Fluorescent sensors of siderophores produced by bacterial pathogens.

Siderophores are iron-chelating molecules that solubilize Fe3+ for microbial utilization and facilitate colonization or infection of eukaryotes by liberating host iron for bacterial uptake. By fluorescently labeling membrane receptors and binding proteins, we created 20 sensors that detect, discriminate, and quantify apo- and ferric siderophores. The sensor proteins originated from TonB-dependent ligand-gated porins (LGPs) of Escherichia coli (Fiu, FepA, Cir, FhuA, IutA, BtuB), Klebsiella pneumoniae (IroN, FepA, FyuA), Acinetobacter baumannii (PiuA, FepA, PirA, BauA), Pseudomonas aeruginosa (FepA, FpvA), and Caulobacter crescentus (HutA) from a periplasmic E. coli binding protein (FepB) and from a human serum binding protein (siderocalin). They detected ferric catecholates (enterobactin, degraded enterobactin, glucosylated enterobactin, dihydroxybenzoate, dihydroxybenzoyl serine, cefidericol, MB-1), ferric hydroxamates (ferrichromes, aerobactin), mixed iron complexes (yersiniabactin, acinetobactin, pyoverdine), and porphyrins (hemin, vitamin B12). The sensors defined the specificities and corresponding affinities of the LGPs and binding proteins and monitored ferric siderophore and porphyrin transport by microbial pathogens. We also quantified, for the first time, broad recognition of diverse ferric complexes by some LGPs, as well as monospecificity for a single metal chelate by others. In addition to their primary ferric siderophore ligands, most LGPs bound the corresponding aposiderophore with ∼100-fold lower affinity. These sensors provide insights into ferric siderophore biosynthesis and uptake pathways in free-living, commensal, and pathogenic Gram-negative bacteria.

Comparative community gene expression analysis of Aquificales-dominated geothermal springs.

Members of Sulfurihydrogenibium are often observed as visible filamentous biomass in circumneutral hot springs and play roles in sulfur-cycling, hydrogen oxidation and iron mineralization. To gain insight into the ecophysiology of Sulfurihydrogenibium populations, we conducted preliminary metatranscriptomic analysis of three distinct thermal springs; Calcite Springs (YNP-CS) and Mammoth Springs (YNP-MHS) in Yellowstone National Park, USA, and Furnas Springs (AZ) in Azores, Portugal. Genes to which transcripts were assigned revealed commonly expressed functions among the sites, while several differences were also observed. All three sites, Sulfurihydrogenibium spp. dominate and are obtaining energy via metabolism of sulfur compounds under microaerophilic conditions. Cell motility was one of the expressed functions in two sites (YNP-CS and AZ) with slower stream flow rates and thicker well-formed biofilms. The transcripts from YNP-CS and -MHS exhibited varying levels of sequence divergence from the reference genomes and corresponding metagenomes, suggesting the presence of microdiversity among Sulfurihydrogenibium populations in situ. Conversely, the majority of the AZ transcripts were identical to the S. azorense genome. Our initial results show that the metatranscriptomes in these similar Aquificales-dominated communities can reveal community-level gene function in geochemically distinct thermal environments.

Thermosulfurimonas dismutans gen. nov., sp. nov., an extremely thermophilic sulfur-disproportionating bacterium from a deep-sea hydrothermal vent.

An extremely thermophilic, anaerobic, chemolithoautotrophic bacterium (strain S95(T)) was isolated from a deep-sea hydrothermal vent chimney located on the Eastern Lau Spreading Center, Pacific Ocean, at a depth of 1910 m. Cells of strain S95(T) were oval to short Gram-negative rods, 0.5-0.6 µm in diameter and 1.0-1.5 µm in length, growing singly or in pairs. Cells were motile with a single polar flagellum. The temperature range for growth was 50-92 °C, with an optimum at 74 °C. The pH range for growth was 5.5-8.0, with an optimum at pH 7.0. Growth of strain S95(T) was observed at NaCl concentrations ranging from 1.5 to 3.5% (w/v). Strain S95(T) grew anaerobically with elemental sulfur as an energy source and bicarbonate/CO(2) as a carbon source. Elemental sulfur was disproportionated to sulfide and sulfate. Growth was enhanced in the presence of poorly crystalline iron(III) oxide (ferrihydrite) as a sulfide-scavenging agent. Strain S95(T) was also able to grow by disproportionation of thiosulfate and sulfite. Sulfate was not used as an electron acceptor. Analysis of the 16S rRNA gene sequence revealed that the isolate belongs to the phylum Thermodesulfobacteria. On the basis of its physiological properties and results of phylogenetic analyses, it is proposed that the isolate represents the sole species of a new genus, Thermosulfurimonas dismutans gen. nov., sp. nov.; S95(T) (=DSM 24515(T)=VKM B-2683(T)) is the type strain of the type species. This is the first description of a thermophilic micro-organism that disproportionates elemental sulfur.

