The program of gene transcription for a single differentiating cell type during sporulation in Bacillus subtilis - PubMed (original) (raw)

The program of gene transcription for a single differentiating cell type during sporulation in Bacillus subtilis

Patrick Eichenberger et al. PLoS Biol. 2004 Oct.

Abstract

Asymmetric division during sporulation by Bacillus subtilis generates a mother cell that undergoes a 5-h program of differentiation. The program is governed by a hierarchical cascade consisting of the transcription factors: sigma(E), sigma(K), GerE, GerR, and SpoIIID. The program consists of the activation and repression of 383 genes. The sigma(E) factor turns on 262 genes, including those for GerR and SpoIIID. These DNA-binding proteins downregulate almost half of the genes in the sigma(E) regulon. In addition, SpoIIID turns on ten genes, including genes involved in the appearance of sigma(K). Next, sigma(K) activates 75 additional genes, including that for GerE. This DNA-binding protein, in turn, represses half of the genes that had been activated by sigma(K) while switching on a final set of 36 genes. Evidence is presented that repression and activation contribute to proper morphogenesis. The program of gene expression is driven forward by its hierarchical organization and by the repressive effects of the DNA-binding proteins. The logic of the program is that of a linked series of feed-forward loops, which generate successive pulses of gene transcription. Similar regulatory circuits could be a common feature of other systems of cellular differentiation.

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Conflict of interest statement

The authors have declared that no conflicts of interest exist.

Figures

Figure 1

Figure 1. The Mother-Cell Line of Gene Transcription

(A) Gene transcription is governed by a hierarchical regulatory cascade that involves gene activation and gene repression. The σE 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 σE. SpoIIID is also an activator, and it turns on genes required for the appearance of pro-σK. The conversion of pro-σK to mature σK is governed by a signal emanating from the forespore as represented by the squiggle. Next, σK 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 σK. 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, σE turns on the synthesis of SpoIIID, and both factors act together to switch on target genes, including genes involved in the appearance of σK. Likewise, in the second coherent FFL, σK directs the synthesis of GerE, and the two factors then act together to switch on target genes (X4). The σE 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 σE. Similar incoherent FFLs are created by the actions of σE and GerR (X1) and by σK and GerE (X3), with GerR and GerE repressing genes that had been switched on by σE and σK, 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, σK and GerE are both required for the activation of X4 genes, whose induction is delayed compared to genes that are turned on by σK alone. Similarly, both σE and the delayed appearance of GerR are anticipated to create a pulse of transcription of X1 genes.

Figure 2

Figure 2. Location of Genes in the σE and σK Regulons and Their Regulation by DNA-Binding Proteins

(A) The σE regulon and its modulation by SpoIIID and GerR. The first gene of each σE-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 σK regulon and its modulation by GerE. The first gene of each σK-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.

Figure 3

Figure 3. Gel Electrophoretic Mobility-Shift Analysis of SpoIIID Binding

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.

Figure 4

Figure 4. Consensus Sequences for SpoIIID, σK, and σE

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 σK-containing RNA polymerase obtained from the compilation of 58 sequences identified as common motifs in regions upstream of σK-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 σK-containing RNA polymerase obtained from the compilation of 23 previously mapped (

http://dbtbs.hgc.jp/

; Helmann and Moran 2002) and 18 newly identified σK-controlled promoters identified by transcription start site mapping. (F) Consensus promoter sequence for σE-containing RNA polymerase obtained from the compilation of 62 σE-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).

Figure 5

Figure 5. Repression of σE -Controlled Genes by GerR

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

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References

    1. Baldus JM, Green BD, Youngman P, Moran CP. Phosphorylation of Bacillus subtilis transcription factor Spo0A stimulates transcription from the spoIIG promoter by enhancing binding to weak 0A boxes. J Bacteriol. 1994;176:296–306. - PMC - PubMed
    1. Ben-Yehuda S, Rudner DZ, Losick R. RacA, a bacterial protein that anchors chromosomes to the cell poles. Science. 2003;299:532–536. - PubMed
    1. Bonsen PP, Spudich JA, Nelson DL, Kornberg A. Biochemical studies of bacterial sporulation and germination. XII. A sulfonic acid as a major sulfur compound of Bacillus subtilis spores. J Bacteriol. 1969;98:62–68. - PMC - PubMed
    1. Britton RA, Eichenberger P, Gonzalez-Pastor JE, Fawcett P, Monson R, et al. Genome-wide analysis of the stationary-phase sigma factor (sigma-H) regulon of Bacillus subtilis . J Bacteriol. 2002;184:4881–4890. - PMC - PubMed
    1. Brown PO, Botstein D. Exploring the new world of the genome with DNA microarrays. Nat Genet. 1999;21(Suppl 1):33–37. - PubMed

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