In vivo depletion of YaeL. (A) Growth of YaeL depletion strain (CAG43509) in yaeL-inducing (LB/0.2% arabinose; ▵) or repressing (LB/0.2% glucose; ▪) media at 30°C. The depletion strain carries yaeL∷kanR on its chromosome and a complementing plasmid encoding yaeL expressed from an arabinose-inducible promoter P_ara. The depletion was performed as described in Materials and Methods. To maintain the cultures in exponential phase, they were subcultured by dilution into the same prewarmed media. Numbers 1–4 designate the subcultures, and letters A–E identify the time points at which samples for Western blotting were removed. (B) σ^E activity during YaeL depletion. Samples from the LB/glucose and LB/arabinose subcultures in (A) were assayed for σ^E activity by monitoring β-galactosidase activity produced from a single-copy [ΦλrpoH P3∷lacZ] fusion. For simplicity, only σ^E activity from the first arabinose subculture is shown, because all subsequent arabinose subcultures had equivalent activities. Assays were performed as described in Materials and Methods. (C) In vivo steady-state levels of YaeL, RseA, and RseA fragment during depletion. At the time points identified in (A), samples were removed from the subcultures and analyzed by Western blotting using anti-YaeL and anti-RseA cytoplasmic domain antisera (see Materials and Methods). Asterisk indicates a nonspecific background band which controls for loading error.

Wild-type YaeL and YaeL E23D, but not the active-site mutant YaeL E23A, restore σ^E activity to a yaeL∷kanR strain. sup⁺ yaeL∷kanR strains carrying pompC and pJAH322 (yaeL; ▪/□, CAG43540) or pJAH340 (yaeL E23A; ●/○, CAG43541) or pJAH325 (yaeL E23D; ▴/▵, CAG43553) were assayed for basal and induced σ^E activity by monitoring β-galactosidase activity produced from a single-copy [ΦλrpoH P3∷lacZ] fusion, as described in Materials and Methods. The σ^E-inducing signal was provided by overexpressing OmpC with 0.2% arabinose, which was added immediately following the first assay time point (OD₆₀₀∼0.15). Solid symbols indicate the β-galactosidase activity of strains growing in the presence of arabinose (and, hence, overexpressing OmpC); open symbols indicate the β-galactosidase activity of strains growing in the absence of arabinose (without OmpC overexpression). The inset shows the very low β-galactosidase activity of the yaeL E23A strain.

YaeL is involved in degradation of RseA. (A) Western blotting (with anti-RseA cytoplasmic domain) of samples harvested from sup⁺ yaeL∷kanR strains carrying pompC and pJAH322 (yaeL; lanes 1,2) or pJAH340 (yaeL E23A; lanes 3,4) or pJAH325 (yaeL E23D; lanes 5,6) grown with or without overexpression of OmpC for 60 min. These samples are from the strains assayed for σ^E activity in Fig. 2. (B) Western blotting (with anti-RseA cytoplasmic domain) of samples harvested from ΔdegS yaeL∷kanR strains with pompC (lanes 1,2; CAG43551) or empty vector (lanes 3,4; CAG43552). Lane 5 shows a reference sample containing both full-length RseA and the RseA fragment. In both panels A and B, arabinose was added to induce the overexpression of OmpC (see Materials and Methods). Cell fractionation experiments confirmed that overexpressed OmpC was present in the outer membrane fraction of the ΔdegS yaeL∷kanR strain carrying pompC (data not shown).

Full-length RseA decreases and the RseA fragment increases following OmpC overexpression. (A) Western blotting (with anti-RseA cytoplasmic domain) of samples harvested from sup⁺ yaeL∷kanR pompC pJAH340 (yaeL E23A; CAG43541) at various time points after OmpC overexpression is induced. (B) Quantitation of full-length RseA and RseA fragment levels. Blots were quantified as described in Materials and Methods. The intensity of each band was normalized to that of a background band (not shown) to control for loading errors.

RseA fragment is localized to the inner membrane. Strain sup⁺ yaeL∷kanR pompC (CAG43514) was grown to mid-log phase, and pompC was induced with arabinose for 1 h prior to fractionation, as detailed in Materials and Methods. The cells were fractionated into four components: periplasm, cytoplasm, and inner and outer membrane. Western blots were probed with anti-HtpG (cytoplasmic protein), anti-FtsH (inner membrane protein), anti-MalE (periplasmic protein), and anti-RseA cytoplasmic domain antisera.

Overexpression of DegS or YaeL separately or together does not affect σ^E activity. Wild-type strains with a yaeL-expressing plasmid (▴/▵; CAG43576) or degS-expressing plasmid plus a yaeL-expressing plasmid (▪/□; CAG43512) were grown to early log phase. At an OD₆₀₀ ∼0.15, arabinose was added to overexpress YaeL, and σ^E activity was by monitored β-galactosidase assays (See Materials and Methods). DegS was constitutively overexpressed in CAG43512. Solid symbols indicate the β-galactosidase activity of strains growing in the presence of arabinose (and, hence, overexpressing YaeL); open symbols indicate the β-galactosidase activity of strains growing in the absence of arabinose (without YaeL overexpression). Strains containing the empty vectors exhibited nearly identical activities and for simplicity are not shown.