Mass spectrometry scores for selected of the proteins purified by affinity chromatography using region A.

<p>Only known proteins identified as binding to region A (A entire), or the cap-site proximal tandem dimer (A dimer, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029895#pone-0029895-g001" target="_blank">Fig. 1B</a>), and with a “Mowse” score of 50 or over are presented.</p

A scheme for the regulation of pspA gene expression.

<p>The scheme hypothesises a general activator of transcription (yellow-boxed) that has the potential to direct transcription in all cells in the slug. However, DimB acts in pstO cells in its repressor form (red-boxed) to prevent the activator functioning. Not shown here is a proposed functionally redundant repressor that can subsume the role of DimB as a repressor of pspA in a dimB- strain. The <i>ecmA</i> promoter is hyper-active in pstO cells of the DimB null strain, so is shown as being co-repressed by the DimB repressor form. In pstB cells the ecmB gene is directly induced by the activating form (green-boxed) of DimB.</p

Expression patterns of <i>pspA</i> reporter fusions.

<p>The <i>pspA</i> promoter region −990 to −122 (pspA:lacZ), and versions of the same region containing a mutation of either the W (pspA-M1:lacZ) or S (pspA-M456:lacZ) sites. Expression patterns were established in standing slugs stained with X-gal.</p

DimB binding to the pspA promoter <i>in vivo</i>.

<p>Cells were incubated with or without DIF and subjected to ChIP analysis. The absolute recoveries from the procedure varied from experiment to experiment, (three independent experiments with triplicate Q-PCR analyses in each). Therefore values are normalized to the induced signal for the <i>ecmA</i> positive control and are shown with their Standard Deviations. Student's paired T test was applied to the <i>pspA</i> analysis with and without DIF-1 and in samples immuno-precipitated from GFP-DimB transformant cells. As indicated by the asterisk the induction by DIF is significant with a P<0.05.</p

DIF repression of <i>pspA</i> expression.

<p>Disaggregated cells at the mound stage were incubated in the presence or absence of DIF-1. Q-PCR analysis of RNA samples was performed and the data is plotted as the mean of 3 independent biological repeats each performed in triplicate. The data is normalized to the expression level of <i>Ig7</i>, a constitutively expressed gene and that for each strain is normalized to the value without DIF. The mean results are shown with their standard deviations.</p

Mapping DimB binding sites in region A by gel retardation analysis.

<p>A) Alignment of regions S and W, the proposed DimB binding sites in region A of the <i>pspA</i> promoter, with the known DimB binding sites within the <i>ecmA</i> promoter: R2 and R1. Also indicated, above the sequence, are the positions of the point mutations used in scanning analysis of DimB binding. B) Total nuclear extracts obtained from Ax-2 and dimB- slug cells used in gel retardation with a region A probe. The competitors are the R2 and R2M sequences from within the <i>ecmA</i> promoter <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029895#pone.0029895-Zhukovskaya1" target="_blank">[8]</a> C) Total nuclear extracts obtained from Ax-2 slug cells used in gel retardation with a region A probe. The competitors are region A itself and scanning mutants M1 to M6. D) Gel retardation with recombinant DimB using an A region probe. Competitors are: A itself, and oligonucleotide M145, containing region A with mutations M1, M4 and M5 that collectively mutate the S and W DimB binding sites. Again, the control competitors are the R2 and R2M sequences from within the <i>ecmA</i> promoter.</p

Identification of proteins that bind to the pspA promoter.

<p>A representation of the minimal promoter sequence required for <i>pspA</i> expression (thick line) showing the sequence of the region used in affinity chromatography, with a proposed DimB binding site underlined. (B) The combined peptide coverage for DimB in the two different purifications, described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029895#pone-0029895-t001" target="_blank">Table 1</a>. is shown in red. (C) Identification of proteins bound to a 16nt tandem dimer containing the proposed DimB site. Only those proteins with a deducible function are indicated and their scores in the mass spectrometry analysis are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029895#pone-0029895-t001" target="_blank">Table 1</a>.</p