Association of nucleoid proteins with coding and non-coding segments of the Escherichia coli genome

Abstract

The Escherichia coli chromosome is condensed into an ill-defined structure known as the nucleoid. Nucleoid-associated DNA-binding proteins are involved in maintaining this structure and in mediating chromosome compaction. We have exploited chromatin immunoprecipitation and high-density microarrays to study the binding of three such proteins, FIS, H-NS and IHF, across the E.coli genome in vivo. Our results show that the distribution of these proteins is biased to intergenic parts of the genome, and that the binding profiles overlap. Hence some targets are associated with combinations of bound FIS, H-NS and IHF. In addition, many regions associated with FIS and H-NS are also associated with RNA polymerase.

Figure 1

Genome-wide DNA-binding profiles of FIS, H-NS and IHF. The figure shows results from ChIP-chip experiments that measure profiles of FIS (A), H-NS (B) and IHF (C) binding across the E.coli genome. Binding signals (y-axis) are plotted against the location on the 4.64 Mb E.coli chromosome (x-axis). The positions of the origin and termini of replication are shown.

Figure 2

Association of FIS, H-NS and IHF with known target sites. The figure shows results from ChIP-chip experiments that measure profiles of FIS, H-NS and IHF binding across segments of the E.coli genome that are illustrated schematically below the profile. Binding signals (y-axis) are plotted against the genomic location (x-axis) for two known targets for each factor. Data for FIS binding at adlB and hns are shown in (A), H-NS binding at adiA and fliC are shown in (B) and IHF binding at sucA and carA are shown in (C).

Figure 3

Relationship between the A:T content of microarray probes and binding signal for RNA polymerase, FIS, H-NS and IHF. The figure shows averaged ChIP-chip binding signals for RNA polymerase, FIS, H-NS and IHF at groups of microarray probes (y-axis) sorted by their A:T content (x-axis).

Figure 4

(A–E) Overlap of FIS, H-NS, IHF and RNA polymerase binding signals at some promoters. The figure shows the results from ChIP-chip experiments that measure profiles of binding of RNA polymerase, FIS, H-NS and IHF across the E.coli genome. Binding signals (y-axis) are plotted against the genomic location (x-axis) and data for six intergenic regions are shown. The binding signals for RNA polymerase, FIS, H-NS and IHF are illustrated in blue, green, cyan and red, respectively.

Figure 5

Association of FIS, H-NS, IHF and RNA polymerase with coding sections of the genome. The figure shows results from ChIP-chip experiments that measure profiles of binding of RNA polymerase, FIS, H-NS and IHF across the E.coli genome. Binding signals (y-axis) are plotted against the genomic location (x-axis) and data for six coding regions are shown. The binding signals for RNA polymerase, FIS, H-NS and IHF are illustrated in blue, green, cyan and red, respectively.

Figure 6

Effect of transcription on the binding of nucleoid-associated proteins to the lac operon. (A) RNA polymerase occupancy of lacZ (closed diamonds), lacY (open squares) or lacA (open triangles) measured by ChIP at intervals (shown on the x-axis) after the addition of IPTG to growing E.coli cultures. (B) RNA polymerase, FIS, IHF and H-NS binding to targets within lacZ, lacY and lacA measured by ChIP, 4 min after the addition of IPTG. Results are shown as ratios of the signal generated ± IPTG.

Figure 7

Changes in FIS and H-NS binding induced by salicylic acid. The figure shows results from ChIP-chip experiments that measure changes in the binding profiles of FIS and H-NS across the genome of E.coli cells that were treated with salicylic acid. The figure shows data for the mar operon (A) and the flg operon (B), which are activated and repressed by salicylic acid, respectively.

Figure 8

Fractionation of the E.coli genome. The figure illustrates the results of an experiment in which phenol–chloroform extraction was used to fractionate formaldehyde cross-linked E.coli nucleoprotein. A microarray was used to compare DNA present in the aqueous phase, following phenol–chloroform extraction, to a control ‘input’ DNA sample. The panel shows the relationship between signal intensity at each probe and the binding of RNA polymerase (A), FIS (B), H-NS (C) or IHF (D) at that locus measured by ChIP-chip.