(A) Gene transcription is governed by a hierarchical regulatory cascade that involves gene activation and gene repression. The σ^E factor turns on a large regulon that includes the genes for GerR and SpoIIID. These DNA-binding proteins, in turn, block further transcription of many of the genes that had been activated by σ^E. SpoIIID is also an activator, and it turns on genes required for the appearance of pro-σ^K. The conversion of pro-σ^K to mature σ^K is governed by a signal emanating from the forespore as represented by the squiggle. Next, σ^K activates the subsequent regulon in the cascade, which includes the gene for the DNA-binding protein GerE. Finally, GerE, which, like SpoIIID, is both an activator and a repressor, turns on the final regulon in the cascade while also repressing many of the genes that had been activated by σ^K. The thickness of lines represents the relative abundance of genes activated (arrows) or repressed (lines ending in bars) by the indicated regulatory proteins.
(B) The regulatory circuit is composed of two coherent FFLs linked in series and three incoherent FFLs. In the first coherent FFL, σ^E turns on the synthesis of SpoIIID, and both factors act together to switch on target genes, including genes involved in the appearance of σ^K. Likewise, in the second coherent FFL, σ^K directs the synthesis of GerE, and the two factors then act together to switch on target genes (X₄). The σ^E factor and SpoIIID also constitute an incoherent FFL in which SpoIIID acts as a repressor to downregulate the transcription of a subset of the genes (X₂) that had been turned on by σ^E. Similar incoherent FFLs are created by the actions of σ^E and GerR (X₁) and by σ^K and GerE (X₃), with GerR and GerE repressing genes that had been switched on by σ^E and σ^K, respectively. The AND symbols indicate that the FFLs operate by the logic of an AND gate in that the output (either gene activation or a pulse of gene expression) requires the action of both transcription factors in the FFL (see Mangan and Alon 2003). For example, σ^K and GerE are both required for the activation of X₄ genes, whose induction is delayed compared to genes that are turned on by σ^K alone. Similarly, both σ^E and the delayed appearance of GerR are anticipated to create a pulse of transcription of X₁ genes.

DNA fragments of interest were amplified by PCR, gel-purified, and end-labeled using [γ-³²P]-ATP and polynucleotide kinase. Purified SpoIIID was added at increasing concentrations (0 nM for lanes 1 and 5, 50 nM for lane 2, 100 nM for lane 3, and 200 nM for lane 4) and incubated at room temperature for 30 min before loading on to a nondenaturing gel containing 6% polyacrylamide. With the exception of (D), the DNA fragments corresponded to the upstream regions of the indicated genes. See Materials and Methods for the identity (coordinates) of the specific DNA sequences used in the analyses.
(A) Gel shifts for known targets of SpoIIID (bofA and spoIVCA), representing positive controls, and genes (abrB, spoIIGA, and racA) under the control of another DNA-binding protein (Spo0A), representing negative controls.
(B) Gel shifts for genes identified as possible targets of SpoIIID by transcriptional profiling.
(C) Gel shift for cotE. Expression of cotE from its P2 promoter is strongly dependent on SpoIIID. No binding of SpoIIID to the upstream sequence for cotE is observed, suggesting that the effect of SpoIIID on transcription from the P2 promoter is indirect.
(D) Gel shifts for chromosomal regions strongly enriched for SpoIIID binding as judged by ChIP-on-chip analysis. For each region, four consecutive DNA fragments of approximately 400 nucleotides in length were analyzed.

Culture samples from strains PE551 (solid triangles, amyE::P_spoIIM–lacZ), SW312 (open triangles, amyE::P_spoIIM–lacZ, ΔgerR), PE511 (solid squares, amyE::spoIIP–lacZ), PE568 (open squares, amyE::spoIIP–lacZ,ΔgerR), PE553 (solid diamonds, amyE::P_yqhV–lacZ), and PE558 (open diamonds, amyE::P_yqhV–lacZ, ΔgerR) were collected at indicated intervals after the start of sporulation in Sterlini–Mandelstam medium and analyzed for β-galactosidase activity.

(A) The σ^E regulon and its modulation by SpoIIID and GerR. The first gene of each σ^E-controlled transcription unit identified by transcriptional profiling is indicated. In the inner circle, genes repressed by SpoIIID are green, and genes repressed by GerR are blue. In the outer circle, genes partially dependent on SpoIIID for expression are orange, and genes strongly dependent on SpoIIID are red. Underlined are SpoIIID-controlled genes for which SpoIIID binding to their upstream sequences has been demonstrated biochemically. Genes unaffected by SpoIIID or GerR are indicated in black.
(B) The σ^K regulon and its modulation by GerE. The first gene of each σ^K-controlled transcription unit identified by transcriptional profiling is indicated. In the inner circle, genes repressed by GerE are green. In the outer circle, genes partially dependent on GerE for expression are orange, and genes strongly dependent on GerE are red. Genes unaffected by GerE are indicated in black.

Consensus sequences are displayed as sequence logos (Schneider and Stephens 1990). The height of the letters in bits represents the information content at each position (the maximum value is two bits).
(A) Consensus binding sequence for SpoIIID as derived from 17 SpoIIID-binding sites mapped by DNAase I footprinting (Halberg and Kroos 1994; Zhang et al 1997; results presented herein).
(B) Consensus binding sequence for SpoIIID obtained by compilation of 68 putative SpoIIID-binding sites identified as common motifs by BioProspector and BioOptimizer analysis in sequences upstream of genes identified by transcriptional profiling or within regions identified by ChIP-on-chip analysis.
(C) Consensus binding sequence for SpoIIID obtained by MDscan analysis of the sequences of 26 SpoIIID-binding regions identified by ChIP-on-chip analysis.
(D) Consensus promoter sequence for σ^K-containing RNA polymerase obtained from the compilation of 58 sequences identified as common motifs in regions upstream of σ^K-regulated genes by a BioProspector/BioOptimizer computational approach (Jensen and Liu 2004). Positions 1–5 on the horizontal axis correspond to the −35 element and positions 21–30 to the −10 element. The optimal spacing between the two regions is 15 bp (± 1 bp).
(E) Consensus promoter sequence for σ^K-containing RNA polymerase obtained from the compilation of 23 previously mapped (http://dbtbs.hgc.jp/; Helmann and Moran 2002) and 18 newly identified σ^K-controlled promoters identified by transcription start site mapping.
(F) Consensus promoter sequence for σ^E-containing RNA polymerase obtained from the compilation of 62 σ^E-controlled promoters identified by transcription start site mapping (Eichenberger et al. 2003). Positions 1–8 on the horizontal axis correspond to the −35 element, and positions 21–30 to the −10 element. The optimal spacing between the two regions is 12 bp (± 1 bp).