high-affinity sites and then spreading along AT-rich DNA. This spreading would be reinforced by the frequent occurrence of the motif in such regions. Our findings suggest that such an organization enables the silencing of extensive regions of the genetic material, thereby providing a coherent framework that unifies studies on the H-NS protein and a concrete molecular basis for the genetic control of H-NS transcriptional silencing.

Figure 1.

(A) Logo representation of the experimentally determined H-NS binding motif derived from sequences in Table 1. (B) Logo representation of the predicted H-NS binding motif derived from identified occurrences in the set of differentially expressed genes responding to H-NS removal under different growth conditions. (C) Average AT content profiles of sequences from −300 to +100 bp relative to the start of translation for sets of genes differentially expressed in hns cells but containing or lacking a predicted H-NS binding site. Logos were generated using enoLOGOS (http://biodev.hgen.pitt.edu/cgi-bin/enologos/enologos.cgi).

Figure 2.

(A) Analysis of known transcriptional regulatory network of H-NS from RegulonDB for occurrence of the binding motif template. Genes containing a predicted strong H-NS binding site are underlined. (B) Potential regulatory interactions between nucleoid-associated proteins. The arrows indicate that one protein may directly affect the expression of another but do not indicate whether such regulation would be positive or negative.

Figure 3.

Maps of occurrence of sequences with close matches to the binding motif template in selected regions of (A) E. coli K12, (B) uropathogenic E. coli 536 (C) plasmids pO157 (top) and R27 (bottom). The predicted sites are oriented with respect to the DNA strands.

Figure 4.

Proposed mechanism of silencing over extended regions. Initial nucleation of H-NS binding at high-affinity sites is following by spreading and condensation of H-NS on supercoiled DNA to generate a silenced fiber. Spreading and condensation could be either consecutive or concomitant. Blue squares represent high-affinity H-NS binding sequences (nucleating sites) and green and yellow circles represent bound H-NS on nominal distal and proximal sides, respectively, of a promoter. The inset of an H-NS-DNA complex is taken from ref. 26 with permission.