Analysis of the Escherichia coli Alp phenotype: heat shock induction in ssrA mutants.
The major phenotypes of lon mutations, UV sensitivity and overproduction of capsule, are due to the stabilization of two substrates, SulA and RcsA. Inactivation of transfer mRNA (tmRNA) (encoded by ssrA), coupled with a multicopy kanamycin resistance determinant, suppressed both lon phenotypes and restored the rapid degradation of SulA. This novel protease activity was named Alp but was never identified further. We report here the identification, mapping, and characterization of a chromosomal mutation, faa (for function affecting Alp), that leads to full suppression of a Deltalon ssrA::cat host and thus bypasses the requirement for multicopy Kan(r); faa and ssrA mutants are additive in their ability to suppress lon mutants. The faa mutation was mapped to the C terminus of dnaJ(G232); dnaJ null mutants have similar effects. The identification of a lon suppressor in dnaJ suggested the possible involvement of heat shock. We find that ssrA mutants alone significantly induce the heat shock response. The suppression of UV sensitivity, both in the original Alp strain and in faa mutants, is reversed by mutations in clpY, encoding a subunit of the heat shock-induced ClpYQ protease that is known to degrade SulA. However, capsule synthesis is not restored by clpY mutants, probably because less RcsA accumulates in the Alp strain and because the RcsA that does accumulate is inactive. Both ssrA effects are partially relieved by ssrA derivatives encoding protease-resistant tags, implicating ribosome stalling as the primary defect. Thus, ssrA and faa each suppress two lon mutant phenotypes but by somewhat different mechanisms, with heat shock induction playing a major role.
Munavar
Hussain
H
Laboratory of Molecular Biology, National Cancer Institute, Bethesda, MD 20892-4255, USA.
Zhou
Yanning
Y
Gottesman
Susan
S
D002876:Chromosomes, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics* / Q000528:radiation effects; D006360:Heat-Shock Proteins / Q000235:genetics; D006358:Hot Temperature; D008741:Methyl Methanesulfonate / Q000494:pharmacology; D016296:Mutagenesis; D009153:Mutagens; D009154:Mutation; D009604:Nitrosoguanidines / Q000494:pharmacology; D010641:Phenotype; D012329:RNA, Bacterial / Q000235:genetics*; D014466:Ultraviolet Rays
D006360:Heat-Shock Proteins; D009153:Mutagens; D009604:Nitrosoguanidines; D012329:RNA, Bacterial; C096222:tmRNA; D008741:Methyl Methanesulfonate