Diazenedicarboxamides as inhibitors of D-alanine-D-alanine ligase (Ddl)

D-Alanine-D-alanine ligase (Ddl) catalyzes the biosynthesis of an essential bacterial peptidoglycan precursor D-alanyl-D-alanine and it represents an important target for development of new antibacterial drugs. A series of semicarbazides, aminocarbonyldiazenecarboxylates, diazenedicarboxamides, and hydrazinedicarboxamides was synthesized and screened for inhibition of DdlB from Escherichia coli. Compounds with good inhibitory activity were identified, enabling us to deduce initial structure-activity relationships. Thirteen diazenedicarboxamides were better inhibitors than D-cycloserine and some of them also possess antibacterial activity, which makes them a promising starting point for further development.     

Cloning and expression of a new bacteriophage (SHPh) DNA ligase isolated from sewage

During the last 50 years, major advances in molecular biology and biotechnology have been attributed to the discovery of enzymes that allow molecular cloning of important genes. One of these enzymes that has been widely acknowledged for its role in the development of biotechnology is the T4 DNA ligase. This enzyme joins the break in the DNA backbone structure by creating a phosphodiester bond between 5′ PO₄ and 3′ OH ends, in an ATP dependent multi-step reaction, thus allowing the ligation of related and foreign DNA sequences. Due to its role in modern DNA recombinant technology, there is a high demand on DNA ligase to allow the ligation of target DNA inserts into a chosen vector as part of DNA cloning technology. To closely look at ligase sequence diversity, a bacteriophage that infects DH5α (commercial lab strain of Escherichia coli) was isolated from sewage system in Hebron, Palestine. The DNA ligase gene of this phage was cloned and its sequence was compared to the NCBI database. The new bacteriophage ligase, named (South Hebron Phage, SHPh) DNA ligase, shows homology to T even bacteriophage DNA ligases posted in the NCBI database with 35 nucleotide differences, an indication of existed diversity among T even DNA ligation enzymes that can be used as markers in phage classification.     

Genetics and function of DNA ligase in Escherichia coli

The characterization of two classes of DNA ligase mutants in Escherichia coli is described. The first class consists of three mutations coding for a temperature-sensitive ligase and defines the structural gene for DNA ligase (lig). The second class of mutants (lop) overproduces an apparently wild-type enzyme; a genetic diploid analysis implies that these are promoter or operator mutations, lig and lop are cotransduced by phage P1 and map at 46 minutes on the E. coli map. Detailed studies of two lig mutants (lig4 and lig ts7) are reported, lig ts7 is a conditionally lethal mutation, proving the essential nature of the ligase gene product. Neither mutant has a major defect in recombination or ultraviolet-repair, but both show retarded sealing of 10 S pulse-labeled DNA (Okazaki fragments).     

Expression,purification and biochemical characterization of Methanocaldococcus jannaschii DNA ligase

We describe the biochemical characterization of Methanocaldococcus jannaschii (M. jannaschii) DNA ligase and its potential application in single nucleotide polymorphism (SNP) genotyping. The recombinant M. jannaschii DNA ligase is an ATP-dependent ligase. The ligase activity was dependent on metal ions of Mg²⁺ and Mn²⁺. The optimal concentrations of ATP cofactor and Mg²⁺ ion were 0.01–2 and 10 mM, respectively. The optimal pH value for DNA ligation was 8.5. High concentrations of NaCl inhibited DNA ligation. The effects of mismatches on joining short oligonucleotides by M. jannaschii DNA ligase were fully characterized. The mismatches at the first position 5′ to the nick inhibited ligation more than those at the first position 3′ to the nick. The mismatches at other positions 5′ to the nick (3rd to 7th sites) exhibited less inhibition on ligation. However, the introduction of a C/C mismatch at the third position 5′ to the nick could completely inhibit the ligation of the terminal-mismatched nick of an oligonucleotide duplex by M. jannaschii DNA ligase. Therefore, introducing an additional mismatch at the third position 5′ to the SNP site is a more effective approach in genotyping by M. jannaschii DNA ligase.     

