研究生基因表达的调控..pptVIP

(A) Plac-lacZ expression was characterized for wild-type NCM3722 cells growing exponentially on glycerol, glucose, or lactose as the sole carbon source (red diamonds, squares and triangles, respectively); 1 mM IPTG was added to deactivate LacI. At time zero, 20 mM oaa was added; a transient repression period of ~30 min is shaded in grey. Strain NQ1053 carrying PlacUV5-lacZ (black squares; right y-axis) was not affected. (B) LacZ expression levels before and during the repression period are quantified and shown as the red and black bars. Also quantified are the expression levels of two PTS deletion strains, NQ721 (Δpts, pink) and NQ506 (Δ5EI Δpts, orange). (C)cAMP concentration in the medium were monitored for wild-type cells grown in glycerol (red diamonds) and the two PTS-deletion strains, NQ721 (Δpts) and NQ506 (Δ5EI Δpts), grown in lactose (pink and orange triangles). 20 mM oaa was added at time zero. (D) Relative cAMP excretion rates which reflect the internal cAMP levels were quantified before and during the repression period; (E) in vitro AC activities in strains with and without PTS, NQ385 (pts+), NQ976 (Δpts) or NQ977 (Δ5EI Δpts), were assayed using permeablized cells in the presence or absence of various metabolites. These strains are also deleted of the cAMP phosphodiesterase which is not primary to catabolite repression; (F) in vitro AC activities in NQ385 were assayed with 0-10 mM of various metabolites. The lines show the bestfit to simple inhibition kinetics y =1/ (1+ x / KI ) , where y is the relative AC activity, x is the metabolite concentration. KI, the half-inhibition concentration, is found to be 0.84±0.04 mM, 0.63±0.02 mM and 1.76±0.05 mM for oaa, pyr, akg respectively. (G) oaa transiently represses PlacZ-lacZ expression in PTS-deleted cells, NQ721 (Δpts) and NQ506 (Δ5EI Δpts), grown exponentially in lactose. 20 mM oaa was supplied at time zero. Relative LacZ activities before and during this transient repression were quantified in (B); (H) Gr