The Staphylococcus aureus ileS gene, encoding isoleucyl-tRNA synthetase, is a member of the T-box family.
ABSTRACT: The Staphylococcus aureus ileS gene, encoding isoleucyl-tRNA synthetase (IleRS), contains a long mRNA leader region. This region exhibits many of the features of the gram-positive synthetase gene family, including the T box and leader region terminator and antiterminator. The terminator was shown to be functional in vivo, and readthrough increased during growth in the presence of mupirocin, an inhibitor of IleRS activity. The S. aureus ileS leader structure includes several critical differences from the other members of the T-box family, suggesting that regulation of this gene in S. aureus may exhibit unique features.
Project description:The mechanisms underlying bacterial tolerance to antibiotics are unclear. A possible adaptation strategy was explored by exposure of drug-naive methicillin-susceptible Staphylococcus aureus strain FDA209P to vancomycin in vitro Strains surviving vancomycin treatment (vancomycin survivor strains), which appeared after 96 h of exposure, were slow-growing derivatives of the parent strain. Although the vancomycin MICs for the survivor strains were within the susceptible range, the cytokilling effects of vancomycin at 20-fold the MIC were significantly lower for the survivor strains than for the parent strain. Whole-genome sequencing demonstrated that ileS, encoding isoleucyl-tRNA synthetase (IleRS), was mutated in two of the three vancomycin survivor strains. The IleRS Y723H mutation is located close to the isoleucyl-tRNA contact site and potentially affects the affinity of IleRS binding to isoleucyl-tRNA, thereby inhibiting protein synthesis and leading to vancomycin tolerance. Introduction of the mutation encoding IleRS Y723H into FDA209P by allelic replacement successfully transferred the vancomycin tolerance phenotype. We have identified mutation of ileS to be one of the bona fide genetic events leading to the acquisition of vancomycin tolerance in S. aureus, potentially acting via inhibition of the function of IleRS.
Project description:Emergence and spread of low-level mupirocin resistance in staphylococci have been increasingly reported in recent years. The aim of this study was to characterize missense mutations within the chromosomal isoleucyl-tRNA synthetase gene (ileS) among clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) with low-level mupirocin resistance. A total of 20 isolates of MRSA with low-level mupirocin resistance (minimal inhibitory concentration, 16-64 microg/mL) were collected from 79 patients in intensive care units for six months. The isolates were analyzed for isoleucyl-tRNA synthetase (IleS) mutations that might affect the binding of mupirocin to the three-dimensional structure of the S. aureus IleS enzyme. All isolates with low-level mupirocin resistance contained the known V588F mutation affecting the Rossman fold, and some of them additionally had previously unidentified mutations such as P187F, K226T, F227L, Q612H, or V767D. Interestingly, Q612H was a novel mutation that was involved in stabilizing the conformation of the catalytic loop containing the KMSKS motif. In conclusion, this study confirms that molecular heterogeneity in ileS gene is common among clinical MRSA isolates with low-level mupirocin resistance, and further study on clinical mutants is needed to understand the structural basis of low-level mupirocin resistance.
Project description:The isoleucyl-tRNA synthetase (ileS) gene was sequenced in toto from 9 and in part from 31 Staphylococcus aureus strains with various degrees of susceptibility to mupirocin. All strains for which the mupirocin MIC was greater than 8 microg/ml contained point mutations affecting the Rossman fold via Val-to-Phe changes at either residue 588 (V588F) or residue 631 (V631F). The importance of the V588F mutation was confirmed by an allele-specific PCR survey of 32 additional strains. Additional mutations of uncertain significance were found in residues clustered on the surface of the IleS protein.
Project description:Bacteria decode the isoleucine codon AUA using a tRNA species that is posttranscriptionally modified at the wobble position of the anticodon with a lysine-containing cytidine derivative called lysidine. The lysidine modification of tRNA(Ile2) is an essential identity determinant for proper aminoacylation by isoleucyl tRNA synthetase (IleRS) and codon recognition on the ribosome. The ATP- and lysine-dependent formation of lysidine is catalyzed by tRNA(Ile)-lysidine synthetase. Using the purified recombinant enzyme from Escherichia coli and an in vitro transcribed tRNA substrate, we have confirmed that lysidine modification is both necessary and sufficient to convert tRNA(Ile2) into a substrate for IleRS. A series of lysine analogs were tested as potential inhibitors during the mechanistic characterization of tRNA(Ile)-lysidine synthetase. Gel electrophoresis revealed that many of these analogs, including some simple alkyl amines, were alternative substrates. Incorporation of these amines into alternative tRNA products was confirmed by mass spectrometry. The availability of tRNA(Ile2) with differential modifications enabled an exploration of the structural requirements of the anticodon for aminoacylation by methionyl tRNA synthetase and IleRS. All of the modifications were effective at creating negative determinants for methionyl tRNA synthetase and positive determinants for IleRS, although the tolerance of IleRS differed between the enzymes from E. coli and Bacillus subtilis.