6-Arylpyrido[2,3-d]pyrimidines as Novel ATP-Competitive Inhibitors of Bacterial D-Alanine:D-Alanine Ligase

Background

ATP-dependent D-alanine:D-alanine ligase (Ddl) is a part of biochemical machinery involved in peptidoglycan biosynthesis, as it catalyzes the formation of the terminal D-ala-D-ala dipeptide of the peptidoglycan precursor UDPMurNAc-pentapeptide. Inhibition of Ddl prevents bacterial growth, which makes this enzyme an attractive and viable target in the urgent search of novel effective antimicrobial drugs. To address the problem of a relentless increase in resistance to known antimicrobial agents we focused our attention to discovery of novel ATP-competitive inhibitors of Ddl.

Methodology/Principal Findings

Encouraged by recent successful attempts to find selective ATP-competitive inhibitors of bacterial enzymes we designed, synthesized and evaluated a library of 6-arylpyrido[2,3-d]pyrimidine-based compounds as inhibitors of Escherichia coli DdlB. Inhibitor binding to the target enzyme was subsequently confirmed by surface plasmon resonance and studied with isothermal titration calorimetry. Since kinetic analysis indicated that 6-arylpyrido[2,3-d]pyrimidines compete with the enzyme substrate ATP, inhibitor binding to the ATP-binding site was additionally studied with docking. Some of these inhibitors were found to possess antibacterial activity against membrane-compromised and efflux pump-deficient strains of E. coli.

Conclusions/Significance

We discovered new ATP-competitive inhibitors of DdlB, which may serve as a starting point for development of more potent inhibitors of DdlB that could include both, an ATP-competitive and D-Ala competitive moiety.     

Determinants for differential effects on D-Ala-D-lactate vs D-Ala-D-Ala formation by the VanA ligase from vancomycin-resistant enterococci.

Bacteria with either intrinsic or inducible resistance to vancomycin make peptidoglycan (PG) precursors of lowered affinity for the antibiotic by switching the PG-D-Ala-D-Ala termini that are the antibiotic-binding target to either PG-D-Ala-D-lactate or PG-D-Ala-D-Ser as a consequence of altered specificity of the D-Ala-D-X ligases in the cell wall biosynthetic pathway. The VanA ligase of vancomycin-resistant enterococci, a D-Ala-D-lactate depsipeptide ligase, has the ability to recognize and activate the weak nucleophile D-lactate selectively over D-Ala(2) to capture the D-Ala(1)-OPO(3)(2)(-) intermediate in the ligase active site. To ensure this selectivity in catalysis, VanA largely rejects the protonated (NH(3)(+)) form of D-Ala at subsite 2 (K(M2) of 210 mM at pH 7.5) but not at subsite 1. In contrast, the deprotonated (NH(2)) form of D-Ala (K(M2) of 0.66 mM, k(cat) of 550 min(-)(1)) is a 17-fold better substrate compared to D-lactate (K(M) of 0.69 mM, k(cat) of 32 min(-)(1)). The low concentration of the free amine form of D-Ala at physiological conditions (i.e., 0.1% at pH 7.0) explains the inefficiency of VanA in dipeptide synthesis. Mutational analysis revealed a residue in the putative omega-loop region, Arg242, which is partially responsible for electrostatically repelling the protonated form of D-Ala(2). The VanA enzyme represents a subfamily of D-Ala-D-X ligases in which two key active-site residues (Lys215 and Tyr216) in the active-site omega-loop of the Escherichia coli D-Ala-D-Ala ligase are absent. To look for functional complements in VanA, we have mutated 20 residues and evaluated effects on catalytic efficiency for both D-Ala-D-Ala dipeptide and D-Ala-D-lactate depsipeptide ligation. Mutation of Asp232 caused substantial defects in both dipeptide and depsipeptide ligase activity, suggesting a role in maintaining the loop position. In contrast, the H244A mutation caused an increase in K(M2) for D-lactate but not D-Ala, indicating a differential role for His244 in the recognition of the weaker nucleophile D-lactate. Replacement of the VanA omega-loop by that of VanC2, a D-Ala-D-Ser ligase, eliminated D-Ala-D-lactate activity while improving by 3-fold the catalytic efficacy of D-Ala-D-Ala and D-Ala-D-Ser activity.     