Molecular cloning of a Cellulomonas fimi cellulose gene in Escherichia coli.

A sensitive and simple immunoassay was developed to screen Escherichia coli transformed with recombinant DNA plasmids carrying a cellulase gene. The assay was used to identify a recombinant DNA plasmid carrying at least one cellulase gene from Cellulomonas fimi. The enzyme present in extracts of E. coli carrying the plasmid was active in catalysing the hydrolysis of carboxymethylcellulose as indicated by the production of reducing sugars.

Evidence for two types of subunits in the bacterioferritin of Magnetospirillum magnetotacticum.

In order to investigate the role of bacterioferritin (Bfr) in the biomineralization of magnetite by microorganisms, we have cloned and sequenced the bfr genes from M. magnetotacticum. The organism has two bfr genes that overlap by one nucleotide. Both encode putative protein products of 18 kDa, the expected size for Bfr subunits, and show a strong similarity to other Bfr subunit proteins. By scanning the DNA sequence databases, we found that a limited number of other organisms, including N. gonorrhea, P. aeruginosa, and Synechocystis PCC6803, also have two bfr genes. When the sequences of a number of microbial Bfrs are compared with each other, they fall into two distinct types with the organisms mentioned above having one of each type. Differences in heme- and metal-binding sites and ferroxidase activities of the two types of subunits are discussed.

A mutation blocking the formation of membrane or periplasmic endogenous and exogenous c-type cytochromes in Escherichia coli permits the cytoplasmic formation of Hydrogenobacter thermophilus holo cytochrome c552.

A mutant of Escherichia coli, JCB606, shown to be pleiotropically deficient in the formation of endogenous membrane and periplasmic c-type cytochromes, synthesised the apo form of the exogenous cytochrome c550 from Paracoccus denitrificans, but not the holo form. In contrast, a cytoplasmically located holo form of Hydrogenobacter thermophilus cytochrome c552 was found in E. coli JCB606. These findings support the proposition that the formation of the cytoplasmic H. thermophilus cytochrome c552 in E. coli does not involve the physiological pathway of c-type cytochrome biosynthesis in E. coli and that the haem insertion may be catalysed.

Genomic organization analysis of acidophilic chemolithotrophic bacteria using pulsed field gel electrophoretic techniques.

The genomic organization of acidophilic chemolithotrophic bacteria belonging to the genus Thiobacillus, Thiomonas and Leptospirillum was studied using pulsed field gel electrophoresis techniques (PFGE). The electrophoretic analysis of intact DNA prepared from different strains showed that all have a circular chromosome, with sizes ranging from 1.9 Mb for Leptospirillum ferrooxidans ATCC 49879, the smallest genome for an acidophilic strict chemolithoautotrophic microorganism, to 3.8 Mb for Thiomonas cuprina DSM 5495, the largest in this study. The number of extrachromosomal elements present varied from none, as observed in several isolates of Leptospirillum ferrooxidan, to five in Thiobacillus thiooxidans ATCC 8085. The mixotroph Thiomonas cuprina DSM 5495 was found to have a linear 50 kb megaplasmid which was inducible when the bacteria was grown in chemolithotrophic conditions. Low-frequency restriction fragment analysis (LFRFA) of different acidophilic chemolithotrophs and related species was carried out by PFGE to determine macrorestriction patterns for rare cutters (SpeI, XbaI, SwaI, PmeI), which were then used for taxonomic identification (karyotyping), genome size determination, and generation of physical and genetic maps.

Archaebacterial ATPases: relationship to other ion-translocating ATPase families examined in terms of immunological cross-reactivity.

