Loss of Elongation Factor P Disrupts Bacterial Outer Membrane Integrity

Abstract

Elongation factor P (EF-P) is posttranslationally modified at a conserved lysyl residue by the coordinated action of two enzymes, PoxA and YjeK. We have previously established the importance of this modification in Salmonella stress resistance. Here we report that, like poxA and yjeK mutants, Salmonella strains lacking EF-P display increased susceptibility to hypoosmotic conditions, antibiotics, and detergents and enhanced resistance to the compound S-nitrosoglutathione. The susceptibility phenotypes are largely explained by the enhanced membrane permeability of the efp mutant, which exhibits increased uptake of the hydrophobic dye 1-N-phenylnaphthylamine (NPN). Analysis of the membrane proteomes of wild-type and efp mutant Salmonella strains reveals few changes, including the prominent overexpression of a single porin, KdgM, in the efp mutant outer membrane. Removal of KdgM in the efp mutant background ameliorates the detergent, antibiotic, and osmosensitivity phenotypes and restores wild-type permeability to NPN. Our data support a role for EF-P in the translational regulation of a limited number of proteins that, when perturbed, renders the cell susceptible to stress by the adventitious overexpression of an outer membrane porin.

(R)-β-Lysine-modified Elongation Factor P Functions in Translation Elongation

“…Many of these phenotypes are consistent with increased membrane permeability, suggesting a role for EF-P in regulating expression of outer membrane proteins, such as KdgM (3). Proteomic analyses indicated that expression of a subset of genes is under posttranscriptional regulation by EF-P and that this activity is dependent on the ␤-Lys modification (3).…”

“…To test the effects of hydroxylation on EF-P activity in vivo, an S. enterica ⌬yfcM strain was characterized with respect to phenotypes previously described for ⌬efp, ⌬poxA, and ⌬yjeK strains (3,8). The phenotypes of the ⌬yfcM mutant were very similar to those of wild type Salmonella when grown on the hypoosmolar antibiotic medium 2 agar and when exposed to gentamicin, lauryl sulfobetaine, or S-nitrosoglutathione (Fig.…”

“…PhenotypesPrevious studies have shown that the pathway encoded by the genes efp, poxA, and yjeK is linked to a number of growth phenotypes in E. coli and S. enterica (3,8). To test the effects of hydroxylation on EF-P activity in vivo, an S. enterica ⌬yfcM strain was characterized with respect to phenotypes previously described for ⌬efp, ⌬poxA, and ⌬yjeK strains (3,8).…”

“…Growth Assays-Sensitivity to hypoosmolarity, gentamicin, lauryl sulfobetaine, and S-nitrosoglutathione was assessed as described previously (3). The migratory ability of the ⌬yfcM mutant was determined using soft agar assays, and accumulation of 1-N-phenylnaphthylamine was conducted to measure membrane permeability, both as described previously (3).…”

A Genomewide Mutagenesis Screen Identifies Multiple Genes Contributing to Vi Capsular Expression in Salmonella enterica Serovar Typhi

“…Mutations in yrfF are lethal in isolates of S. Typhimurium (29), but we did not find this to be the case in S. Typhi BRD948. The oxyR gene, which encodes a LysR family member, was also identified in the screen (30,31), as were the transcription elongation factor-encoding gene greA (32,33) and the elongation factor P-encoding gene efp (34). Construction of defined S. Typhi BRD948 mutant derivatives.…”