Distribution and roles of p-hydroxycinnamate: CoA ligase in lignin biosynthesis

p-Hydroxycinnamate:CoA ligases were extracted from the xylems of angiosperms and gymnosperms, and the substrate specificities toward ferulate and sinapate were examined. Most of angiosperm and gymnosperm CoA ligases examined were active with ferulate but not with sinapate; however, the enzymes of Erythrina crista-galli, Robinia pseudoacacia and bamboo showed considerable activity with sinapate. The other enzymes, although inactive with sinapate, showed no inhibitory effect on the Erythrina CoA ligase reaction with sinapate. The K_ms for sinapate and ferulate of the Erythrina enzyme were 1.0 and 2.1 μM, respectively, and p-hydroxycinnamate was the best substrate among cinnamates examined. The MW of the CoA ligase was 40 000 and the pH optimum was between 7.2 and 7.6. The possible roles of p-hydroxycinnamate:CoA ligase in lignin biosynthesis are discussed.     

Glycopeptide resistance mediated by enterococcal transposon Tn 1546 requires production of VanX for hydrolysis of D-alanyl-D-alanine

Cloning and nucleotide sequencing indicated that transposon Tn 1546 from Enterococcus faecium BM4147 encodes a 23365 Da protein, VanX, required for glycopeptide resistance. The vanX gene was located downstream from genes encoding the VanA ligase and the VanH dehydrogenase which synthesize the depsipeptide D-alanyl-D-lactate (D-Ala-D-Lac). In the presence of ramoplanin, an Enterococcus faecalis JH2-2 derivative producing VanH, VanA and VanX accumulated mainly UDP-MurNAc-L-Ala-γ-D-Glu-L-Lys-D-Ala-D-Lac (pentadepsipeptide) and small amounts of UDP-MurNAc-L-Ala-γ-D-Glu-L-Lys-D-Ala-D-Ala (pentapeptide) in the ratio 49:1. Insertional inactivation of vanX led to increased synthesis of pentapeptide with a resulting change in the ratio of pentadepsipeptide: pentapeptide to less than 1:1. Expression of vanX in E. faecalis and Escherichia coli resulted in production of a D,D-dipeptidase that hydrolysed D-Ala-D-Ala. Pentadepsipeptide, pentapeptide and D-Ala-D-Lac were not substrates for the enzyme. These results establish that VanX is required for production of a D,D-dipeptidase that hydrolyses D-Ala-D-Ala, thereby preventing pentapeptide synthesis and subsequent binding of glycopeptides to D-Ala-D-Ala-containing peptidoglycan precursors at the cell surface.     

Evolution of structure and substrate specificity ind-alanine:d-Alanine ligases and related enzymes

Thed-alanine:d-alanine-ligase-related enzymes can have three preferential substrate specificities. Usually, these enzymes synthesized-alanyl-d-alanine. In vancomycin-resistant Gram-positive bacteria, structurally related enzymes synthesized-alanyl-d-lactate or Dalanyl-d-serine. The sequence of internal fragments of eight structurald-alanine:d-alanine ligase genes from enterococci has been determined. Alignment of the deduced amino acid sequences with those of other related enzymes from Gram-negative and Gram-positive bacteria revealed the presence of four distinct sequence patterns in the putative substrate-binding sites, each correlating with specificity to a particular substrate (d-alanine:d-lactate ligases exhibited two patterns). Phylogenetic analysis showed different clusters. The enterococcal subtree was largely superimposable on that derived from 16S rRNA sequences. In lactic acid bacteria, structural divergence due to differences in substrate specificity was observed. Glycopeptide resistance proteins VanA and VanB, the VanC-type ligases, and Dd1A and DdlB from enteric bacteria andHaemophilus influenzae constituted separate clusters. Correspondence to: P. Courvalin     

