Several methanogenic archaea lack cysteinyl-transfer RNA (tRNA) synthetase (CysRS), the essential enzyme that provides Cys-tRNA(Cys) for translation in most organisms. Partial purification of the corresponding activity from Methanocaldococcus jannaschii indicated that tRNA(Cys) becomes acylated with O-phosphoserine (Sep) but not with cysteine. Further analyses identified a class II-type O-phosphoseryl-tRNA synthetase (SepRS) and Sep-tRNA:Cys-tRNA synthase (SepCysS). SepRS specifically forms Sep-tRNA(Cys), which is then converted to Cys-tRNA(Cys) by SepCysS. Comparative genomic analyses suggest that this pathway, encoded in all organisms lacking CysRS, can also act as the sole route for cysteine biosynthesis. This was proven for Methanococcus maripaludis, where deletion of the SepRS-encoding gene resulted in cysteine auxotrophy. As the conversions of Sep-tRNA to Cys-tRNA or to selenocysteinyl-tRNA are chemically analogous, the catalytic activity of SepCysS provides a means by which both cysteine and selenocysteine may have originally been added to the genetic code.

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.

Translation of the genetic code requires attachment of tRNAs to their cognate amino acids. Errors during amino-acid activation and tRNA esterification are corrected by aminoacyl-tRNA synthetase-catalyzed editing reactions, as extensively described for aliphatic amino acids. The contribution of editing to aromatic amino-acid discrimination is less well understood. We show that phenylalanyl-tRNA synthetase misactivates tyrosine and that it subsequently corrects such errors through hydrolysis of tyrosyl-adenylate and Tyr-tRNA(Phe). Structural modeling combined with an in vivo genetic screen identified the editing site in the B3/B4 domain of the beta subunit, 40 angstroms from the active site in the alpha subunit. Replacements of residues within the editing site had no effect on Phe-tRNA(Phe) synthesis, but abolished hydrolysis of Tyr-tRNA(Phe) in vitro. Expression of the corresponding mutants in Escherichia coli significantly slowed growth, and changed the activity of a recoded beta-galactosidase variant by misincorporating tyrosine in place of phenylalanine. This loss in aromatic amino-acid discrimination in vivo revealed that editing by phenylalanyl-tRNA synthetase is essential for faithful translation of the genetic code.

Multiple peptide resistance (MprF) virulence factors control cellular permeability to cationic antibiotics by aminoacylating inner membrane lipids. It has been shown previously that one class of MprF can use Lys-tRNA Lys to modify phosphatidylglycerol (PG), but the mechanism of recognition and possible role of other MprFs are unknown. Here, we used an in vitro reconstituted lipid aminoacylation system to investigate the two phylogenetically distinct MprF paralogs (MprF1 and MprF2) found in the bacterial pathogen Clostridium perfringens. Although both forms of MprF aminoacylate PG, they do so with different amino acids; MprF1 is specific for Ala-tRNA Ala , and MprF2 utilizes Lys-tRNA Lys . This provides a mechanism by which the cell can fine tune the charge of the inner membrane by using the neutral amino acid alanine, potentially providing resistance to a broader range of antibiotics than offered by lysine modification alone. Mutation of tRNA Ala and tRNA Lys had little effect on either MprF activity, indicating that the aminoacyl moiety is the primary determinant for aminoacyl-tRNA recognition. The lack of discrimination of the tRNA is consistent with the role of MprF as a virulence factor, because species-specific differences in tRNA sequence would not present a barrier to horizontal gene transfer. Taken together, our findings reveal how the MprF proteins provide a potent virulence mechanism by which pathogens can readily acquire resistance to chemically diverse antibiotics.alanine ͉ lysine ͉ tRNA

Aminoacylphosphatidylglycerol synthases (aaPGSs) are multiple peptide resistance factors that transfer amino acids from aminoacyl-tRNAs to phosphatidylglycerol (PG) in the cytoplasmic membrane. Aminoacylation of PG is used by bacteria to decrease the net negative charge of the cell envelope, diminishing affinity for charged molecules and allowing for adaptation to environmental changes. Lys-PGS, which transfers lysine to PG, is essential for the virulence of certain pathogens, providing resistance to both host cationic antimicrobial peptides and therapeutic antibiotics. Ala-PGS was also recently described, but little is known about the possible activities of other members of the highly diverse aaPGS family of proteins. Systematic deletion of the predicted membrane-inserted domains of several aaPGSs revealed that the carboxyl-terminal hydrophilic domain alone is sufficient for aminoacylphosphatidylglycerol transferase catalytic activity. In contrast to previously characterized aaPGSs, the Enterococcus faecium enzyme used an expanded repertoire of amino acids to modify PG with Ala, Arg, or Lys. Reexamination of previously characterized aaPGSs also revealed broader than anticipated substrate specificity, for example Bacillus subtilis Lys-PGS was shown to also catalyze Ala-PG synthesis. The relaxed substrate specificities of these aaPGSs allows for more elaborate remodeling of membrane lipids than previously thought, potentially providing bacteria that harbor these enzymes resistance to a broad spectrum of antibiotics and environmental stresses.

