Post-transcriptional modification in archaeal tRNAs: identities and phylogenetic relations of nucleotides from mesophilic and hyperthermophilic Methanococcales.

Post-transcriptional modifications in archaeal RNA are known to be phylogenetically distinct but relatively little is known of tRNA from the Methanococci, a lineage of methanogenic marine euryarchaea that grow over an unusually broad temperature range. Transfer RNAs from Methanococcus vannielii, Methanococcus maripaludis, the thermophile Methanococcus thermolithotrophicus, and hyperthermophiles Methanococcus jannaschii and Methanococcus igneus were studied to determine whether modification patterns reflect the close phylogenetic relationships inferred from small ribosomal subunit RNA sequences, and to examine modification differences associated with temperature of growth. Twenty-four modified nucleosides were characterized, including the complex tricyclic nucleoside wyosine characteristic of position 37 in tRNA(Phe) and known previously only in eukarya, plus two new wye family members of presently unknown structure. The hypermodified nucleoside 5-methylaminomethyl-2-thiouridine, reported previously only in bacterial tRNA at the first position of the anticodon, was identified by liquid chromatography-electrospray ionization mass spectrometry in four of the five organisms. The ribose-methylated nucleosides, 2'-O-methyladenosine, N(2),2'-O-dimethylguanosine and N(2),N(2),2'-O-trimethylguanosine, were found only in hyperthermophile tRNA, consistent with their proposed roles in thermal stabilization of tRNA.

Structural modifications of Methanococcus jannaschii flagellin proteins revealed by proteome analysis.

Methanococcus jannaschii is an autotrophic archaeon originally isolated from an oceanic thermal vent. The primary metabolic pathway for energy production in this hyperthermophilic microbe is methanogenesis from H2 and CO2. As an autotroph, M. jannaschii requires only CO2 as a carbon source for synthesizing all necessary biomolecules. Changes in the environmental availability of these molecules can be expected to activate regulatory mechanisms manifested as the up and down regulation of specific genes and the concomitant increase and decrease in abundance of the corresponding proteins. In our analysis of the proteome of M. jannaschii, we have observed significant changes in the abundance of a common subset of predominant proteins in response to reduced H2 concentration, limited ammonium availability, and the stage of cell growth (exponential compared with stationary). The masses of tryptic peptides from these proteins match those predicted by M. jannaschii genome open reading frames annotated as flagellin B1 (MJ0891) and flagellin B2 (MJ0892). Multiple proteins with different isoelectric points and molecular weights match each of these proteins, and the abundance of these protein variants changes with growth conditions. These data indicate that structural modifications altering both the isoelectric point and size of the M. jannaschii flagellin B1 and B2 proteins occur in response to growth conditions and growth stage of M. jannaschii and further suggest the regulation of M. jannaschii motility through structural modifications of the building blocks of the flagella.

Archaeal RecA homologues: different response to DNA-damaging agents in mesophilic and thermophilic Archaea.

Two archaeal proteins, RadA and RadB, share similarity with the RecA/Rad51 family of recombinases, with RadA being the functional homologue. We have studied and compared the RadA and RadB proteins of mesophilic and thermophilic Archaea. In growing cells, RadA levels are similar in mesophilic Methanococcus species and the hyperthermophile Methanococcus jannaschii. Treatment of cells with mutagenic agents (methylmethane sulfonate or UV light) increased the expression of RadA (as evidenced by higher levels of both mRNA and protein) in all organisms tested, but the increase was greater in the mesophiles than in the thermophiles M. jannaschii and Sulfolobus solfataricus. Recombinantly expressed RadA proteins from the mesophile M. voltae and the thermophile M. jannaschii were similar in their ATPase- and DNA-binding activities. All the data are consistent with proposals that RadA plays the same role as eukaryotic Rad51. Surprisingly, the data also suggested that the thermophiles do not need more RadA protein or activity than the mesophiles. On the other hand, RadB is not coregulated with RadA, and its role remains unclear. Neither RadA nor RadB from a mesophile or from a thermophile rescued the UV-sensitive phenotype of an Escherichia coli recA- host.

Similar subunit architecture of archaeal and eukaryal RNA polymerases.