Structure-function analysis of the kinase-CPD domain of yeast tRNA ligase (Trl1) and requirements for complementation of tRNA splicing by a plant Trl1 homolog

Trl1 is an essential 827 amino acid enzyme that executes the end-healing and end-sealing steps of tRNA splicing in Saccharomyces cerevisiae. Trl1 consists of two domains—an N-terminal ligase component and a C-terminal 5′-kinase/2′,3′-cyclic phosphodiesterase (CPD) component—that can function in tRNA splicing in vivo when expressed as separate polypeptides. To understand the structural requirements for the kinase-CPD domain, we performed an alanine scan of 30 amino acids that are conserved in Trl1 homologs from other fungi. We thereby identified four residues (Arg463, His515, Thr675 and Glu741) as essential for activity in vivo. Structure–function relationships at these positions, and at four essential or conditionally essential residues defined previously (Asp425, Arg511, His673 and His777), were clarified by introducing conservative substitutions. Biochemical analysis showed that lethal mutations of Asp425, Arg463, Arg511 and His515 in the kinase module abolished polynucleotide kinase activity in vitro. We report that a recently cloned 1104 amino acid Arabidopsis RNA ligase functions in lieu of yeast Trl1 in vivo and identify essential side chains in the ligase, kinase and CPD modules of the plant enzyme. The plant ligase, like yeast Trl1 but unlike T4 RNA ligase 1, requires a 2′-PO₄ end for tRNA splicing in vivo.     

Structural and Functional Characterization of VanG d-Ala:d-Ser Ligase Associated with Vancomycin Resistance in Enterococcus faecalis

d-Alanyl:d-lactate (d-Ala:d-Lac) and d-alanyl:d-serine ligases are key enzymes in vancomycin resistance of Gram-positive cocci. They catalyze a critical step in the synthesis of modified peptidoglycan precursors that are low binding affinity targets for vancomycin. The structure of the d-Ala:d-Lac ligase VanA led to the understanding of the molecular basis for its specificity, but that of d-Ala:d-Ser ligases had not been determined. We have investigated the enzymatic kinetics of the d-Ala:d-Ser ligase VanG from Enterococcus faecalis and solved its crystal structure in complex with ADP. The overall structure of VanG is similar to that of VanA but has significant differences mainly in the N-terminal and central domains. Based on reported mutagenesis data and comparison of the VanG and VanA structures, we show that residues Asp-243, Phe-252, and Arg-324 are molecular determinants for d-Ser selectivity. These residues are conserved in both enzymes and explain why VanA also displays d-Ala:d-Ser ligase activity, albeit with low catalytic efficiency in comparison with VanG. These observations suggest that d-Ala:d-Lac and d-Ala:d-Ser enzymes have evolved from a common ancestral d-Ala:d-X ligase. The crystal structure of VanG showed an unusual interaction between two dimers involving residues of the omega loop that are deeply anchored in the active site. We constructed an octapeptide mimicking the omega loop and found that it selectively inhibits VanG and VanA but not Staphylococcus aureus d-Ala:d-Ala ligase. This study provides additional insight into the molecular evolution of d-Ala:d-X ligases and could contribute to the development of new structure-based inhibitors of vancomycin resistance enzymes.     

