Expression profiles of σ³²-regulon members after overexpressing rpoH. (A) Activity of σ³² following overexpression of rpoH. An exponential phase culture of strain CAG50002 (which carries an IPTG-inducible copy of rpoH) growing at 30°C in M9 complete–methionine was induced with IPTG at OD₄₅₀ = 0.3 (t = 0). At various times, pulse chase analysis was used to determine the rate of synthesis of two σ³²-dependent hsps, DnaK, and GroEL. Data is normalized to their synthesis rates at induction (0 min). (B) Hierarchical clustering of 105 genes whose expression is significantly altered following rpoH overexpression (CAG50002) vs. wild type (CAG50001). The color chart illustrates the average expression level at each time point for each gene from three time course experiments. Red denotes increased and green denotes decreased mRNA expression in CAG50002 vs. CAG50001: Maximum intensity represents greater than fourfold change. Time in minutes after induction of rpoH in the time-course experiments is indicated at the top of the figure; genes are identified by their unique ID and name. (C,D) SOM analysis of significantly induced genes following rpoH overexpression. The expression ratios for each gene across three time courses were averaged for each time point. Genes were partitioned based on their induction kinetics (fast/slow; C) or magnitude of induction (strong/weak; D). Each line represents an average trace of expression pattern for that group of genes. Relative Expression Level indicates mean and variance normalized (C) or raw (D) log₂ (rpoH overexpressed/wild type) expression ratios.

5′RACE identified 22 σ³²-dependent transcription starts. To identify σ³²-specific transcripts, mRNA from rpoH⁺ and rpoH⁻ strains (CAG50002 and CAG50003, respectively) was 5′-labeled with an RNA oligo, reverse-transcribed, amplified by PCR, and then visualized by 7.5% PAGE (see Materials and Methods). Bands present in rpoH⁺ but not in rpoH⁻ reactions were regarded as σ³² specific. Two known σ³² promoters, dnaK and ibpA, were tested; 22 new σ³²-dependent promoters were identified from the 54 newly identified σ³²-induced TUs. Displayed are duplicate examples of two new (ibpA, ileS) and two known (dnaK, ibpA) σ³²-specific promoters.

In vitro transcription assays identified seven new promoters transcribed by σ³². Multiround in vitro transcription assays were performed to test 11 predicted and six previously documented promoters. Each promoter template was tested with RNAP containing either σ⁷⁰ or σ³²: σ³²-dependent transcripts were obtained from seven promoters, five of which also generated σ⁷⁰-dependent transcripts (yadF, ybeD, glnS, yceJ, and xerD).

Core promoter motifs of 50 σ³² promoters. Fifty of the 51 validated σ³²-dependent promoters were used to derive sequence logos of the core motifs (see Table 1A,B) (the repE promoter was excluded due to the large number of multiple start points that made it difficult to confidently identify the upstream −10 and −35 motifs). The sequences were initially aligned by their start sites and WCONSENSUS was used to search small windows upstream for the conserved −10 and −35 motifs (see Materials and Methods). (A) Sequence logos (http://weblogo.berkeley.edu; Crooks et al. 2004) of the −35, −10, and +1 start site motifs. Note that only 43 promoters were used to derive the start site motif. Seven promoters were excluded that had either multiple starts with no clearly preferred position (b0473 P1 and P2, b1057, b3179, and b3400) or no defined starts (b0209 and b2954). The information content (I_seq) of each conserved sequence window is indicated. (B) Frequency distribution of distance between −35 (TTGAAA) and −10 (CCCATAT) motifs. (C) Frequency distribution of distance between −10 (CCCATAT) motif and +1 (A/G) start. (D) Histogram of the 50 σ³²-promoter start sites upstream of the gene translation start.

Sequence logos of A/T-rich motifs upstream of the σ³² promoters. WCONSENSUS was used to search sequences upstream of the −35 element of 50 σ³² promoters (excluding repE). Distal, Proximal, and Complete motifs were identified from the search windows −46 to −60, −36 to −51, and −36 to −60, respectively, by aligning the promoters with respect to their −35 elements and assigning the first “T” of the −35 motif as position −35.

Location of A/T-rich motifs in σ³² promoters. The location and arrangement of the Distal, Proximal, and Complete A/T-rich motifs identified in Figure 5 are shown for the 50 σ³² promoters.