Constantinos Stathopoulos

Cysteinyl-tRNA synthetase is not essential for viability of the archaeon Methanococcus maripaludis

The methanogenic archaea Methanocaldococcus jannaschii and Methanothermobacter thermautotrophicus contain a dual-specificity prolyl-tRNA synthetase (ProCysRS) that accurately forms both prolyl-tRNA (Pro-tRNA) and cysteinyl-tRNA (Cys-tRNA) suitable for in vivo translation. This intriguing enzyme may even perform its dual role in organisms that possess a canonical single-specificity cysteinyl-tRNA synthetase (CysRS), raising the question as to whether this latter aminoacyl-tRNA synthetase is indeed required for cell viability. To test the postulate that all synthetase genes are essential, we disrupted the cysS gene (encoding CysRS) of Methanococcus maripaludis. The knockout strain was viable under normal growth conditions. Biochemical analysis showed that the pure M. maripaludis ProCysRS was capable of forming Cys-tRNA, implying that the dual-specificity enzyme compensates in vivo for the loss of CysRS. The canonical CysRS has a higher affinity for cysteine than ProCysRS, a reason why M. maripaludis may have acquired cysS by a late lateral gene transfer. These data challenge the notion that all twenty aminoacyl-tRNA synthetases are essential for the viability of a cell.

In Vitro and In Vivo Evaluations of Oxacillin Efficiency against mecA-Positive Oxacillin-Susceptible Staphylococcus aureus

ABSTRACT Community-type Staphylococcus aureus strains that are positive for mecA and PBP2a but appear phenotypically susceptible to oxacillin are increasingly reported worldwide. Four S. aureus clinical isolates carrying the mecA gene with oxacillin MICs of <2 μg/ml were tested for oxacillin efficiency by population analyses and experimental thigh infections. These isolates harbored staphylococcal cassette chromosome mec type IV and belonged to two genotypes. Two of the four isolates were found by population analysis to be truly oxacillin susceptible. All four isolates exhibited significant reductions in the numbers of colonies grown after dicloxacillin treatment of experimental thigh infections, as also did a mecA-negative S. aureus control strain. These observations indicate that some of the phenotypically oxacillin susceptible mecA-positive Staphylococcus aureus isolates may be at least partially responsive to oxacillin.

A single tRNA base pair mediates bacterial tRNA-dependent biosynthesis of asparagine

In many prokaryotes and in organelles asparagine and glutamine are formed by a tRNA-dependent amidotransferase (AdT) that catalyzes amidation of aspartate and glutamate, respectively, mischarged on tRNAAsn and tRNAGln. These pathways supply the deficiency of the organism in asparaginyl- and glutaminyl-tRNA synthtetases and provide the translational machinery with Asn-tRNAAsn and Gln-tRNAGln. So far, nothing is known about the structural elements that confer to tRNA the role of a specific cofactor in the formation of the cognate amino acid. We show herein, using aspartylated tRNAAsn and tRNAAsp variants, that amidation of Asp acylating tRNAAsn is promoted by the base pair U1–A72 whereas the G1–C72 pair and presence of the supernumerary nucleotide U20A in the D-loop of tRNAAsp prevent amidation. We predict, based on comparison of tRNAGln and tRNAGlu sequence alignments from bacteria using the AdT-dependent pathway to form Gln-tRNAGln, that the same combination of nucleotides also rules specific tRNA-dependent formation of Gln. In contrast, we show that the tRNA-dependent conversion of Asp into Asn by archaeal AdT is mainly mediated by nucleotides G46 and U47 of the variable region. In the light of these results we propose that bacterial and archaeal AdTs use kingdom-specific signals to catalyze the tRNA-dependent formations of Asn and Gln.