Synthesis and inhibitory activity of mechanism-based 4-coumaroyl-CoA ligase inhibitors

4-Coumaroyl-CoA ligase (4CL) is ubiquitous in the plant kingdom, and plays a central role in the biosynthesis of phenylpropanoids such as lignins, flavonoids, and coumarins. 4CL catalyzes the formation of the coenzyme A thioester of cinnamates such as 4-coumaric, caffeic, and ferulic acids, and the regulatory position of 4CL in the phenylpropanoid pathway renders the enzyme an attractive target that controls the composition of phenylpropanoids in plants. In this study, we designed and synthesized mechanism-based inhibitors for 4CL in order to develop useful tools for the investigation of physiological functions of 4CL and chemical agents that modulate plant growth with the ultimate goal to produce plant biomass that exhibits features that are beneficial to humans. The acylsulfamide backbone of the inhibitors in this study was adopted as a mimic of the acyladenylate intermediates in the catalytic reaction of 4CL. These acylsulfamide inhibitors and the important synthetic intermediates were fully characterized using two-dimensional NMR spectroscopy. Five 4CL proteins with distinct substrate specificity from four plant species, i.e., Arabidopsis thaliana, Glycine max (soybean), Populus trichocarpa (poplar), and Petunia hybrida (petunia), were used to evaluate the inhibitory activity, and the half-maximum inhibitory concentration (IC₅₀) of each acylsulfamide in the presence of 4-coumaric acid (100?µM) was determined as an index of inhibitory activity. The synthetic acylsulfamides used in this study inhibited the 4CLs with IC₅₀ values ranging from 0.10 to 722?µM, and the IC₅₀ values of the most potent inhibitors for each 4CL were 0.10–2.4?µM. The structure–activity relationship observed in this study revealed that both the presence and the structure of the acyl group of the synthetic inhibitors strongly affect the inhibitory activity, and indicates that 4CL recognizes the acylsulfamide inhibitors as acyladenylate mimics.     

Stephania suberosa Forman extract synergistically inhibits ampicillin- and vancomycin-resistant Enterococcus faecium

Increasing antibiotic resistance in enterococci is among the most serious public health problems worldwide. The new naturally occurring antibacterial agents were explored. This study, therefore, investigated the antibacterial potential of Stephania suberosa extract (SSE) and its synergism with ampicillin (AMP) or vancomycin (VAN) against AMP- and VAN-resistant Enterococcus faecium. Disc diffusion assay revealed that SSE inhibited E. faecium DMST 12829, 12852, 12970, and a reference strain of Enterococcus faecalis ATCC 29,212 in a dose-dependent manner. The minimum inhibitory concentration (MIC) of SSE against all E. faecium isolates was 0.5 mg/mL. E. faecium DMST 12,829 and 12,852 were highly resistant to AMP, as indicated by high MIC values, and E. faecium DMST 12,829 and 12,970 were resistant to VAN. Enterococcus spp. were killed by SSE at the minimum bactericidal concentrations (MBCs) ranging from 0.5 to 4 mg/mL. Checkerboard determination showed that SSE plus AMP and SSE plus VAN combinations exhibited synergistic interaction against E. faecium isolates. The killing curve assay of E. faecium isolates confirmed the antibacterial and synergistic activities of combined agents by dramatically reducing the viable counts compared to a single agent. Scanning electron microscope elucidated the cell damage and abnormal cell division. Enterococcal proteases were also inhibited by SSE. These findings support that SSE could reverse the activity of AMP and VAN. Moreover, it can synergistically inhibit AMP- and VAN-resistant E. faecium. Our combined agents could be attractive candidates for developing new combinatorial agents to resurrect the efficacy of antibiotics for treating AMP- and VAN-resistant E. faecium infections.     

Sequencing of the ddl gene and modeling of the mutated D-alanine:D-alanine ligase in glycopeptide-dependent strains of Enterococcus faecium