Protein interactions among RNA polymerase small subunits from the archaeon Methanococcus jannaschii were investigated using affinity pulldown assays in pairwise and higher-order combinations. In the most extensive study of archaeal RNA polymerase subunit interactions to date, including 37 pairs of proteins, 10 ternary combinations, and three quaternary combinations, we found evidence for pairwise interactions of subunit D with subunits L and N, and a ternary complex of subunits D, L and N. No other small subunit interactions occurred. These results are consistent with interactions observed in a crystal structure of eukaryotic RNA polymerase II and support a common archaeal/eukaryal RNA polymerase architecture. We further propose that subunit E" is not an integral member of archaeal RNA polymerases. Finally, we discuss the relative accuracy of the various methods that have been used to predict protein-protein interactions in RNA polymerase.

The RDP-II (Ribosomal Database Project).

The Ribosomal Database Project (RDP-II), previously described by Maidak et al. [Nucleic Acids Res. (2000), 28, 173-174], continued during the past year to add new rRNA sequences to the aligned data and to improve the analysis commands. Release 8.0 (June 1, 2000) consisted of 16 277 aligned prokaryotic small subunit (SSU) rRNA sequences while the number of eukaryotic and mitochondrial SSU rRNA sequences in aligned form remained at 2055 and 1503, respectively. The number of prokaryotic SSU rRNA sequences more than doubled from the previous release 14 months earlier, and approximately 75% are longer than 899 bp. An RDP-II mirror site in Japan is now available (http://wdcm.nig.ac.jp/RDP/html/index.h tml). RDP-II provides aligned and annotated rRNA sequences, derived phylogenetic trees and taxonomic hierarchies, and analysis services through its WWW server (http://rdp.cme.msu.edu/). Analysis services include rRNA probe checking, approximate phylogenetic placement of user sequences, screening user sequences for possible chimeric rRNA sequences, automated alignment, production of similarity matrices and services to plan and analyze terminal restriction fragment polymorphism experiments. The RDP-II email address for questions and comments has been changed from curator@cme.msu.edu to rdpstaff@msu.edu.

Aminoacyl-tRNA synthetases, the genetic code, and the evolutionary process.

The aminoacyl-tRNA synthetases (AARSs) and their relationship to the genetic code are examined from the evolutionary perspective. Despite a loose correlation between codon assignments and AARS evolutionary relationships, the code is far too highly structured to have been ordered merely through the evolutionary wanderings of these enzymes. Nevertheless, the AARSs are very informative about the evolutionary process. Examination of the phylogenetic trees for each of the AARSs reveals the following. (i) Their evolutionary relationships mostly conform to established organismal phylogeny: a strong distinction exists between bacterial- and archaeal-type AARSs. (ii) Although the evolutionary profiles of the individual AARSs might be expected to be similar in general respects, they are not. It is argued that these differences in profiles reflect the stages in the evolutionary process when the taxonomic distributions of the individual AARSs became fixed, not the nature of the individual enzymes. (iii) Horizontal transfer of AARS genes between Bacteria and Archaea is asymmetric: transfer of archaeal AARSs to the Bacteria is more prevalent than the reverse, which is seen only for the "gemini group. " (iv) The most far-ranging transfers of AARS genes have tended to occur in the distant evolutionary past, before or during formation of the primary organismal domains. These findings are also used to refine the theory that at the evolutionary stage represented by the root of the universal phylogenetic tree, cells were far more primitive than their modern counterparts and thus exchanged genetic material in far less restricted ways, in effect evolving in a communal sense.

An archaeal genomic signature.

Comparisons of complete genome sequences allow the most objective and comprehensive descriptions possible of a lineage's evolution. This communication uses the completed genomes from four major euryarchaeal taxa to define a genomic signature for the Euryarchaeota and, by extension, the Archaea as a whole. The signature is defined in terms of the set of protein-encoding genes found in at least two diverse members of the euryarchaeal taxa that function uniquely within the Archaea; most signature proteins have no recognizable bacterial or eukaryal homologs. By this definition, 351 clusters of signature proteins have been identified. Functions of most proteins in this signature set are currently unknown. At least 70% of the clusters that contain proteins from all the euryarchaeal genomes also have crenarchaeal homologs. This conservative set, which appears refractory to horizontal gene transfer to the Bacteria or the Eukarya, would seem to reflect the significant innovations that were unique and fundamental to the archaeal "design fabric." Genomic protein signature analysis methods may be extended to characterize the evolution of any phylogenetically defined lineage. The complete set of protein clusters for the archaeal genomic signature is presented as supplementary material (see the PNAS web site, www.pnas.org).

