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.

a b s t r a c tThe role of tRNA in translating the genetic code has received considerable attention over the last 50 years, and we now know in great detail how particular amino acids are specifically selected and brought to the ribosome in response to the corresponding mRNA codon. Over the same period, it has also become increasingly clear that the ribosome is not the only destination to which tRNAs deliver amino acids, with processes ranging from lipid modification to antibiotic biosynthesis all using aminoacyl-tRNAs as substrates. Here we review examples of alternative functions for tRNA beyond translation, which together suggest that the role of tRNA is to deliver amino acids for a variety of processes that includes, but is not limited to, protein synthesis. Ó 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. tRNA-dependent amino acid biosynthesisThe attachment of amino acids to the 3 0 -end of tRNAs is catalyzed by the aminoacyl-tRNA synthetase (aaRS) family of proteins [1]. aaRSs are ubiquitous and essential but only eukaryotes and a handful of bacteria have the full set of 20 enzymes, one for each canonical amino acid in the genetic code. Most bacteria and archaea lack asparaginyl-tRNA synthetase (AsnRS) and/or glutaminyl-tRNA synthetase (GlnRS) and some methanogenic archaea lack cysteinyl-tRNA synthetase (CysRS) [2]. Also, no aaRS for the rare amino acid selenocysteine has been found in any domain of life [3]. These organisms instead use indirect pathways to synthesize a number of amino acids (Asn, Cys, Gln and Sec) directly on their cognate tRNA: non-discriminating aaRSs first form misacylated aminoacyl-tRNA (aa-tRNA), which is not used by the ribosome but instead converted to cognate aa-tRNA by various RNA-dependent modifying enzymes [4].In organisms lacking GlnRS and AsnRS, Glu-tRNA Gln and AsptRNA Asn are synthesized by non-discriminating aaRSs and converted to cognate Gln-tRNA Gln and Asn-tRNA Asn by tRNA-dependent amidotransferases (AdT). Two types of AdT exist, the heterotrimeric GatCAB present in both bacteria and archaea and the homodimeric GatDE present in archaea [5,6]. The tRNA moiety is recognized by the B and E kinase subunits of GatCAB and GatDE, respectively, which phosphorylate the mischarged tRNAs to form activated intermediates [7][8][9]. The glutaminase subunit (GatA/D) liberates ammonia from an amide donor and amidates Glu or Asp on the tRNA to form Gln or Asn, respectively. In both types of AdT, a 40 Å-long hydrophilic channel connects the glutaminase and kinase subunits [9,10]. It has been proposed, but remains to be proven, that ammonia liberated in the glutaminase active site is transported through the channel via a series of protonations and deprotonations to the kinase active site, and that binding of mischarged tRNA is required for opening the channel. Another open question concerns the precise in vivo mechanism by which misacylated aa-tRNA species are stabilized and escape detection, and subsequent delivery to the ribosome, by e...

Recent research into various aspects of bacterial metabolism such as cell wall and antibiotic synthesis, degradation pathways, cellular stress, and amino acid biosynthesis has elucidated roles of aminoacyl-transfer ribonucleic acid (aa-tRNA) outside of translation. Although the two enzyme families responsible for cell wall modifications, aminoacyl-phosphatidylglycerol synthases (aaPGSs) and Fem, were discovered some time ago, they have recently become of intense interest for their roles in the antimicrobial resistance of pathogenic microorganisms. The addition of positively charged amino acids to phosphatidylglycerol (PG) by aaPGSs neutralizes the lipid bilayer making the bacteria less susceptible to positively charged antimicrobial agents. Fem transferases utilize aa-tRNA to form peptide bridges that link strands of peptidoglycan. These bridges vary among the bacterial species in which they are present and play a role in resistance to antibiotics that target the cell wall. Additionally, the formation of truncated peptides results in shorter peptide bridges and loss of branched linkages which makes bacteria more susceptible to antimicrobials. A greater understanding of the structure and substrate specificity of this diverse enzymatic family is necessary to aid current efforts in designing potential bactericidal agents. These two enzyme families are linked only by the substrate with which they modify the cell wall, aa-tRNA; their structure, cell wall modification processes and the physiological changes they impart on the bacterium differ greatly.

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