Glycopeptide dependence for growth in enterococci results from mutations in the ddl gene that inactivate the host D-Ala:D-Ala ligase. The strains require glycopeptides as inducers for synthesis of resistance proteins, which allows for the production of peptidoglycan precursors ending in D-Ala-D-Lac instead of D-Ala-D-Ala. The sequences of the ddl gene from nine glycopeptide-dependent Enterococcus faecium clinical isolates were determined. Each one had a mutation consisting either in a 5-bp insertion at position 41 leading to an early stop codon, an in-frame 6-bp deletion causing the loss of two residues (KDVA243-246 to KA), or single base-pair changes resulting in an amino acid substitution (E13 --> G, G99 --> R, V241 --> D, D295 --> G, P313 --> L). The potential consequences of the deletion and point mutations on the 3-D structure of the enzyme were evaluated by comparative molecular modeling of the E. faecium enzyme, using the X-ray structure of the homologous Escherichia coli D-Ala:D-Ala ligase DdlB as a template. All mutated residues were found either to interact directly with one of the substrates of the enzymatic reaction (E13 and D295) or to stabilize the position of critical residues in the active site. Maintenance of the 3-D structure in the vicinity of these mutations in the active site appears critical for D-Ala:D-Ala ligase activity.     

The p-coumaryl CoA ligase of potato tubers

The demonstration of activity of p-coumaryl CoA ligase in extracts of aged potato disks proved difficult owing to the presence of extremely high levels of apyrase which caused rapid hydrolysis of ATP, a co-factor for ligase activity. This problem was largely overcome by including an inhibitor of apyrase, sodium fluoride in the ligase assay and by initiation of the reaction with ATP. A method for the separation of apyrase and p-coumaryl CoA ligase by chromatography on DEAE-cellulose is described. p-Coumaryl CoA ligase was not detectable in freshly prepared disks of potato tubers. However on ageing in the light a large increase in the activity of this enzyme occurs, The enzyme of aged potato disks shows high activity with p-coumaric, ferulic, caffeic and with m- and p-methoxycinnamic acids. However the affinity of the enzyme for the methoxy derivatives is much lower than for cinnamic acids bearing free hydroxyl groups.     

Characterization of an ATP-dependent DNA ligase from the thermophilic archaeon Methanobacterium thermoautotrophicum

We report the production, purification and characterization of a DNA ligase encoded by the thermophilic archaeon Methanobacterium thermoautotrophicum. The 561 amino acid Mth ligase catalyzed strand-joining on a singly nicked DNA in the presence of a divalent cation (magnesium, manganese or cobalt) and ATP (K_m 1.1 µM). dATP can substitute for ATP, but CTP, GTP, UTP and NAD⁺ cannot. Mth ligase activity is thermophilic in vitro, with optimal nick-joining at 60°C. Mutational analysis of the conserved active site motif I (KxDG) illuminated essential roles for Lys251 and Asp253 at different steps of the ligation reaction. Mutant K251A is unable to form the covalent ligase–adenylate intermediate (step 1) and hence cannot seal a 3′-OH/5′-PO₄ nick. Yet, K251A catalyzes phosphodiester bond formation at a pre-adenylated nick (step 3). Mutant D253A is active in ligase–adenylate formation, but defective in activating the nick via formation of the DNA–adenylate intermediate (step 2). D253A is also impaired in phosphodiester bond formation at a pre-adenylated nick. A profound step 3 arrest, with accumulation of high levels of DNA–adenylate, could be elicited for the wild-type Mth ligase by inclusion of calcium as the divalent cation cofactor. Mth ligase sediments as a monomer in a glycerol gradient. Structure probing by limited proteolysis suggested that Mth ligase is a tightly folded protein punctuated by a surface-accessible loop between nucleotidyl transferase motifs III and IIIa.     

