Characterization of six lipoproteins in the sigmaE regulon

Abstract

In Escherichia coli, sigma(E) regulon functions are required for envelope homeostasis during stress and are essential for viability under all growth conditions. The E. coli genome encodes approximately 100 lipoproteins, and 6 of these are regulated by sigma(E). Phenotypes associated with deletion of each of these lipoproteins are the subject of this report. One lipoprotein, YfiO, is essential for cellular viability. However, overexpression of this protein is not sufficient to alleviate the requirement of sigma(E) for viability, suggesting that the sigma(E) regulon provides more than one essential function. The remaining five lipoproteins in the sigma(E) regulon are nonessential; cells are viable even when all five are removed simultaneously. Deletion of three nonessential lipoprotein genes (nlpB, yraP, ygfL) results in the exhibition of phenotypes that suggest they are important for maintenance of the integrity of the cell envelope. deltanlpB cells are selectively sensitive to rifampin; deltayraP cells are selectively sensitive to sodium dodecyl sulfate. Such selective sensitivity has not been previously reported. Both deltayraP and deltanlpB are synthetically lethal with surA::Cm, which encodes a periplasmic chaperone and PPIase, suggesting that NlpB and YraP play roles in a periplasmic folding pathway that functions in parallel with that of SurA. Finally, the deltayfgL mutant exhibits a broad range of envelope defects, including sensitivity to several membrane-impermeable agents, an altered outer membrane protein profile, synthetic lethality with both surA::Cm and deltafkpA::Cm strains, and sensitivity to a bactericidal permeability-increasing peptide. We suggest that this lipoprotein performs a very important but as-yet-unknown function in maintaining the integrity of the cell envelope.

FIG. 1.

The lipoboxes of the six σ^E-transcribed lipoproteins. Displayed are the N-terminal sequences of each lipoprotein in the σ^E regulon. The lipobox motifs are depicted in large bold type. The conserved cysteine residue in each lipobox, which is destined for lipid modification, is shaded. For each sequence, the number in parentheses to the right refers to the position of the cysteine from the N-terminal methionine.

FIG. 2.

Effects of deleting nonessential lipoprotein genes on sensitivity to BPI-derived P2 peptide. Stationary-phase bacteria grown without agitation in 5 ml TSB were diluted to ∼2 × 10⁷ CFU/ml in 20 mM sodium phosphate buffer, pH 6.0, in a final volume of 50 μl. Samples were incubated with 4 mg/ml BPI for 90 min at 37°C. Data represent the mean CFU per ml from serial dilutions of triplicate samples plated on LB agar and incubated for 18 h at 37°C.

FIG. 3.

σ^E activity of strains in which each of the nonessential lipoproteins has been deleted. Relative σ^E activities in WT (CAG45114), ΔyeaY (CAG41241), ΔyraP (CAG41251), ΔnlpB (CAG41258), ΔyfeY (CAG41413), ΔyfgL (CAG41445), and surA::Cm (CAG41265) strains growing at 30°C in LB were assayed by monitoring β-galactosidase activity from a single-copy Φλ[rpoHP3-lacZ] fusion as described in Materials and Methods. The increased σ^E activity observed in the ΔyfgL mutant can be complemented by pyfgL.

FIG.4.

The OMP profiles of wild type, ΔyfgL, and ΔsurA::Cm strains. (A) Outer membrane fractions were prepared from cells that had been growing exponentially at 37°C in LB plus 0.2% maltose, as described in Materials and Methods. The fractions were analyzed on a 12% SDS-polyacrylamide gel with 50% urea and stained with Coomassie blue. The positions of LamB, OmpC, OmpF, and OmpA are indicated. (B) The levels of LamB, OmpC, OmpF, and OmpA were quantified via spot densitometry as described in Materials and Methods. (C) The OMP profile defect in the ΔyfgL mutant can be complemented by pyfgL.

FIG.4.

The OMP profiles of wild type, ΔyfgL, and ΔsurA::Cm strains. (A) Outer membrane fractions were prepared from cells that had been growing exponentially at 37°C in LB plus 0.2% maltose, as described in Materials and Methods. The fractions were analyzed on a 12% SDS-polyacrylamide gel with 50% urea and stained with Coomassie blue. The positions of LamB, OmpC, OmpF, and OmpA are indicated. (B) The levels of LamB, OmpC, OmpF, and OmpA were quantified via spot densitometry as described in Materials and Methods. (C) The OMP profile defect in the ΔyfgL mutant can be complemented by pyfgL.