Domain-specific recruitment of amide amino acids for protein synthesis.

The formation of aminoacyl-transfer RNA is a crucial step in ensuring the accuracy of protein synthesis. Despite the central importance of this process in all living organisms, it remains unknown how archaea and some bacteria synthesize Asn-tRNA and Gln-tRNA. These amide aminoacyl-tRNAs can be formed by the direct acylation of tRNA, catalysed by asparaginyl-tRNA synthetase and glutaminyl-tRNA synthetase, respectively. A separate, indirect pathway involves the formation of mis-acylated Asp-tRNA(Asn) or Glu-tRNA(Gln), and the subsequent amidation of these amino acids while they are bound to tRNA, which is catalysed by amidotransferases. Here we show that all archaea possess an archaea-specific heterodimeric amidotransferase (encoded by gatD and gatE) for Gln-tRNA formation. However, Asn-tRNA synthesis in archaea is divergent: some archaea use asparaginyl-tRNA synthetase, whereas others use a heterotrimeric amidotransferase (encoded by the gatA, gatB and gatC genes). Because bacteria primarily use transamidation, and the eukaryal cytoplasm uses glutaminyl-tRNA synthetase, it appears that the three domains use different mechanisms for Gln-tRNA synthesis; as such, this is the only known step in protein synthesis where all three domains have diverged. Closer inspection of the two amidotransferases reveals that each of them recruited a metabolic enzyme to aid its function; this provides direct evidence for a relationship between amino-acid metabolism and protein biosynthesis.

The adaptor hypothesis revisited.

As originally postulated in Crick's Adaptor hypothesis, the faithful synthesis of proteins from messenger RNA is dependent on the presence of perfectly acylated tRNAs. The hypothesis also suggested that each aminoacyl-tRNA would be made by a unique enzyme. Recent data have now forced a revision of this latter point, with an increasingly diverse array of enzymes and pathways being implicated in aminoacyl-tRNA synthesis. These unexpected findings have far-reaching implications for our understanding of protein synthesis and its origins.

Cysteine biosynthesis pathway in the archaeon Methanosarcina barkeri encoded by acquired bacterial genes?

The pathway of cysteine biosynthesis in archaea is still unexplored. Complementation of a cysteine auxotrophic Escherichia coli strain NK3 led to the isolation of the Methanosarcina barkeri cysK gene [encoding O-acetylserine (thiol)-lyase-A], which displays great similarity to bacterial cysK genes. Adjacent to cysK is an open reading frame orthologous to bacterial cysE (serine transacetylase) genes. These two genes could account for cysteine biosynthesis in this archaeon. Analysis of recent genome data revealed the presence of bacteria-like cysM genes [encoding O-acetylserine (thiol)-lyase-B] in Pyrococcus spp., Sulfolobus solfataricus, and Thermoplasma acidophilum. However, no orthologs for these genes can be found in Methanococcus jannaschii, Methanobacterium thermoautotrophicum, and Archaeoglobus fulgidus, implying the existence of unrecognizable genes for the same function or a different cysteine biosynthesis pathway.

Archaeal aminoacyl-tRNA synthesis: diversity replaces dogma.

Accurate aminoacyl-tRNA synthesis is essential for faithful translation of the genetic code and consequently has been intensively studied for over three decades. Until recently, the study of aminoacyl-tRNA synthesis in archaea had received little attention. However, as in so many areas of molecular biology, the advent of archaeal genome sequencing has now drawn researchers to this field. Investigations with archaea have already led to the discovery of novel pathways and enzymes for the synthesis of numerous aminoacyl-tRNAs. The most surprising of these findings has been a transamidation pathway for the synthesis of asparaginyl-tRNA and a novel lysyl-tRNA synthetase. In addition, seryl- and phenylalanyl-tRNA synthetases that are only marginally related to known examples outside the archaea have been characterized, and the mechanism of cysteinyl-tRNA formation in Methanococcus jannaschii and Methanobacterium thermoautotrophicum is still unknown. These results have revealed completely unexpected levels of complexity and diversity, questioning the notion that aminoacyl-tRNA synthesis is one of the most conserved functions in gene expression. It has now become clear that the distribution of the various mechanisms of aminoacyl-tRNA synthesis in extant organisms has been determined by numerous gene transfer events, indicating that, while the process of protein biosynthesis is orthologous, its constituents are not.

