FIG. 7.
Heat shock induction of ykgR. (A) Primer extension analysis of ykgR-SPA mRNA (top) and Western blot analysis of YkgR-SPA protein (bottom) levels in MG1655 without and with heat shock. Thirty-milliliter LB, M63 glucose, or M63 glycerol cultures were inoculated with a dilution of overnight LB cultures and grown to an OD600 of 0.3 to 0.4 before being split into three 10-ml aliquots. Two aliquots were kept at 30°C while the other was incubated at 45°C. Cells were harvested before transfer (T0) and after 20 min (30°C or 45°C). (B) Heat shock induction of ykgR transcriptional and translational fusions. Extracts from strains containing the SPA-tagged ykgR allele (ykgR-SPA), a transcriptional fusion to the ykgR promoter (PykgR-5′+SPA), a translational fusion to the ykgR promoter and 5′-UTR (P + 5′ykgR-SPA), or a control transcriptional fusion to the azuC promoter (PazuC-5′+SPA) were probed for SPA expression in cells without or with heat shock. In the transcriptional fusions, the ykgR and azuC 5′-UTRs were replaced by the MCS 5′-UTR from pBAD24, and the ORFs were replaced by the SPA tag. In the translational fusion, the ykgR ORF was replaced by the SPA tag. Cultures grown overnight in LB were diluted into 30 ml LB and grown to an OD600 of 0.4 to 0.5 before being split into two 10-ml aliquots. One aliquot was kept at 30°C while the other was incubated at 45°C. (C) YkgR-SPA expression in wild-type cells and cells with altered sigma factor levels. YkgR-SPA levels were assayed in wild-type and ΔrpoS cells without and with heat shock, as well as in ykgR-SPA cells in which σH or σE synthesis was induced by the addition of isopropyl-β-d-thiogalactopyranoside (IPTG) to half of the sample. MG1655 and ΔrpoS cells grown overnight in LB were diluted into 30 ml LB and incubated at 30°C until the OD600 reached 0.4. Cultures were then split into two 10-ml aliquots. One set of samples was transferred to 45°C while the other was kept at 30°C. Cells were harvested before transfer (T0) and after 20 min of induction (30°C and 45°C). YkgR-SPA cells containing rpoH (pSAKTtrc) and rpoE (pCL245) overexpression plasmids were grown overnight in LB plus 100 μg/ml carbenicillin and were diluted into 30 ml of LB plus 100 μg/ml carbenicillin. At an OD600 of 0.4, cultures were split into two 10-ml aliquots, and sigma factor expression was induced in one set of samples by adding IPTG to 1 mM. Cells were harvested before induction (T0) and 20 min after induction (− and +, respectively). Primer extension assays were as conducted as described for Fig. 6. Western blot analysis was performed as stated for Fig. 2. A single asterisk denotes a band corresponding to the full-length SPA-tagged YkgR protein, and a double asterisk denotes a band corresponding to the SPA peptide.