PMC

Schematic representation of a biochemical pathway under the control of the rpoS gene: arginine degradation to succinate via putrescine (adapted from EcoCyc; http://ecocyc.org/). Genes belonging to this pathway and found to be preferentially transcribed by Eσ^S in ROMA experiments are boxed with solid lines (speB, puuB and gabD); genes co-transcribed with either puuB or gabD (i.e. belonging to the puuCBE and gabDTP operons; see also Supplementary Table 1) are boxed with dashed lines. Other genes of the pathway known to be dependent on the rpoS gene in vivo (puuA and puuD) (,) are underlined.

In vitro transcription experiments on supercoiled plasmids. Transcription from the ilvY, speB, xapA, ydhY and ssrS P1 promoter regions cloned into pJCD01 plasmid was performed in the presence of either Eσ^S or Eσ⁷⁰; transcript amounts were determined by quantitative real-time PCR as described in ‘Materials and Methods’ section, using the RNA-I transcript as reference as previously described (). Relative transcript levels are shown as Eσ^S/Eσ⁷⁰ ratio. Experiments were performed three times in duplicate, and standard errors are shown.

In vitro transcription experiments on pJCD01 plasmid derivatives in which either the ssrS P1 promoter (ssrS P1 WT) or a mutated derivative (ssrS P1 mut) had been cloned. The ssrS P1 mut carries a double substitution (CG to TA) at positions −3/−2 (shown in the figure). In vitro transcription was performed in the presence of either Eσ^S or Eσ⁷⁰; transcript amounts were determined by quantitative real-time PCR as described in ‘Materials and Methods’ section, using the RNA-I transcript as reference as previously described (). Experiments were performed three times in duplicate, and standard errors are shown.

Conserved sequence features in promoters of genes showing preferential transcription by either Eσ⁷⁰ or Eσ^S, shown as a sequence logo derived from multiple sequence alignments. (A) Comparison of alignments in the −60 to −18 promoter regions. (B) Comparison of alignments in the −17 to +2 promoter regions. Note that different y-axis scales were used in the two panels to account for the different levels of sequence conservation. Multiple alignment included 31 promoters controlling genes preferentially transcribed by Eσ^S in ROMA experiments (listed in Supplementary File S1) and 29 promoters preferentially recognized by Eσ⁷⁰ (listed in Supplementary File S2).