Immunological cross-reactivity among three types of H(+)-ATPases, that is, three archaebacterial ATPases, the F1-ATPase from thermophilic bacterium PS3 (TF1) and the vacuolar membrane ATPase from Saccharomyces cerevisiae, was examined by means of immunoblot analyses. The three archaebacterial ATPases were very similar in immunological cross-reactivity, suggesting that they belong to the same family of ATPases. Cross-reaction was also observed between the ATPase from Sulfolobus acidocaldarius, one of the three archaebacteria, and TF1. S. cerevisiae vacuolar ATPase reacted with the antibodies prepared against each of the three archaebacterial ATPases, but did not react with the antibody against TF1. Electron microscopic examination revealed that the oligomeric structure of Sulfolobus ATPase was very similar to that of F1-ATPase. These results, taken together, suggest that the archaebacterial ATPases share close structural similarities with the vacuolar ATPases, and, to a lesser degree, with the F0F1-ATPases.

Spontaneous mutation of Thiosphaera pantotropha enabling growth on methanol correlates with synthesis of a 26 kDa c-type cytochrome.

Thiosphaera pantotropha grows on methanol as carbon and energy source following spontaneous mutation to a Mox+ phenotype after incubation in media containing methanol. Acquisition of ability to grow on methanol correlates with the appearance of a c-type cytochrome, molecular mass 26 kDa, which is suggested to substitute for the product of the moxG gene, which is the electron acceptor from methanol in related bacteria, but which is absent from T. pantotropha. Mutation leading to growth on methylamine as carbon and energy source was not observed despite the presence of in vitro methylamine dehydrogenase activity in cells grown on choline. Lack of growth on methylamine may correlate with the absence of amicyanin, which is the obligatory electron acceptor from methylamine dehydrogenase in other organisms.

Phylogenetic position of the menaquinone-containing acidophilic chemo-organotroph Acidobacterium capsulatum.

The phylogenetic position of an acidophilic chemo-organotrophic menaquinone-containing bacterium, Acidobacterium capsulatum, was studied on the basis of 16S rRNA gene sequence information. A. capsulatum showed the highest level of sequence similarity to Heliobacterium chlorum, a member of the Gram-positive group, yet this level was only 81%. Distance matrix tree analysis suggested that A. capsulatum belongs to a unique lineage deeply branching from the Chlamydia-Planctomyces group or from the Gram-positive line.

Multiple copies of ammonia monooxygenase (amo) operons have evolved under biased AT/GC mutational pressure in ammonia-oxidizing autotrophic bacteria.

The recent availability of complete sequences of ammonia monooxygenase (16 amoA, 5 amoB and 5 amoC gene sequences) and particulate methane monooxygenase (2 pmoA, pmoB and pmoC gene sequences each) genes allowed for a detailed analysis of their relatedness. Nucleotide sequence analysis was performed in order to identify the origins of the nearly identical operon copies within a given nitrosofier/methanotroph strain. Our data suggest that amo-homologous gene evolution has occurred in individual strains (orthology) under biased AT/GC pressure rather than by horizontal transfer. The multiple operon copies within individual strains are the result of operon duplication (paralogy). While the near identity of the multiple operon copies makes it impossible to determine whether paralogous gene expansion occurred in the last common ancestor of ammonia oxidizers or after speciation took place, we conclude that the duplication events were not recent events. We propose that the elimination of third basepair degeneracy between copies within one organism is implemented by a rectification mechanism resulting in concerted evolution.

A qualitative evaluation of the published oligonucleotides specific for the 16S rRNA gene sequences of the ammonia-oxidizing bacteria.

Over the past few years, there has been an increasing interest in making oligonucleotides specific for ammonia-oxidizing bacteria (AOB), in order to detect and monitor these slow growing bacteria in environmental samples, in enrichment cultures and in wastewater treatment plants. Based on 16S rDNA sequences, a broad selection of oligonucleotides have been designed, either encompassing all known AOB in the beta-subgroup of the Proteobacteria (beta AOB), or subclasses within beta AOB. Thirty different oligonucleotides have so far been published, with varying specificity. The first AOB-specific oligonucleotides published were obtained as a result of an alignment of only eleven 16S rDNA sequences from AOB. Including the present study, there are now forty nearly full length 16S rDNA sequences available from these bacteria, in addition to a number of partial sequences, so that an improved evaluation of the published oligonucleotides can be done. Two new 16S rRNA gene sequences from Nitrosospira are presented here, in a phylogenetic analysis containing every 16S rRNA gene sequences (> 1 kb) available from AOB. On the basis of an alignment of all these sequences, combined with searches in the nucleotide sequence databases, an evaluation of the thirty published oligonucleotides is presented. The analysis expose the strength and weakness of each oligonucleotide and discuss the use of oligonucleotides specific for 16S rRNA genes in future studies of AOB. The present work also identifies one new, broad range primer, specific for the AOB in the beta-subgroup of the Proteobacteria.

