Location and consequences of a Tn10dCam insertion in the σ^E-dependent rpoE P2 promoter. A. The Tn10dCam insertion site determined by sequence analysis, the transcription start site (*), and the –10 and –35 sequences (underlined) are indicated (Rouviere et al., 1995). P₁ and P₂ denote the two previously mapped σ^E promoters (Rouviere et al., 1995). Also shown is the location of the promoter between rpoE and rseA (Rhodius et al., 2006). (+) indicates positive transcriptional regulation by σ^E. B. A putative –35 sequence (underlined) is supplied by the transposon. Location of the Tn10dCam insertion site is indicated by symbol (▴). Sequence to left of symbol (Italics) is Tn10 sequence and to the right (large type) is 5′ sequence of rpoE.

Stress induction of a σ^E-regulated promoter is inhibited in rpoE2072::Tn10dCam cells. β-Galactosidase activity expressed from the rpoH P3 promoter was measured following induction of the OMP C-terminal peptide, YYF, from pBA166, or with the control vector plasmid pTrc99. The experiment was repeated twice with similar results. A. β-Galactosidase activity plotted over time following IPTG addition to LBH cultures. Strains are: SMR8843, rpoE::Tn[pYYF] (); SMR8844 rpoE::Tn[pTrc] (▴); SMR8846, rpoE⁺[pYYF] (•); SMR8845, rpoE⁺[pTrc] (♦). B. Growth curve following IPTG addition. Symbols as in (A).

The rpoE2072::Tn10dCam mutation decreases stress-induced Lac⁺ reversion. Strains are rpoE⁺, SMR4562 (); rpoE2072::Tn10dCam, SMR5236 (♦). A. Representative experiment performed at 30°C. Values are means ± one standard error of the mean (SEM) for eight independent cultures of each strain. Where not visible, error bars are smaller than the symbol. A second experiment at 30°C gave similar results. B. Relative viability of the Lac^- population monitored per Harris et al. (1996) beginning on the day after plating (day 1) for the experiment presented in (A). Values are means ± SEM for data from six selection plates. Because Lac⁺ mutant cells form colonies that are visible 2 days later (McKenzie et al., 1998), the day 3 Lac⁺ colony counts pertain to the day 1 viable cell measurements, and day 5 Lac⁺ colonies to the day 3 viable cells, etc. To make this comparison easier, we have shifted the viability data (B) 2 days rightward (the day 1 viability data are presented on day 3, etc.) for easier comparison with (A). C. Lac⁺ colony formation rates at 37° from multiple experiments. Lac⁺ colonies per day were calculated from colonies appearing from days 3–5 for seven independent stress-induced mutation assays and fold-difference between rates for SMR4562, rpoE⁺ and SMR5236, rpoE2072::Tn10dCam presented. Viability of all cultures was monitored per Harris et al. (1996). Mean ± SEM for the seven experiments is shown in last row of table. As observed previously, overall mutation rates are higher at 30°C than 37°C, although mutations that decrease mutagenesis do so similarly at both temperatures [Ponder et al. (2005) and A versus C].

RpoE is required for stress-induced lac amplification. A.lac-amplified colonies, identified by sectored-colony morphology on LBH medium with X-gal, during a stress-induced mutagenesis experiment. Strains, rpoE⁺ SMR4562; rpoE::Tn SMR5236. Means for four cultures ± 1 SEM. Similar results were obtained from two additional experiments. B.Viable cell measurements were performed as described in Fig. 3. Means for four cultures ± 1 SEM. C.lac amplification rates from days 2–7 in three experiments. D.Lac⁺ point mutation rates from days 2–7 in three experiments. Point mutants were distinguished from lac-amplified clones by their pure blue colony morphology on LBH X-gal.