The structural basis of cysteine aminoacylation of tRNAPro by prolyl-tRNA synthetases

Cysteinyl-tRNA synthetase is an essential enzyme required for protein synthesis. Genes encoding this protein have not been identified in Methanocaldococcus jannaschii, Methanothermobacter thermautotrophicus, or Methanopyrus kandleri. It has previously been proposed that the prolyl-tRNA synthetase (ProRS) enzymes in these organisms recognize either proline or cysteine and can aminoacylate their cognate tRNAs through a dual-specificity mechanism. We report five crystal structures at resolutions between 2.6 and 3.2 Å: apo M. jannaschii ProRS, and M. thermautotrophicus ProRS in apo form and in complex with cysteinyl-sulfamoyl-, prolyl-sulfamoyl-, and alanyl-sulfamoyl-adenylates. These aminoacyl-adenylate analogues bind to a single active-site pocket and induce an identical set of conformational changes in loops around the active site when compared with the ligand-free conformation of ProRS. The cysteinyl- and prolyl-adenylate analogues have similar, nanomolar affinities for M. thermautotrophicus ProRS. Homology modeling of tRNA onto these adenylate complexes places the 3′-OH of A76 in an appropriate position for the transfer of any of the three amino acids to tRNA. Thus, these structures explain recent biochemical experiments showing that M. jannaschii ProRS misacylates tRNAPro with cysteine, and argue against the proposal that these archaeal ProRS enzymes possess the dual capacity to aminoacylate both tRNAPro and tRNACys with their cognate amino acids.

Mitochondrial tRNA Mutations: Clinical and Functional Perturbations

During the last decade, there has been a progressive accumulation of reports that connect the identification of specific mitochondrial tRNA gene mutations to severe disorders in human. As a result, mitochondrial tRNA genes and their products have emerged as novel and essential molecular markers for wide biochemical and genetic screenings among different human populations. So far, 139 pathogenic and 243 polymorphic mt tRNA mutations have been described and they have become the foreground of numerous case reports. Given the complexity of mitochondrial genetics and biochemistry, the clinical manifestations of mitochondrial disorders are extremely heterogeneous. They range from lesions of single tissues or structures to more severe impairements including myopathies, encephalomyopathies, cardiomyopathies, or complex multisystem syndromes. Moreover, the exact mechanisms by which biochemical cascades can be dramatically affected by mitochondrial tRNA mutations still remain uncharacterized. However and regardless of the vast amount of information that daily emerges, only few efforts have been carried out to systematically record all the mitochondrial tRNA-associated pathogenic mutations or polymorphisms. In this report, we summarize all the clinical phenotypes associated with mitochondrial tRNA pathogenic mutations that have been reported so far. In a next step we describe in detail all the pathogenic and polymorphic mutations that have been recorded so far and we categorize them per tRNA species and per associated disease. Finally, we discuss the impact of the frequency of mitochondrial tRNA mutations in general population surveys and we preview any relevant implications on the essential functional integrity of mitochondrial biochemical pathways.

Cysteinyl-tRNA formation: The last puzzle of aminoacyl-tRNA synthesis

With the exception of the methanogenic archaea Methanococcus jannaschii and Methanobacterium thermoautotrophicum ΔH, all organisms surveyed contain orthologs of Escherichia coli cysteinyl‐tRNA synthetase (CysRS). The characterization of CysRS‐encoding (cysS) genes and the demonstration of their ability to complement an E. coli cysS ts mutant reveal that Methanococcus maripaludis and Methanosarcina barkeri, two other methanogenic archaea, possess canonical CysRS proteins. A molecular phylogeny inferred from 40 CysRS sequences indicates that the CysRS of M. maripaludis and Methanosarcina spp. are specific relatives of the CysRS of Pyrococcus spp. and Chlamydia, respectively. This result suggests that the CysRS gene was acquired by lateral gene transfer in at least one euryarchaeotic lineage.

Intensive care unit dissemination of multiple clones of linezolid-resistant Enterococcus faecalis and Enterococcus faecium

OBJECTIVES Outbreaks caused by linezolid-resistant (LR) enterococci remain rare. We report the epidemiological and molecular characteristics of the multiclonal dissemination of LR enterococci in the intensive care unit (ICU) of a Greek hospital. METHODS All LR enterococcal isolates recovered from patients hospitalized in the ICU of the University Hospital of Larissa, Greece, between January 2007 and October 2008 were included. Isolates were tested by PFGE and PCR followed by sequence analysis of the entire 23S rRNA gene. Patient records were retrieved to access patterns of acquisition and outcome. RESULTS Sixteen separate patients were infected and/or colonized by 22 LR enterococcal isolates (17 Enterococcus faecium and 5 Enterococcus faecalis). Linezolid MICs varied from 8 to 16 mg/L; 12 isolates showed cross-resistance to vancomycin. Genotyping revealed as many as seven and three PFGE types among E. faecium and E. faecalis isolates, respectively, indicating multiclonal spread of LR enterococci. Nine patients had received linezolid prior to the recovery of LR enterococci, while the remaining seven patients were not exposed to the drug. All isolates carried the mutation G2576T; the mutated position was heterogeneous in 12 isolates and homogeneous in 10. CONCLUSIONS The multiclonal composition of LR enterococci indicates that linezolid resistance possibly occurred on several independent occasions. Its acquisition was often not related to linezolid administration; patients might have acquired their LR isolate from another patient that had received linezolid or, alternatively, resistance may have arisen by mutation that occurred independently.