Isolation and characterization of a moderately thermophilic nitrite-oxidizing bacterium from a geothermal spring.

Geothermal environments are a suitable habitat for nitrifying microorganisms. Conventional and molecular techniques indicated that chemolithoautotrophic nitrite-oxidizing bacteria affiliated with the genus Nitrospira are widespread in environments with elevated temperatures up to 55 °C in Asia, Europe, and Australia. However, until now, no thermophilic pure cultures of Nitrospira were available, and the physiology of these bacteria was mostly uncharacterized. Here, we report on the isolation and characterization of a novel thermophilic Nitrospira strain from a microbial mat of the terrestrial geothermal spring Gorjachinsk (pH 8.6; temperature 48 °C) from the Baikal rift zone (Russia). Based on phenotypic properties, chemotaxonomic data, and 16S rRNA gene phylogeny, the isolate was assigned to the genus Nitrospira as a representative of a novel species, for which the name Nitrospira calida is proposed. A highly similar 16S rRNA gene sequence (99.6% similarity) was detected in a Garga spring enrichment grown at 46 °C, whereas three further thermophilic Nitrospira enrichments from the Garga spring and from a Kamchatka Peninsula (Russia) terrestrial hot spring could be clearly distinguished from N. calida (93.6-96.1% 16S rRNA gene sequence similarity). The findings confirmed that Nitrospira drive nitrite oxidation in moderate thermophilic habitats and also indicated an unexpected diversity of heat-adapted Nitrospira in geothermal hot springs.

Sulfurihydrogenibium rodmanii sp. nov., a sulfur-oxidizing chemolithoautotroph from the Uzon Caldera, Kamchatka Peninsula, Russia, and emended description of the genus Sulfurihydrogenibium.

Four thermophilic, sulfur-oxidizing, chemolithoautotrophic strains with >99 % 16S rRNA gene sequence similarity were isolated from terrestrial hot springs in the Geyser Valley and the Uzon Caldera, Kamchatka, Russia. One strain, designated UZ3-5T, was characterized fully. Cells of UZ3-5T were Gram-negative, motile, slightly oval rods (about 0.7 microm wide and 1.0 microm long) with multiple polar flagella. All four strains were obligately microaerophilic chemolithoautotrophs and could use elemental sulfur or thiosulfate as electron donors and oxygen (1-14 %, v/v) as the electron acceptor. Strain UZ3-5T grew at temperatures between 55 and 80 degrees C (optimally at 75 degrees C; 1.1 h doubling time), at pH 5.0-7.2 (optimally at pH 6.0-6.3) and at 0-0.9 % NaCl (optimally in the absence of NaCl). The G+C content of the genomic DNA of strain UZ3-5T was 35 mol%. Phylogenetic analysis revealed that strain UZ3-5T was a member of the genus Sulfurihydrogenibium, its closest relative in culture being Sulfurihydrogenibium azorense Az-Fu1T (98.3 % 16S rRNA gene sequence similarity). On the basis of its physiological and molecular characteristics, strain UZ3-5T represents a novel species of the genus Sulfurihydrogenibium, for which the name Sulfurihydrogenibium rodmanii sp. nov. is proposed. The type strain is UZ3-5T (=OCM 900T =ATCC BAA-1536T =DSM 19533T).

Venenivibrio stagnispumantis gen. nov., sp. nov., a thermophilic hydrogen-oxidizing bacterium isolated from Champagne Pool, Waiotapu, New Zealand.

