Fig 5. Removal of the membrane anchor enables RNase Y to suppress the phenotypes of a ΔcshA mutant.

(A) In the left panel, over-night cultures of single mutants ΔcshA, ΔY and Y^Δ2–24 and double mutants ΔYΔcshA and Y^Δ2–24ΔcshA were diluted, spotted on agar-plates, and incubated at the indicated temperatures and times. In the right panel, over-night cultures were spotted on horse-blood-agar. (B) Transformants of strain ΔYΔcshA with plasmids expressing variants of RNase Y were selected at 42°C, then restreaked and grown over night at 42°C. Finally the cultures were diluted and spotted at the indicated temperatures. ΔYΔcshA with pY^Δ2–24 grows significantly better than the other strains at 24°C. (C) Cartoon showing the four versions of RNase Y expressed from the plasmids; wild-type RNase Y (pY), anchorless RNase Y (pY^Δ2–24), RNase Y active site mutant (pY^367AA), and anchorless RNase Y active site mutant (pY^{Δ2–24,367AA}). (D) The Y^Δ2–24ΔcshA strain was transformed with the plasmids expressing the wild-type RNase Y, Y^367AA or Y^{Δ2–24,367AA}. Overnight cultures were diluted, spotted on agar-plates and incubated at the indicated temperatures for the indicated period of time. Both pY and pY^367AA inhibit growth at 24°C. (E) Cartoon showing how the wild-type RNase Y and Y^367AA can anchor the Y^Δ2–24 protein back to the membrane, via dimer-formation.