Molecular basis for the temperature sensitivity of Escherichia coli pth(Ts).
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.
Cruz-Vera
L R
LR
Departamento de Genética y Biología Molecular, Centro de Investigación y de Estudios Avanzados del IPN, México City, Mexico.
Toledo
I
I
Hernández-Sánchez
J
J
Guarneros
G
G
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D002265:Carboxylic Ester Hydrolases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D015169:Colony Count, Microbial; D004795:Enzyme Stability; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D010957:Plasmids / Q000235:genetics; D013696:Temperature
D000900:Anti-Bacterial Agents; D002265:Carboxylic Ester Hydrolases; C019674:aminoacyl-tRNA hydrolase