Analysis of theEscherichia coliAlp Phenotype: Heat Shock Induction inssrAMutants

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Abstract
The major phenotypes oflonmutations, UV sensitivity and overproduction of capsule, are due to the stabilization of two substrates, SulA and RcsA. Inactivation of transfer mRNA (tmRNA) (encoded byssrA), coupled with a multicopy kanamycin resistance determinant, suppressed bothlonphenotypes 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(forfunctionaffectingAlp), that leads to full suppression of a Δlon ssrA::cathost and thus bypasses the requirement for multicopy Kanr;faaandssrAmutants are additive in their ability to suppresslonmutants. Thefaamutation was mapped to the C terminus ofdnaJ(G232);dnaJnull mutants have similar effects. The identification of alonsuppressor indnaJsuggested the possible involvement of heat shock. We find thatssrAmutants alone significantly induce the heat shock response. The suppression of UV sensitivity, both in the original Alp strain and infaamutants, is reversed by mutations inclpY, encoding a subunit of the heat shock-induced ClpYQ protease that is known to degrade SulA. However, capsule synthesis is not restored byclpYmutants, probably because less RcsA accumulates in the Alp strain and because the RcsA that does accumulate is inactive. BothssrAeffects are partially relieved byssrAderivatives encoding protease-resistant tags, implicating ribosome stalling as the primary defect. Thus,ssrAandfaaeach suppress twolonmutant phenotypes but by somewhat different mechanisms, with heat shock induction playing a major role.