A novel thermophilic, hydrogen-oxidizing bacterium, designated strain CP.B2(T), was isolated from a terrestrial hot spring in Waiotapu, New Zealand. Cells were motile, slightly rod-shaped, non-spore-forming and Gram-negative. Isolate CP.B2(T) was an obligate chemolithotroph, growing by utilizing H(2) as electron donor and O(2) as corresponding electron acceptor. Elemental sulfur (S(0)) or thiosulfate ( ) was essential for growth. Microbial growth occurred under microaerophilic conditions in 1.0-10.0 % (v/v) O(2) [optimum 4-8 % (v/v) O(2)], between 45 and 75 degrees C (optimum 70 degrees C) and at pH values of 4.8-5.8 (optimum pH 5.4). The DNA G+C content was 29.3 mol%. 16S rRNA gene sequence analysis demonstrated that strain CP.B2(T) belonged to the order Aquificales, with a close phylogenetic relationship to Sulfurihydrogenibium azorense (94 % sequence similarity to the type strain). However, genotypic and metabolic characteristics differentiated the novel isolate from previously described genera of the Aquificales. Therefore, CP.B2(T) represents a novel species in a new genus, for which the name Venenivibrio stagnispumantis gen. nov., sp. nov. is proposed. The type strain of Venenivibrio stagnispumantis is CP.B2(T) (=JCM 14244(T) =DSM 18763(T)).

Physiological and phylogenetic characterization of a novel lithoautotrophic nitrite-oxidizing bacterium, 'Candidatus Nitrospira bockiana'.

A new isolate of a lithoautotrophic nitrite-oxidizing bacterium was obtained from internal corrosion deposits from a steel pipeline of the Moscow heating system. The organism oxidized nitrite as the sole energy source and fixed carbon dioxide as the only carbon source. The cells were extremely pleomorphic: loosely wound spirals, slightly curved and even straight rods were detected, as well as coccoid cells. The highest rate of nitrite consumption (1.5 mM nitrite as substrate) was measured at 42 degrees C, with a temperature range of 28-44 degrees C. In enrichment cultures with Nocardioides sp. as an accompanying organism, optimal oxidation of 5.8 mM nitrite occurred at 45 degrees C, with a range of 28-48 degrees C. Neither pyruvate nor yeast extract stimulated nitrification. Organotrophic growth was not observed. Phylogenetic analysis of 16S rRNA gene sequences revealed that the novel isolate represents a new sublineage of the genus Nitrospira. On the basis of physiological, chemotaxonomic and molecular characteristics, the name 'Candidatus Nitrospira bockiana' is proposed.

Thermosulfidibacter takaii gen. nov., sp. nov., a thermophilic, hydrogen-oxidizing, sulfur-reducing chemolithoautotroph isolated from a deep-sea hydrothermal field in the Southern Okinawa Trough.

A novel thermophilic, sulfur-reducing chemolithoautotroph, strain ABI70S6(T), was isolated from a deep-sea hydrothermal field at the Yonaguni Knoll IV, Southern Okinawa Trough. Cells of strain ABI70S6(T) were motile rods, 0.9-2.0 microm in length and 0.4-0.8 microm in width. Strain ABI70S6(T) was an obligately anaerobic chemolithotroph, exhibiting hydrogen oxidation coupled with sulfur reduction. Growth was observed at 55-78 degrees C (optimum, 70 degrees C), pH 5.0-7.5 (optimum, pH 5.5-6.0) and 0.5-4.5 % NaCl (optimum, 3.0 % NaCl). H(2) and elemental sulfur were utilized as electron donor and acceptor, respectively. The major fatty acids were C(16 : 0) (40.0 %) and C(20 : 1) (60.0 %). The G+C content of genomic DNA was 44.2 mol%. The physiological attributes of strain ABI70S6(T) are similar to those of species of genera within the family Desulfurobacteriaceae, most of which are thermophilic and chemolithoautotrophic sulfur reducers. However, 16S rRNA gene sequence similarities between the novel isolate and type strains of all species within the family Desulfurobacteriaceae were <87 %, which is close to the similarities found between the novel isolate and members of the family Thermodesulfobacteriaceae (<85 %). Based on physiological and phylogenetic features of the novel isolate, it is proposed that it represents a novel species in a novel genus, Thermosulfidibacter takaii gen. nov., sp. nov., within the phylum Aquificae. The type strain of T. takaii is ABI70S6(T) (=JCM 13301(T)=DSM 17441(T)).

Hydrogenivirga okinawensis sp. nov., a thermophilic sulfur-oxidizing chemolithoautotroph isolated from a deep-sea hydrothermal field, Southern Okinawa Trough.

