Molecular basis for the temperature sensitivity of Escherichia coli pth(Ts)

Abstract

The gene pth, encoding peptidyl-tRNA hydrolase (Pth), is essential for protein synthesis and viability of Escherichia coli. Two pth mutants have been studied in depth: a pth(Ts) mutant isolated as temperature sensitive and a pth(rap) mutant selected as nonpermissive for bacteriophage lambda vegetative growth. Here we show that each mutant protein is defective in a different way. The Pth(Ts) protein was very unstable in vivo, both at 43 degrees C and at permissive temperatures, but its specific activity was comparable to that of the wild-type enzyme, Pth(wt). Conversely, the mutant Pth(rap) protein had the same stability as Pth(wt), but its specific activity was low. The thermosensitivity of the pth(Ts) mutant, presumably, ensues after Pth(Ts) protein levels are reduced at 43 degrees C. Conditions that increased the cellular Pth(Ts) concentration, a rise in gene copy number or diminished protein degradation, allowed cell growth at a nonpermissive temperature. Antibiotic-mediated inhibition of mRNA and protein synthesis, but not of peptidyl-tRNA drop-off, reduced pth(Ts) cell viability even at a permissive temperature. Based on these results, we suggest that Pth(Ts) protein, being unstable in vivo, supports cell viability only if its concentration is maintained above a threshold that allows general protein synthesis.

FIG. 1

Variant Pth protein stabilities. Pth protein concentrations in C600 pth(wt), C600 pth(rap), or C600 pth(Ts) at various times were measured after rifampin and chloramphenicol addition (see Materials and Methods). The arrow indicates Pth(Ts) protein position.

FIG. 2

In vitro aggregation of Pth(Ts) protein. S30 extracts of the indicated strains were incubated for 20 min at 30 or 43°C and centrifuged at 10,000 × g for 10 min. Pth protein concentration was visualized by immunoblot assay in equivalent amounts of total extract (E), pellet (P), or supernatant (S) fractions. The intensity of the cross-reacted materials above Pth is stronger than that in Fig. 1 because of increased protein concentrations loaded on the gels.

FIG. 3

Effect of pth(Ts) gene overexpression on the survival of pth(Ts) mutant cells after shift to 43°C. (A) Colony-forming ability of strain AA7852 untransformed (open circles) or transformed with plasmid pGI03 with (closed squares) or without (closed triangles) IPTG added at 0 min. (B) Concentration of Pth(Ts) protein, as estimated by immunoblot analysis, in the cells at the indicated times after the temperature shift. The arrows indicate Pth(Ts) position.

FIG. 4

Effect of antibiotic addition on pth(Ts) mutant cell growth at 30°C. The figure shows colony-forming ability of AA7852 cells after addition of rifampin (100 μg/ml) (open circles), erythromycin (80 μg/ml) (open squares), rifampin plus chloramphenicol (50 μg/ml) (open triangles), or rifampin plus erythromycin (closed circles).

FIG. 5

Effect of dnaJ259 mutation on pth(Ts) mutant cell growth at 30 and 41°C. (A) C600 dnaJ259 pth(Ts). (B) C600 dnaJ259. (C) C600 pth(Ts). (D) C600. Cells from a single colony were streaked on LB-agar plates and incubated overnight at the indicated temperatures. In this case, 41°C was used as the nonpermissive temperature because the dnaJ259 strain is not viable at 43°C.