Fig 7. αRBS sequences in the open reading frame are necessary for fHbp thermoregulation.

(A) Western blot analysis of fHbp-GFP levels in E. coli harbouring pfHbp-9C-gfp (WT) or the plasmid with modified αRBS-1, αRBS-2 or both (αRBS-1&2). Bacteria were grown at the temperatures indicated to mid-log phase, and cell lysates were subjected to Western blot analysis. The membranes were incubated with antibodies recognising fHbp or RecA. (B) In vitro translation of RNA from E. coli harbouring pfHbp-9C-gfp or plasmids containing altered RBSs (indicated). in vitro translation was performed at the temperatures indicated and subject to Western analysis with antibodies recognising GFP and RecA (loading control). mut-αRBS-1 and mut-αRBS-2 showed reduced thermosensing ability whereas modification of both αRBS-1 and 2 (mut-αRBS-1&2) almost completely abolishes thermoregulation both in E. coli and in in vitro translation assays. Relative values (rel. values) of GFP are indicated below each lane.