The RDP (Ribosomal Database Project) continues.

The Ribosomal Database Project (RDP-II), previously described by Maidak et al., continued during the past year to add new rRNA sequences to the aligned data and to improve the analysis commands. Release 7.1 (September 17, 1999) included more than 10 700 small subunit rRNA sequences. More than 850 type strain sequences were identified and added to the prokaryotic alignment, bringing the total number of type sequences to 3324 representing 2460 different species. Availability of an RDP-II mirror site in Japan is also near completion. RDP-II provides aligned and annotated rRNA sequences, derived phylogenetic trees and taxonomic hierarchies, and analysis services through its WWW server (http://rdp.cme.msu.edu/ ). Analysis services include rRNA probe checking, approx-i-mate phylogenetic placement of user sequences, screening user sequences for possible chimeric rRNA sequences, automated alignment, production of similarity matrices and services to plan and analyze terminal restriction fragment length polymorphism (T-RFLP) experiments.

CRITICA: coding region identification tool invoking comparative analysis.

Gene recognition is essential to understanding existing and future DNA sequence data. CRITICA (Coding Region Identification Tool Invoking Comparative Analysis) is a suite of programs for identifying likely protein-coding sequences in DNA by combining comparative analysis of DNA sequences with more common noncomparative methods. In the comparative component of the analysis, regions of DNA are aligned with related sequences from the DNA databases; if the translation of the aligned sequences has greater amino acid identity than expected for the observed percentage nucleotide identity, this is interpreted as evidence for coding. CRITICA also incorporates noncomparative information derived from the relative frequencies of hexanucleotides in coding frames versus other contexts (i.e., dicodon bias). The dicodon usage information is derived by iterative analysis of the data, such that CRITICA is not dependent on the existence or accuracy of coding sequence annotations in the databases. This independence makes the method particularly well suited for the analysis of novel genomes. CRITICA was tested by analyzing the available Salmonella typhimurium DNA sequences. Its predictions were compared with the DNA sequence annotations and with the predictions of GenMark. CRITICA proved to be more accurate than GenMark, and moreover, many of its predictions that would seem to be errors instead reflect problems in the sequence databases. The source code of CRITICA is freely available by anonymous FTP (rdp.life.uiuc.edu in/pub/critica) and on the World Wide Web (http:/(/)rdpwww.life.uiuc.edu).

Thermal adaptation analyzed by comparison of protein sequences from mesophilic and extremely thermophilic Methanococcus species.

The genome sequence of the extremely thermophilic archaeon Methanococcus jannaschii provides a wealth of data on proteins from a thermophile. In this paper, sequences of 115 proteins from M. jannaschii are compared with their homologs from mesophilic Methanococcus species. Although the growth temperatures of the mesophiles are about 50 degrees C below that of M. jannaschii, their genomic G+C contents are nearly identical. The properties most correlated with the proteins of the thermophile include higher residue volume, higher residue hydrophobicity, more charged amino acids (especially Glu, Arg, and Lys), and fewer uncharged polar residues (Ser, Thr, Asn, and Gln). These are recurring themes, with all trends applying to 83-92% of the proteins for which complete sequences were available. Nearly all of the amino acid replacements most significantly correlated with the temperature change are the same relatively conservative changes observed in all proteins, but in the case of the mesophile/thermophile comparison there is a directional bias. We identify 26 specific pairs of amino acids with a statistically significant (P < 0.01) preferred direction of replacement.

A new version of the RDP (Ribosomal Database Project).

The Ribosomal Database Project (RDP-II), previously described by Maidak et al. [ Nucleic Acids Res. (1997), 25, 109-111], is now hosted by the Center for Microbial Ecology at Michigan State University. RDP-II is a curated database that offers ribosomal RNA (rRNA) nucleotide sequence data in aligned and unaligned forms, analysis services, and associated computer programs. During the past two years, data alignments have been updated and now include >9700 small subunit rRNA sequences. The recent development of an ObjectStore database will provide more rapid updating of data, better data accuracy and increased user access. RDP-II includes phylogenetically ordered alignments of rRNA sequences, derived phylogenetic trees, rRNA secondary structure diagrams, and various software programs for handling, analyzing and displaying alignments and trees. The data are available via anonymous ftp (ftp.cme.msu. edu) and WWW (http://www.cme.msu.edu/RDP). The WWW server provides ribosomal probe checking, approximate phylogenetic placement of user-submitted sequences, screening for possible chimeric rRNA sequences, automated alignment, and a suggested placement of an unknown sequence on an existing phylogenetic tree. Additional utilities also exist at RDP-II, including distance matrix, T-RFLP, and a Java-based viewer of the phylogenetic trees that can be used to create subtrees.