Project description:The correct amino acid sequence of E. coli isoleucyl-tRNA synthetase (IleRS) was established by means of peptide mapping by MALDI mass spectrometry, using a set of four endoproteases (trypsin, LysC, AspN and GluC). Thereafter, the active site of IleRS was mapped by affinity labeling with reactive analogs of the substrates. For the ATP binding site, the affinity labeling reagent was pyridoxal 5'-diphospho-5'-adenosine (ADP-PL), whereas periodate-oxidized tRNA(Ile), the 2',3'-dialdehyde derivative of tRNA(Ile) was used to label the binding site for the 3'-end of tRNA on the synthetase. Incubation of either reagent with IleRS resulted in a rapid loss of both the tRNA(Ile) aminoacylation and isoleucinedependent isotopic ATP-PPi exchange activities. The stoichiometries of IleRS labeling by ADP-PL or tRNA(Ile)ox corresponded to 1 mol of reagent incorporated per mol of enzyme. Altogether, the oxidized 3'-end of tRNA(Ile) and the pyridoxal moiety of the ATP analog ADP-PL react with the lysyl residues 601 and 604 of the consensus sequence (601)KMSKS(605). Identification of the binding site for L-isoleucine or for non cognate amino acids on E. coli IleRS was achieved by qualitative comparative labeling of the synthetase with bromomethyl ketone derivatives of L-isoleucine (IBMK) or of the non-cognate amino acids valine (VBMK), phenylalanine (FBMK) and norleucine (NleBMK). Labeling of the enzyme with IBMK resulted in a complete loss of isoleucine-dependent isotopic [(32)P]PPi-ATP exchange activity. VBMK, NleBMK and FBMK were also capable of abolishing the activity of IleRS, FBMK being the less efficient in inactivating the synthetase. Analysis by MALDI mass spectrometry designated cysteines-462 and -718 as the target residues of the substrate analog IBMK on E. coli IleRS, whereas VBMK, NleBMK and FBMK labeled in common His-394, His-478 and Cys-718. In addition, VBMK and NleBMK, which are chemically similar to IBMK, were found covalently bound to Cys-462, and VBMK was specifically attached to His-332 (or His-337) of the synthetase. The amino acid residues labeled by the substrate analogs are mainly distributed between three regions in the primary structure of E. coli IleRS: these are segments [325-394], [451-479] and [591-604]. In the 3-D structures of IleRS from T. thermophilus and S. aureus, the [325-394] stretch is part of the editing domain, while fragments [451-479] and [591-604] representing the isoleucine binding domain and the dinucleotide (or Rossmann) fold domain, respectively, are located in the catalytic core. His-332 of E. coli IleRS, that is strictly conserved among all the available IleRS sequences is located in the editing active site of the synthetase. It is proposed that His-332 of E. coli IleRS participates directly in hydrolysis, or helps to deprotonate the hydroxyl group of threonine at the hydrolytic site.
Project description:Aminoacyl-tRNA synthetases catalyze the aminoacylation of tRNAs with their cognate amino acids. They are an essential part of each translation system and in eukaryotes are therefore found in both the cytosol and mitochondria. Thus, eukaryotes either have two distinct genes encoding the cytosolic and mitochondrial isoforms of each of these enzymes or a single gene encoding dually localized products. Trypanosomes require trans-splicing of a cap containing leader sequence onto the 5'-untranslated region of every mRNA. Recently we speculated that alternative trans-splicing could lead to the expression of proteins having amino-termini of different lengths that derive from the same gene. We now demonstrate that alternative trans-splicing, creating a long and a short spliced variant, is the mechanism for dual localization of trypanosomal isoleucyl-tRNA synthetase (IleRS). The protein product of the longer spliced variant possesses an amino-terminal presequence and is found exclusively in mitochondria. In contrast, the shorter spliced variant is translated to a cytosol-specific isoform lacking the presequence. Furthermore, we show that RNA stability is one mechanism determining the differential abundance of the two spliced isoforms.