Broad nucleotide cofactor specificity of DNA ligase from the hyperthermophilic crenarchaeon Hyperthermus butylicus and its evolutionary significance

The nucleotide cofactor specificity of the DNA ligase from the hyperthermophilic crenarchaeon Hyperthermus butylicus (Hbu) was studied to investigate the evolutionary relationship of DNA ligases. The Hbu DNA ligase gene was expressed under control of the T7lac promoter of pTARG in Escherichia coli BL21-CodonPlus(DE3)-RIL. The expressed enzyme was purified using the IMPACT?-CN system (intein-mediated purification with an affinity chitin-binding tag) and cation-ion (Arg-tag) chromatography. The optimal temperature for Hbu DNA ligase activity was 75 °C, and the optimal pH was 8.0 in Tris–HCl. The activity was highly dependent on MgCl₂ or MnCl₂ with maximal activity above 5 mM MgCl₂ and 2 mM MnCl₂. Notably, Hbu DNA ligase can use ADP and GTP in addition to ATP. The broad nucleotide cofactor specificity of Hbu DNA ligase might exemplify an undifferentiated ancestral stage in the evolution of DNA ligases. This study provides new evidence for possible evolutionary relationships among DNA ligases.     

Efficient Transfer of the Pheromone-Independent Enterococcus faecium Plasmid pMG1 (Gmr) (65.1 Kilobases) to Enterococcus Strains during Broth Mating

Plasmid pMG1 (65.1 kb) was isolated from a gentamicin-resistant Enterococcus faecium clinical isolate and was found to encode gentamicin resistance. EcoRI restriction of pMG1 produced five fragments, A through E, with molecular sizes of 50.2, 11.5, 2.0, 0.7, and 0.7 kb, respectively. The clockwise order of the fragments was ACDEB. pMG1 transferred at high frequency to Enterococcus strains in broth mating. pMG1 transferred between Enterococcus faecalis strains, between E. faecium strains, and between E. faecium and E. faecalis strains at a frequency of approximately 10⁻⁴ per donor cell after 3 h of mating. The pMG1 transfers were not induced by the exposure of the donor cell to culture filtrates of plasmid-free E. faecalis FA2-2 or an E. faecium strain. Mating aggregates were not observed by the naked eye during broth mating. Small mating aggregates of several cells in the broth matings were observed by microscopy, while no aggregates of donor cells which had been exposed to a culture filtrate of E. faecalis FA2-2 or an E. faecium strain were observed, even by microscopy. pMG1 DNA did not show any homology in Southern hybridization with that of the pheromone-responsive plasmids and broad-host-range plasmids pAMβ1 and pIP501. These results indicate that there is another efficient transfer system in the conjugative plasmids of Enterococcus and that this system is different from the pheromone-induced transfer system of E. faecalis plasmids.     

Vancomycin-Resistant Enterococcus faecium (VREF) from Norwegian Poultry Cluster with VREF from Poultry from the United Kingdom and The Netherlands in an Amplified Fragment Length Polymorphism Genogroup

The genetic relationship between 197 vancomycin-resistant Enterococcus faecium (VREF) isolates and 21 vancomycin-susceptible E. faecium isolates from Norwegian poultry was analyzed by amplified fragment length polymorphism (AFLP). The isolates were compared to 255 VREF isolates from various sources and countries. The Norwegian isolates constituted a relatively homogeneous population of E. faecium and clustered in a previously defined poultry AFLP genogroup.     

Evidence for in vivo compartmentation of phenylalanyl-tRNA ligase in amphibian oocytes

The in vivo activity of phenylalanyl-tRNA ligase of Xenopus laevis oocytes was assayed by measuring the esterification of microinjected yeast tRNA^Phe with [¹⁴C]phenylalanine added to the extracellular medium. The three enzyme substrates, ATP, phenylalanine, and tRNA^Phe, are present in the in vivo assay at saturating concentrations as seen by the fact that microinjection into the cell of additional amounts of these compounds does not increase the quantity of [¹⁴C]Phe-tRNA^Phe formed. The in vivo activity of Phe-tRNA ligase in oocytes at several stages of development is less than 10% of the in vitro activity measured in homogenates of the same cells. The in vivo assay of Phe-tRNA ligase in oocytes that have been microinjected with this enzyme partially purified from X. laevis ovary shows that the enzyme is not inhibited by the cellular conditions. The conclusion drawn from these experiments is that a large fraction of the Phe-tRNA ligase present in oocytes is in a cellular compartment which is not available to the injected tRNA.