Figure 2

The steps used to map RRs are illustrated using an example. (a) DVU1419 is a σ54-dependent RR encoded in a three-gene operon. (b) RR DVU1419 shifts the upstream region of its own operon. (c) Quantitative PCR shows that the upstream region of DVU1418 is enriched in the RR-bound DNA fraction (E) compared to the input DNA (I). As a negative control (-C), quantitative PCR of the upstream region of an unrelated kinase gene, DVU0013, shows no enrichment. (d) Artemis plots [52] of the three most highly enriched peaks obtained by DAP-chip. The corresponding gene is highlighted in the red oval. The vertical line corresponds to the location of the binding site motif (highlighted in yellow) within the peak. (e) An 18-bp motif was predicted by applying MEME (MicrobesOnline, using PATSER [53]) on the upstream regions of orthologs of DVU1418 in the closely related species D. vulgaris Miyazaki (DvM) and D. desulfuricans G20. (f) A similar motif was found by applying MEME to the upstream regions of the three DAP-chip gene targets. (g) Electrophoretic mobility shift assay was used to validate the motif. Changing selected bases within the motif eliminated the shift. DvH, D. vulgaris Hildenborough; HK, histidine kinase, Hpt, histidine phosphotransfer domain; mut, modified motif; RR, response regulator; wt, wild-type motif.