Spot 42 and SpfP have different effects at different temperatures. (A) Immunoblotting analysis of GalK–HA-His6 levels in a Δspf galK-HA-His6::kan strain (GSO1060) transformed with pRI, pRI-Spot 42, or pRI-Spot 42STOP and grown in LB at 30 °C, 37 °C, and 42 °C. (B) Immunoblotting analysis of GalK–HA-His6 levels in a Δspf galK-HA-His6::kan strain (GSO1060) transformed with pKK, pKK–SpfP-recoded, or pKK–SpfP-recodedSTOP and grown in LB at 30 °C, 37 °C, and 42 °C. For A and B, samples were collected at an OD600 of ∼0.5. Anti-His antibody was used to detect the HA-His6 tag. The Ponceau S–stained membrane documents approximately equal loading of the samples. (C) Immunoblotting analysis of GalK–HA-His6 levels in galK-HA-His6::kan (GSO1057), Δspf galK-HA-His6::kan (GSO1060), spf-recoded galK-HA-His6::kan (GSO1077), or spfSTOP::kan
galK-HA-His6 (GSO1075) grown in M63 glucose to an OD600 of ∼0.4 at 30 °C (Left) or 42 °C (Right). Cells were collected and resuspended in M63 galactose at 30 °C or 42 °C, respectively, with samples collected at the indicated times. (D) The small protein SpfP reinforces the multioutput feedforward loop between CRP and Spot 42. For cells grown in the absence of glucose, CRP directly increases transcription of targets and represses Spot 42. When glucose is available (shown here), the Spot 42 RNA represses CRP-activated targets through base pairing, particularly at lower temperatures (Left), and the small protein SpfP blocks CRP-dependent activation, particularly at higher temperature (Right) (adapted from ref. 16).