A novel extremely thermophilic sulfur-oxidizing bacterium, strain LS12-2(T), was isolated from a deep-sea hydrothermal field at the Yonaguni Knoll IV, Southern Okinawa Trough. Cells of strain LS12-2(T) were motile rods, 1.5-4.0 microm in length and 0.4-0.5 microm in width. Strain LS12-2(T) was an obligate chemolithoautotroph that could utilize elemental sulfur or thiosulfate as an electron donor and nitrate or oxygen as an electron acceptor. Growth was observed at 65-85 degrees C (optimum 70-75 degrees C), pH 5.8-8.3 (optimum pH 6.9-7.5), 1.0-4.0 % (w/v) NaCl (optimum 2.5 %) and 1.0-7.0 % O(2) in the gas phase (optimum 3.0 %). Fatty acids detected were C(16 : 0) (8.0 %), C(18 : 0) (9.0 %), C(18 : 1) (62.5 %) and C(20 : 1) (20.5 %). The genomic DNA G+C content was 51.3 mol%. 16S rRNA gene sequence analysis indicated that strain LS12-2(T) belonged to the genus Hydrogenivirga. Based on physiological and phylogenetic characteristics of the isolate, it is proposed that this strain represents a novel species in the genus Hydrogenivirga, Hydrogenivirga okinawensis sp. nov. The type strain of Hydrogenivirga okinawensis is LS12-2(T) (=JCM 13302(T)=DSM 17378(T)).

Sulfurihydrogenibium kristjanssonii sp. nov., a hydrogen- and sulfur-oxidizing thermophile isolated from a terrestrial Icelandic hot spring.

Three thermophilic, aerobic, hydrogen- and sulfur-oxidizing bacteria were isolated from an Icelandic hot spring near the town of Hveragerdi and share >99 % 16S rRNA gene sequence similarity. One of these isolates, designated strain I6628T, was selected for further characterization. Strain I6628T is a motile rod, 1.5-2.5 microm long and about 0.5 microm wide. Growth occurred between 40 and 73 degrees C (optimally at 68 degrees C), at pH 5.3-7.8 (optimally at pH 6.6) and at NaCl concentrations between 0 and 0.5 % (w/v). Strain I6628T grew with H2, S0 or S2O3(2-) as an electron donor with O2 (up to 25 %, v/v; optimally at 4-9 %) as the sole electron acceptor. CO2 and succinate were utilized as carbon sources but no organic compounds, including succinate, could be used as an energy source. The G+C content of the genomic DNA was determined to be 28.1 mol%. Phylogenetic analysis of the 16S rRNA gene sequence indicated that strain I6628T is a member of the genus Sulfurihydrogenibium, the closest cultivated relative being the recently described strain Sulfurihydrogenibium rodmanii UZ3-5T (98.2 % sequence similarity). On the basis of the physiology and phylogeny of this organism, strain I6628T represents a novel species of the genus Sulfurihydrogenibium, for which the name Sulfurihydrogenibium kristjanssonii sp. nov. is proposed. The type strain is I6628T (=DSM 19534T =OCM 901T =ATCC BAA-1535T).

Expression, purification, and characterization of a new heterotetramer structure of leucyl-tRNA synthetase from Aquifex aeolicus in Escherichia coli.

Aminoacyl-tRNA synthetases are key players in the interpretation of the genetic code. They constitute a textbook example of multi-domain proteins including insertion and terminal functional modules appended to one of the two class-specific active site domains. The non-catalytic domains usually have distinct roles in the aminoacylation reaction. Aquifex aeolicus leucyl-tRNA synthetase (LeuRS) is composed of a separated catalytic site and tRNA anticodon-binding site, which would represent one of the closest relics of the primordial aminoacyl-tRNA synthetase. Moreover, the essential catalytic site residues are split into the two different subunits. In all other class-I aminoacyl-tRNA synthetases, those two functional polypeptides are nowadays fused into a single protein chain. In this work, we report the isolation and the characterization, in Escherichia coli, of a novel oligomeric form (alphabeta)2 for A. aeolicus LeuRS, which is present in addition to the alphabeta heterodimer. A. aeolicus (alphabeta)2 LeuRS has been characterized by biochemical and biophysical methods. Native gel electrophoresis, mass spectrometry, analytical ultracentrifugation, and kinetic analysis confirmed that the (alphabeta)2 enzyme was a stable and active entity. By mass spectrometry we confirmed that the heterodimer alphabeta can bind one tRNALeu molecule whereas the heterotetramer (alphabeta)2 can bind two tRNALeu molecules. Active site titration and aminoacylation assays showed that two functional active sites are found per heterotetramer, suggesting that this molecular species might exist and be active in vivo. All those data suggest that the existence of the heterotetramer is certainly not an artifact of overexpression in E. coli.