Detection of mutations in the FemXAB protein family in oxacillin-susceptible mecA-positive Staphylococcus aureus clinical isolates

Objectives Methicillin-resistant Staphylococcus aureus (MRSA) strains that express the mecA gene but are oxacillin susceptible (OS-MRSA; oxacillin MIC </=2 mg/L) are increasingly reported. To gain molecular and functional insights on this observation, we focused on additional factors possibly contributing to phenotypic susceptibility. Methods The nucleotide content of mecA, femA, femB and femX genes, which are considered essential for methicillin resistance, was determined in four OS-MRSA clinical isolates and a genetically similar low-level MRSA control (oxacillin MIC 6 mg/L). Gene expression was quantified compared with the low- and a high-level MRSA (MIC 256 mg/L) control. The tertiary structure of Fem proteins was predicted based on protein structure homology modelling, using web-based automated comparative protein modelling. Growth kinetics were tested for the study and control isolates, to determine whether FemXAB mutations lead to reduced fitness. Results Genes mecA, femA, femB and femX were expressed similarly in the study and the control isolates. Mutations in the gene mecA were not present in any isolate. However, several mutations leading to amino acid substitutions in positions possibly affecting Fem enzyme activity were detected in all fem genes. Two OS-MRSA that had no oxacillin heteroresistance had more mutations in the Fem proteins compared with the remaining isolates that were heteroresistant. The low-level MRSA control had considerably fewer mutations. No differences between growth rates of the OS-MRSA and the MRSA controls were observed. Conclusions Accumulation of amino acid changes in Fem proteins might affect intact cell wall synthesis, even though not causing reduced viability, thus contributing to atypical oxacillin responsiveness.

Methanocaldococcus jannaschii Prolyl-tRNA Synthetase Charges tRNAPro with Cysteine

Methanocaldococcus jannaschiiprolyl-tRNA synthetase (ProRS) was previously reported to also catalyze the synthesis of cysteinyl-tRNACys(Cys-tRNACys) to make up for the absence of the canonical cysteinyl-tRNA synthetase in this organism (Stathopoulos, C., Li, T., Longman, R., Vothknecht, U. C., Becker, H., Ibba, M., and Söll, D. (2000) Science 287, 479–482; Lipman, R. S., Sowers, K. R., and Hou, Y. M. (2000)Biochemistry 39, 7792–7798). Here we show by acid urea gel electrophoresis that pure heterologously expressed recombinantM. jannaschii ProRS misaminoacylates M. jannaschii tRNAPro with cysteine. The enzyme is unable to aminoacylate purified mature M. jannaschiitRNACys with cysteine in contrast to facile aminoacylation of the same tRNA with cysteine by Methanococcus maripaludiscysteinyl-tRNA synthetase. Although M. jannaschii ProRS catalyzes the synthesis of Cys-tRNAPro readily, the enzyme is unable to edit this misaminoacylated tRNA. We discuss the implications of these results on the in vivo activity of the M. jannaschii ProRS and on the nature of the enzyme involved in the synthesis of Cys-tRNACys in M. jannaschii.

Linezolid Dependence in Staphylococcus epidermidis Bloodstream Isolates

We document linezolid dependence among 5 highly linezolid-resistant (LRSE) Staphylococcus epidermidis bloodstream isolates that grew substantially faster at 32 µg/mL linezolid presence. These isolates carried the mutations T2504A and C2534T in multiple 23S rRNA copies and 2 mutations leading to relevant amino acid substitutions in L3 protein. Linezolid dependence could account for increasing LRSE emergence.