SummaryThe bacterial envelope is a semi-permeable barrier that protects the cell from the hostilities of the environment. To survive the ever-changing conditions of their surroundings, bacteria need to rapidly adjust the biochemical properties of their cellular envelope. Amino acid (aa) addition to phosphatidylglycerol (PG) of the membrane is one of the mechanisms used by bacteria to lower the net negative charge of their cellular envelope, thereby decreasing its affinity for several antibacterial agents such as the cationic antimicrobial peptides (CAMPs) produced by the innate immune response during host infection. This process requires the activity of an integral membrane protein, called aa-PG synthase (aaPGS), to transfer the aa of aminoacyltRNA (aa-tRNA) onto the PG of the membrane. aaPGSs constitute a new family of virulence factors that are found in a wide range of microorganisms. aa-PGs not only provide resistance to CAMPs but also to other classes of antibacterial agents and to environmental stresses such as those encountered during extreme osmotic or acidic conditions. This review will describe the known biochemical properties of aa-PGSs, their specificity for aa-tRNAs and phospholipids, and the growing repertoire of aa used as substrates by these enzymes. Their prevalence in bacteria and the phenotypes and modulations of membrane properties associated with these molecules will be addressed, as well as their regulation as a component of the envelope stress response system in certain bacteria.

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.

Accurate selection of amino acids is essential for faithful translation of the genetic code. Errors during amino acid selection are usually corrected by the editing activity of aminoacyl-tRNA synthetases such as phenylalanyl-tRNA synthetases (PheRS), which edit misactivated tyrosine. Comparison of cytosolic and mitochondrial PheRS from the yeast Saccharomyces cerevisiae suggested that the organellar protein might lack the editing activity. Yeast cytosolic PheRS was found to contain an editing site, which upon disruption abolished both cis and trans editing of Tyr-tRNA Phe . Wild-type mitochondrial PheRS lacked cis and trans editing and could synthesize Tyr-tRNA Phe , an activity enhanced in active site variants with improved tyrosine recognition. Possible trans editing was investigated in isolated mitochondrial extracts, but no such activity was detected. These data indicate that the mitochondrial protein synthesis machinery lacks the tyrosine proofreading activity characteristic of cytosolic translation. This difference between the mitochondria and the cytosol suggests that either organellar protein synthesis quality control is focused on another step or that translation in this compartment is inherently less accurate than in the cytosol.The aminoacyl-tRNA synthetases (aaRS) 2 are a ubiquitous and essential protein family required for protein synthesis (1-3). Structurally and functionally the aaRSs are divided into two unrelated but biochemically analogous groups, class I and class II (4 -6). The aaRSs attach amino acids to the 3Ј-ends of tRNA containing the corresponding anticodon sequence, and the resulting aminoacyl-tRNAs (aa-tRNAs) are used as substrates for ribosomal translation of mRNA. The accuracy of aa-tRNA synthesis is generally assured by the existence of aaRSs specific for each particular amino acid-tRNA pair. Cognate tRNA recognition and discrimination of non-cognate RNAs are achieved by sequencespecific direct and indirect readout of the numerous combinations of bases present in tRNAs (7-10). The relative structural simplicity and inherent similarity between the amino acid substrates makes their accurate recognition and discrimination more challenging. Although some amino acids such as cysteine and tyrosine are different enough to allow their specific recognition by a particular aaRS (11, 12), others such as valine and isoleucine are less easily distinguished. For example the class I aaRS isoleucyl-tRNA synthetase (IleRS) is only able to poorly discriminate against valine, which has a misactivation rate of about 1:200 compared with the cognate substrate isoleucine. Despite this significant rate of misactivation and misaminoacylation, the accuracy of translation is not compromised because of the existence of an intrinsic proofreading and editing mechanism in IleRS that specifically hydrolyzes both misactivated Val-AMP and misaminoacylated Val-tRNA Ile (13,14). In addition to IleRS, many other class I and class II aaRSs also employ editing to prevent release of non-cognate aa-tRNA and subsequent l...