SUMMARY We report an interaction between poxA, encoding a paralog of lysyl tRNA-synthetase, and the closely linked yjeK gene, encoding a putative 2,3-β-lysine aminomutase, that is critical for virulence and stress resistance in Salmonella enterica. Salmonella poxA and yjeK mutants share extensive phenotypic pleiotropy including attenuated virulence in mice, an increased ability to respire under nutrient limiting conditions, hypersusceptibility to a variety of diverse growth inhibitors and altered expression of multiple proteins including several encoded on the SPI-1 pathogenicity island. PoxA mediates post-translational modification of bacterial elongation factor P (EF-P), analogous to the modification of the eukaryotic EF-P homologue, eIF5A, with hypusine. The modification of EF-P is a mechanism of regulation whereby PoxA acts as an aminoacyl-tRNA synthetase that attaches an amino acid to a protein resembling tRNA rather than to a tRNA.

Elongation factor P (EF-P) is a universally conserved bacterial translation factor homologous to eukaryotic/archaeal initiation factor 5A. In Salmonella, deletion of the efp gene results in pleiotropic phenotypes, including increased susceptibility to numerous cellular stressors. Only a limited number of proteins are affected by the loss of EF-P, and it has recently been determined that EF-P plays a critical role in rescuing ribosomes stalled at PPP and PPG peptide sequences. Here we present an unbiased in vivo investigation of the specific targets of EF-P by employing stable isotope labeling of amino acids in cell culture (SILAC) to compare the proteomes of wild-type and efp mutant Salmonella. We found that metabolic and motility genes are prominent among the subset of proteins with decreased production in the Δefp mutant. Furthermore, particular tripeptide motifs are statistically overrepresented among the proteins downregulated in efp mutant strains. These include both PPP and PPG but also additional motifs, such as APP and YIRYIR, which were confirmed to induce EF-P dependence by a translational fusion assay. Notably, we found that many proteins containing polyproline motifs are not misregulated in an EF-P-deficient background, suggesting that the factors that govern EF-P-mediated regulation are complex. Finally, we analyzed the specific region of the PoxB protein that is modulated by EF-P and found that mutation of any residue within a specific GSCGPG sequence eliminates the requirement for EF-P. This work expands the known repertoire of EF-P target motifs and implicates factors beyond polyproline motifs that are required for EF-P-mediated regulation.

CA1 pyramidal neurons degenerate after transient global ischemia, whereas neurons in other regions of the hippocampus remain intact. A step in this selective injury is Ca(2+) and/or Zn(2+) entry through Ca(2+)-permeable AMPA receptor channels; reducing Ca(2+) permeability of AMPA receptors via expression of Ca(2+)-impermeable GluR2(R) channels or activation of CRE transcription in the hippocampus of adult rats in vivo using shutoff-deficient pSFV-based vectors rescues vulnerable CA1 pyramidal neurons from forebrain ischemic injury. Conversely, the induction of Ca(2+) and/or Zn(2+) influx through AMPA receptors by expressing functional Ca(2+)-permeable GluR2(Q) channels causes the postischemic degeneration of hippocampal granule neurons that otherwise are insensitive to ischemic insult. Thus, the AMPA receptor subunit GluR2 gates entry of Ca(2+) and/or Zn(2+) that leads to cell death following transient forebrain ischemia.

The lysyl-tRNA synthetase paralog PoxA modifies elongation factor P (EF-P) with α-lysine at low efficiency. Cell-free extracts contained non-α-lysine substrates of PoxA that modified EF-P by a change in mass consistent with β–lysine, a substrate also predicted by genomic analyses. EF-P was efficiently, functionally, modified with (R)-β-lysine but not (S)-β-lysine or genetically encoded α-amino acids, indicating that PoxA has evolved an activity orthogonal to that of the canonical aminoacyl-tRNA synthetases.

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.