The role of the σ^E response in stress-induced mutagenesis is independent of SOS induction and dinB upregulation. A. Expression from the dinB promoter is not reduced in rpoE mutant cells. β-Galactosidase activity assayed in rpoE⁺ and rpoE2072::Tn10dCam cells carrying a plasmid-encoded P_dinB::lacZ fusion. Strains are: lexA(Def)rpoE::Tn, SMR10475; lexA(Def), SMR10474; rpoE⁺, SMR10472; rpoE::Tn, SMR10479. β-Galactosidase activity is expressed as Miller units per 0.5 ml culture and is the average of two experiments ± range. B. DinB protein levels are not reduced in rpoE2072::Tn10dCam cells. Western immunoblot of rpoE2072::Tn10dCam (rpoE::Tn, SMR5236) and rpoE⁺ (SMR4562) cells using antibodies against DinB. Proteins separated by SDS-PAGE were blotted to PVDF membranes and reacted with antibodies as described in Experimental procedures. A separate experiment gave similar results. C. Constitutive expression of SOS/LexA regulon genes does not alleviate the requirement for an inducible σ^E response in stress-induced mutagenesis. Rates of stress-induced Lac⁺ colony formation at 37°C calculated from colonies arising from days 3–5 from three separate experiments. Mean ± SEM. Strains are: lexA(Def), SMR10369; lexA(Def)rpoE::Tn, SMR10370; rpoE⁺, SMR4562; rpoE::Tn, SMR5236. lexA(Def) strains also carry mutations in sulA (required for cell viability) and psiB (inactivating an SOS-upregulated inhibitor of mutation) per McKenzie et al. (2000). D. SOS-induced levels of DinB do not substitute for a stress-inducible σ^E in stress-induced mutagenesis. Experimental details as in (C). Strains are: rpoE⁺, SMR4562; rpoE::Tn, SMR5236; dinBO^c1, SMR10464; dinBO^c2, SMR10465; rpoE::Tn dinBO^c1, SMR10466; rpoE::Tn dinBO^c2, SMR10467.

rpoE2072::Tn10dCam inhibits stress-induced mutagenesis independently of effects on the σ³² cytoplasmic heat-shock- or RpoS-stress responses. A. Constitutive activation of the σ³² response genes in dnaK mutant cells does not substitute for a functional σ^E response in stress-induced mutagenesis. Strains are: SMR4562 rpoE⁺ (); SMR8862 dnaK (▴); SMR5236 rpoE::Tn (♦); SMR8863 rpoE::Tn dnaK (•). Assay was performed at 37°C as described in Experimental procedures. Values are means ± one SEM for six independent cultures of each strain in one experiment. Three experiments gave similar results. B. Viability of all cultures was monitored per Harris et al. (1996). Strains and symbols are as in (A) but with open symbols. C. Activity of the RpoS-dependent katE promoter is not diminished by rpoE2072::Tn10dCam. β-Galactosidase activity from a katE::lacZ fusion was measured in saturated LBH cultures in strains SL590, rpoE⁺; SMR8919, rpoE2072::Tn; and CH1761, rpoS::FRTKan. The means ± range of two experiments are shown. Error bars are too small to see for the rpoS strain.

I-SceI-generated DSBs relieve much of the rpoE2072::Tn10dCam defect in stress-induced mutagenesis. A. Stress-induced mutation rates in the presence of I-SceI generated DSBs. Expression of I-SceI enzyme in cells with no I-SceI cutsite (left panel) does not affect the requirement for functional rpoE in stress-induced mutagenesis. However when I-SceI is expressed in cells with a cutsite near lac (DSBs, right panel), the rpoE2072::Tn10dCam phenotype is greatly reduced. Rates are Lac⁺ cfu per 10⁸ cells per day between days 3 and 5 and represent the averages of four experiments ± SEM. Experiments were performed at 37°C as described in Experimental procedures, and viability monitored per Harris et al. (1996). Strains are: rpoE⁺, SMR4562; rpoE::Tn, SMR5236; rpoE⁺ I-SceI (enzyme, no cutsite), SMR6276; rpoE::Tn I-SceI (enzyme, no cutsite), SMR9191; rpoE⁺ DSB (enzyme + cutsite), SMR6280; and rpoE::Tn DSB (enzyme + cutsite), SMR10168. B. Western immunoblot using antibodies against TraI. Proteins separated by SDS-PAGE as described in Experimental procedures were blotted to nitrocellulose membranes and probed with anti-TraI. MC4100 [F⁺]; rpoE⁺[F′], SMR4562; rpoE::Tn[F′], SMR5236. Two separate experiments gave similar results.