Genetics of Methanococcus: possibilities for functional genomics in Archaea.

Although the genomic sequences of a number of Archaea have been completed in the last three years, genetic systems in the sequenced organisms are absent. In contrast, genetic studies of the mesophiles in the archaeal genus Methanococcus have become commonplace following the recent developments of antibiotic resistance markers, DNA transformation methods, reporter genes, shuttle vectors and expression vectors. These developments have led to investigations of the transcription of the genes for hydrogen metabolism, nitrogen fixation and flagellin assembly. These genetic systems can potentially be used to analyse the genomic sequence of the hyperthermophile Methanococcus jannaschii, addressing questions of its physiology and the function of its many uncharacterized open reading frames. Thus, the sequence of M. jannaschii can serve as a starting point for gene isolation, while in vivo genetics in the mesophilic methanococci can provide the experimental systems to test the predictions from genomics.

Glutamyl-tRNA(Gln) amidotransferase in Deinococcus radiodurans may be confined to asparagine biosynthesis.

Asparaginyl-tRNA (Asn-tRNA) and glutaminyl-tRNA (Gln-tRNA) are essential components of protein synthesis. They can be formed by direct acylation by asparaginyl-tRNA synthetase (AsnRS) or glutaminyl-tRNA synthetase (GlnRS). The alternative route involves transamidation of incorrectly charged tRNA. Examination of the preliminary genomic sequence of the radiation-resistant bacterium Deinococcus radiodurans suggests the presence of both direct and indirect routes of Asn-tRNA and Gln-tRNA formation. Biochemical experiments demonstrate the presence of AsnRS and GlnRS, as well as glutamyl-tRNA synthetase (GluRS), a discriminating and a nondiscriminating aspartyl-tRNA synthetase (AspRS). Moreover, both Gln-tRNA and Asn-tRNA transamidation activities are present. Surprisingly, they are catalyzed by a single enzyme encoded by three ORFs orthologous to Bacillus subtilis gatCAB. However, the transamidation route to Gln-tRNA formation is idled by the inability of the discriminating D. radiodurans GluRS to produce the required mischarged Glu-tRNAGln substrate. The presence of apparently redundant complete routes to Asn-tRNA formation, combined with the absence from the D. radiodurans genome of genes encoding tRNA-independent asparagine synthetase and the lack of this enzyme in D. radiodurans extracts, suggests that the gatCAB genes may be responsible for biosynthesis of asparagine in this asparagine prototroph.

Ribose biosynthesis and evidence for an alternative first step in the common aromatic amino acid pathway in Methanococcus maripaludis.

An acetate-requiring mutant of Methanococcus maripaludis allowed efficient labeling of riboses following growth in minimal medium supplemented with [2-(13)C]acetate. Nuclear magnetic resonance and mass spectroscopic analysis of purified cytidine and uridine demonstrated that the C-1' of the ribose was about 67% enriched for 13C. This value was inconsistent with the formation of erythrose 4-phosphate (E4P) exclusively by the carboxylation of a triose. Instead, these results suggest that either (i) E4P is formed by both the nonoxidative pentose phosphate and triose carboxylation pathways or (ii) E4P is formed exclusively by the nonoxidative pentose phosphate pathway and is not a precursor of aromatic amino acids.

Characterization of pURB500 from the archaeon Methanococcus maripaludis and construction of a shuttle vector.

The complete sequence of the 8,285-bp plasmid pURB500 from Methanococcus maripaludis C5 was determined. Sequence analysis identified 18 open reading frames as well as two regions of potential iterons and complex secondary structures. The shuttle vector, pDLT44, for M. maripaludis JJ was constructed from the entire pURB500 plasmid and pMEB.2, an Escherichia coli vector containing a methanococcal puromycin-resistance marker (P. Gernhardt, O. Possot, M. Foglino, L. Sibold, and A. Klein, Mol. Gen. Genet. 221:273-279, 1990). By using polyethylene glycol transformation, M. maripaludis JJ was transformed at a frequency of 3.3 x 10(7) transformants per microg of pDLT44. The shuttle vector was stable in E. coli under ampicillin selection but was maintained at a lower copy number than pMEB.2. Based on the inability of various restriction fragments of pURB500 to support maintenance in M. maripaludis JJ, multiple regions of pURB500 were required. pDLT44 did not replicate in Methanococcus voltae.