Project description:The T box riboswitch regulates many amino acid-related genes in Gram-positive bacteria. T box riboswitch-mediated gene regulation was shown previously to occur at the level of transcription attenuation via structural rearrangements in the 5' untranslated (leader) region of the mRNA in response to binding of a specific uncharged tRNA. In this study, a novel group of isoleucyl-tRNA synthetase gene (ileS) T box leader sequences found in organisms of the phylum Actinobacteria was investigated. The Stem I domains of these RNAs lack several highly conserved elements that are essential for interaction with the tRNA ligand in other T box RNAs. Many of these RNAs were predicted to regulate gene expression at the level of translation initiation through tRNA-dependent stabilization of a helix that sequesters a sequence complementary to the Shine-Dalgarno (SD) sequence, thus freeing the SD sequence for ribosome binding and translation initiation. We demonstrated specific binding to the cognate tRNA(Ile) and tRNA(Ile)-dependent structural rearrangements consistent with regulation at the level of translation initiation, providing the first biochemical demonstration, to our knowledge, of translational regulation in a T box riboswitch.
Project description:Isoleucyl-tRNA synthetase (IleRS) is unusual among aminoacyl-tRNA synthetases in having a tRNA-dependent pre-transfer editing activity. Alongside the typical bacterial IleRS (such as Escherichia coli IleRS), some bacteria also have the enzymes (eukaryote-like) that cluster with eukaryotic IleRSs and exhibit low sensitivity to the antibiotic mupirocin. Our phylogenetic analysis suggests that the ileS1 and ileS2 genes of contemporary bacteria are the descendants of genes that might have arisen by an ancient duplication event before the separation of bacteria and archaea. We present the analysis of evolutionary constraints of the synthetic and editing reactions in eukaryotic/eukaryote-like IleRSs, which share a common origin but diverged through adaptation to different cell environments. The enzyme from the yeast cytosol exhibits tRNA-dependent pre-transfer editing analogous to E. coli IleRS. This argues for the presence of this proofreading in the common ancestor of both IleRS types and an ancient origin of the synthetic site-based quality control step. Yet surprisingly, the eukaryote-like enzyme from Streptomyces griseus IleRS lacks this capacity; at the same time, its synthetic site displays the 10(3)-fold drop in sensitivity to antibiotic mupirocin relative to the yeast enzyme. The discovery that pre-transfer editing is optional in IleRSs lends support to the notion that the conserved post-transfer editing domain is the main checkpoint in these enzymes. We substantiated this by showing that under error-prone conditions S. griseus IleRS is able to rescue the growth of an E. coli lacking functional IleRS, providing the first evidence that tRNA-dependent pre-transfer editing in IleRS is not essential for cell viability.
Project description:In the thermophilic archaeon Methanobacterium thermoautotrophicum Marburg, the structural gene for isoleucyl-tRNA synthetase (ileS) is flanked upstream by orf401 and downstream by purL. orf401 encodes a 43.5-kDa protein with an unknown function. Northern (RNA) hybridization and S1 nuclease protection experiments showed that the orf401, ileS, and purL genes are cotranscribed from an archael consensus promoter in front of orf401. The corresponding transcript was about eightfold increased in cells that had been exposed to pseudomonic acid A, a specific inhibitor of isoleucyl-tRNA synthetase. Growth inhibition by puromycin, tryptophan starvation, or starvation for hydrogen did not affect the level of this transcript. The level of a trpE transcript, however, was drastically elevated upon tryptophan starvation, while inhibition by pseudomonic acid A had no effect on the level of this transcript. Expression of ileS thus appears to be controlled by a regulatory mechanism which specifically responds to the availability of isoleucyl-tRNA. Extensive decay of the orf401-ileS-purL message was observed. Degradation occurred, presumably by endonucleolytic cleavage, within the orf401 region.
Project description:Mupirocin is a topical antibiotic used for the treatment of skin infections and the eradication of methicillin-resistant Staphylococcus aureus carriage. It inhibits bacterial protein synthesis by interfering with isoleucyl-tRNA synthetase activity. High-level mupirocin resistance (MIC of ≥ 512 μg/ml) is mediated by the expression of mupA (ileS2), which encodes an alternate isoleucyl-tRNA synthetase. In this study, we describe high-level mupirocin resistance mediated by a novel locus, mupB. The mupB gene (3,102 bp) shares 65.5% sequence identity with mupA but only 45.5% identity with ileS. The deduced MupB protein shares 58.1% identity (72.3% similarity) and 25.4% identity (41.8% similarity) with MupA and IleS, respectively. Despite this limited homology, MupB contains conserved motifs found in class I tRNA synthetases. Attempts to transfer high-level mupirocin resistance via conjugation or transformation (using plasmid extracts from an mupB-containing strain) were unsuccessful. However, by cloning the mupB gene into a shuttle vector, it was possible to transfer the resistance phenotype to susceptible S. aureus by electroporation, proving that mupB was responsible for the high-level mupirocin resistance. Further studies need to be done to determine the prevalence of mupB and to understand risk factors and outcomes associated with resistance mediated by this gene.