Analysis of the Escherichia coli Alp phenotype: heat shock induction in ssrA mutants

Abstract

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.

FIG. 1.

Isolation of faa mutants. The capsule synthesis and UV sensitivity of lon mutants were used to first isolate Alp strains (17, 35) and, in this paper, faa mutants that proved to mimic the Alp phenotype. The status of capsule synthesis was determined from expression of a cps-lac fusion on MacConkey plates; UV sensitivity was determined as described in Materials and Methods. pUC4K is a plasmid derivative of pUC18 carrying kan.

FIG. 2.

Reversal of UV resistance phenotype in clpY mutants. Cells were grown and exposed to UV light as described in Materials and Methods. The UV dose is expressed in seconds. EOP is shown for four pairs of clpY⁺ (filled symbols) and clpY::cat (open symbols) strains. Circles, SG22787 (Δlon-510 clpY⁺) and SG22789 (Δlon-510 clpY::cat); squares, SG22786/pUC4K (Δlon-510 ssrA::cat clpY⁺/pUC4K, original Alp strain) and SG22788/pUC4K (Δlon-510 ssrA::cat clpY::cat/pUC4K); triangles, SG22808 (Δlon-510 faa clpY⁺) and SG22805 (Δlon-510 faa clpY::cat); and diamonds, SG22809 (Δlon-510 dnaJ::kan clpY⁺) and SG22807 (Δlon-510 dnaJ::kan clpY::cat). Results are representative of three separate trials on these same strains.

FIG. 3.

ClpQ levels in UV-resistant strains. Strains were grown in LB broth at 30°C to an OD₆₀₀ of 0.5, and samples were taken for SDS gel electrophoresis and Western blotting, as described in Materials and Methods. Levels in the gels were quantitated as described in Materials and Methods, and the results were normalized to the level of ClpQ in the lon mutant grown at 30°C, set equal to 1 (column 1). lon, heat; results after 10 min of heat treatment. Results are the averages of at least two determinations. The following strains were used: SG22787 (Δlon-510), SG22808 (Δlon-510 faa), SG22809 (Δlon-510 dnaJ::kan), SG22786 (Δlon-510 ssrA::cat), SG22786/pUC18, SG22786/pUC4K, SG22810 (Δlon-510 faa ssrA::cat), and SG22774 (lon⁺ ssrA::cat).

FIG. 4.

SulA turnover. Cells were grown, induced, and treated as described in Materials and Methods. (A) SulA turnover, normalized to that at 15 min after spectinomycin addition, from the data in Fig. 5B. Filled squares, SG22787 (Δlon-510 clpY⁺); open squares, SG22789 (Δlon-510 clpY::cat); filled triangles, SG22808 (Δlon-510 faa clpY⁺); and open triangles, SG22805 (Δlon-510 faa clpY::cat). (B) Western blots of protein turnover after addition of spectinomycin. Because the antibiotic takes a few minutes to work, the first point shown is 15 min after spectinomycin addition. Bottom left, SG22786/pUC4K (Δlon-510 ssrA::cat⁺/pUC4K) (original Alp strain); bottom right, SG22788/pUC4K (Δlon-510 ssrA::cat clpY::cat/pUC4K). Other strains were as defined for panel A.

FIG. 5.

RcsA accumulation and activity. Activity of cps-lac fusion after growth at 30°C (A) and at 32°C (B) in M63 glucose CAA and Western blot of RcsA accumulation. Cells were grown to an OD₆₀₀ of 0.5, and samples were taken for Western blotting and for an assay of the cps-lac fusion. Lanes 1, SG22771 (lon⁺); lanes 2, SG22787 (lon mutant); lanes 3, SG22789 (lon clpY); lanes 4, SG22786/pUC4K (lon ssrA/pUC4K); lanes 5, SG22788/pUC4K (lon ssrA clpY/pUC4K); lanes 6, SG22808 (lon faa); lanes 7, SG22805 (lon faa clpY); lanes 8, SG22809 (lon dnaJ::kan); lanes 9, SG22807 (lon dnaJ::kan clpY); lanes 10, SG22810 (lon faa ssrA); and lanes 11, SG22804 (lon faa ssrA clpY). β gal, β-galactosidase.

FIG. 6.

Complementation of ssrA::cat/pUC4K. A set of plasmids carrying ssrA derivatives was introduced into SG22786/pJK2-2 (Δlon-510 ssrA::cat/pACYC184-Kan^r) and assayed for cps-lac expression, RcsA levels, and UV sensitivity, as described in Materials and Methods. Top left panel, cps-lac activity. Lane 1, ssrA⁺ plasmid; lane 2, ssrA(UG) plasmid; lane 3, ssrA(DD) plasmid; and lane 4, ssrA⁰ plasmid. The ssrA(UG) allele cannot be charged and acts as a negative control that does not interact with stalled ribosomes at all (40). The ssrA(DD) and ssrA⁰ derivatives add nondegradable tags to proteins but should release stalled ribosomes (40). Bottom left panel, RcsA levels (determined as described in Materials and Methods). Right panel, UV sensitivity. Cells were grown and exposed to UV light as described in Materials and Methods. Open squares, ssrA⁺ plasmid; filled diamonds, ssrA(UG) plasmid; open circles, ssrA(DD) plasmid; and filled circles, ssrA⁰ plasmid. β gal, β-galactosidase.

FIG. 6.

Complementation of ssrA::cat/pUC4K. A set of plasmids carrying ssrA derivatives was introduced into SG22786/pJK2-2 (Δlon-510 ssrA::cat/pACYC184-Kan^r) and assayed for cps-lac expression, RcsA levels, and UV sensitivity, as described in Materials and Methods. Top left panel, cps-lac activity. Lane 1, ssrA⁺ plasmid; lane 2, ssrA(UG) plasmid; lane 3, ssrA(DD) plasmid; and lane 4, ssrA⁰ plasmid. The ssrA(UG) allele cannot be charged and acts as a negative control that does not interact with stalled ribosomes at all (40). The ssrA(DD) and ssrA⁰ derivatives add nondegradable tags to proteins but should release stalled ribosomes (40). Bottom left panel, RcsA levels (determined as described in Materials and Methods). Right panel, UV sensitivity. Cells were grown and exposed to UV light as described in Materials and Methods. Open squares, ssrA⁺ plasmid; filled diamonds, ssrA(UG) plasmid; open circles, ssrA(DD) plasmid; and filled circles, ssrA⁰ plasmid. β gal, β-galactosidase.