Cloning and phylogenetic analysis of the genes encoding acetohydroxyacid synthase from the archaeon Methanococcus aeolicus.

The gene for acetohydroxyacid synthase (AHAS) was cloned from the archaeon Methanococcus aeolicus. Contrary to biochemical studies [Xing, R. and Whitman, W.B. (1994) J. Bacteriol. 176, 1207-1213] the enzyme was encoded by two open reading frames (ORFs). Based on sequence homology, these ORFs were designated ilvB and ilvN for the large and small subunits of AHAS, respectively. A putative methanogen promoter preceded ilvB-ilvN, and a potential internal promoter was found upstream of ilvN. ilvB encoded a 65-kDa protein, which agreed well with the measured value for the purified enzyme. ilvN encoded a 19-kDa protein, which fell within the range of M(r) of small subunits from other sources. Phylogenetic analysis of the deduced amino acid sequence of ilvB showed a close relationship between the AHAS of Bacteria and Archaea, to the exclusion of other enzymes in this family, including pyruvate oxidase, glyoxylate carboligase, pyruvate decarboxylase, and the acetolactate synthase found in fermentative Bacteria. Thus, this family of enzymes probably arose prior to the divergence of the Bacteria and Archaea. Moreover, the higher plant AHAS and the red algal AHAS were related to the AHAS II of Escherichia coli and the cyanobacterial AHAS, respectively. For this reason, these genes appear to have been acquired by the Eucarya during the endosymbiosis that gave rise to the mitochondrion and chloroplast, respectively. One of the ORFs in the Methanococcus jannaschii genome possesses high similarity to the M. aeolicus ilvB, indicating that it is an authentic AHAS.

Development of genetic approaches for the methane-producing archaebacterium Methanococcus maripaludis.

Methanococcus maripaludis is a strict anaerobe that utilizes H2 or formate as an electron donor for CO2 reduction to methane. Recent progress in development of genetic systems in this archaebacterium makes it an excellent model system for molecular and biochemical studies. This progress includes development of methods for growth on solid medium, enriching auxotrophic mutants, efficient transformation, and random insertional inactivation of genes. Genetic markers for both puromycin and neomycin resistance are available. Lastly, a shuttle vector has been constructed from a cryptic methanococcal plasmid. These technical advances made it possible to utilize genetic approaches for the study of autotrophic CO2 assimilation in methanococci.

Aminoacyl-tRNA synthesis in Archaea.

The mechanism of aminoacyl-tRNA synthesis differs substantially between Archaea, Bacteria and Eukarya. Sequencing of archaeal genomes has suggested that the asparaginyl-, cysteinyl-, glutaminyl- and lysyl-tRNA synthetases are absent from a number of organisms in this kingdom. The absence of the asparaginyl- and glutaminyl-tRNA synthetases is in agreement with the observation that Asn-tRNA and Gln-tRNA are synthesized by tRNA-dependent transamidation of Asp-tRNA and Glu-tRNA respectively in the archaeon Haloferax volcanii. Biochemical and genetic studies have now shown that while the cysteinyl- and lysyl-tRNA synthetases are present, the enzymes responsible for these activities are unique to Archaea.

Neomycin resistance as a selectable marker in Methanococcus maripaludis.

We cloned the aminoglycoside phosphotransferase genes APH3'I and APH3'II between the Methanococcus voltae methyl reductase promoter and terminator in a plasmid containing a fragment of Methanococcus maripaludis chromosomal DNA. The resulting plasmids encoding neomycin resistance transformed M. maripaludis at frequencies similar to those observed for pKAS102 encoding puromycin resistance. The antibiotic geneticin was not inhibitory to M. maripaludis.