The complete genome of the hyperthermophilic bacterium Aquifex aeolicus.

Aquifex aeolicus was one of the earliest diverging, and is one of the most thermophilic, bacteria known. It can grow on hydrogen, oxygen, carbon dioxide, and mineral salts. The complex metabolic machinery needed for A. aeolicus to function as a chemolithoautotroph (an organism which uses an inorganic carbon source for biosynthesis and an inorganic chemical energy source) is encoded within a genome that is only one-third the size of the E. coli genome. Metabolic flexibility seems to be reduced as a result of the limited genome size. The use of oxygen (albeit at very low concentrations) as an electron acceptor is allowed by the presence of a complex respiratory apparatus. Although this organism grows at 95 degrees C, the extreme thermal limit of the Bacteria, only a few specific indications of thermophily are apparent from the genome. Here we describe the complete genome sequence of 1,551,335 base pairs of this evolutionarily and physiologically interesting organism.

The complete genome sequence of the hyperthermophilic, sulphate-reducing archaeon Archaeoglobus fulgidus.

Archaeoglobus fulgidus is the first sulphur-metabolizing organism to have its genome sequence determined. Its genome of 2,178,400 base pairs contains 2,436 open reading frames (ORFs). The information processing systems and the biosynthetic pathways for essential components (nucleotides, amino acids and cofactors) have extensive correlation with their counterparts in the archaeon Methanococcus jannaschii. The genomes of these two Archaea indicate dramatic differences in the way these organisms sense their environment, perform regulatory and transport functions, and gain energy. In contrast to M. jannaschii, A. fulgidus has fewer restriction-modification systems, and none of its genes appears to contain inteins. A quarter (651 ORFs) of the A. fulgidus genome encodes functionally uncharacterized yet conserved proteins, two-thirds of which are shared with M. jannaschii (428 ORFs). Another quarter of the genome encodes new proteins indicating substantial archaeal gene diversity.

A reconstruction of the metabolism of Methanococcus jannaschii from sequence data.

The interpretation of the Methanococcus jannaschii genome will inevitably require many years of effort. This initial attempt to connect the sequence data to aspects of known biochemistry and to provide an overview of what is already apparent from the sequence data will be refined. Numerous issues remain that can be resolved only by direct biochemical analysis. Let us draw the reader's attention to just a few that might be considered central: (1) We are still missing key enzymes from the glycolytic pathway, and the conjecture is that this is due to ADP-dependency. The existence of glycolytic activity in the cell-free extract should be tested. (2) The issue of whether the Calvin cycle is present needs to be examined. (3) We need to determine whether the 2-oxoglutarate synthase (ferredoxin-dependent) (EC 1.2.7.3) activity is present. (4) The issue of whether cyclic 2,3-bisphosphate is detectable in the cell-free extracts needs to be checked. If it is, this result would confirm our assertion of the two pathways controlling synthesis and degradation of cyclic 2,3-bisphosphate.

Compilation and analysis of intein sequences.

We have compiled a list of all the inteins (protein splicing elements) whose sequences have been published or were available from on-line sequence databases as of September 18, 1996. Analysis of the 36 available intein sequences refines the previously described intein motifs and reveals the presence of another intein motif, Block H. Furthermore, analysis of the new inteins reshapes our view of the conserved splice junction residues, since three inteins lack the intein penultimate His seen in prior examples. Comparison of intein sequences suggests that, in general, (i) inteins present in the same location within extein homologs from different organisms are very closely related to each other in paired sequence comparison or phylogenetic analysis and we suggest that they should be considered intein alleles; (ii) multiple inteins present in the same gene are no more similar to each other than to inteins present in different genes; (iii) phylogenetic analysis indicates that inteins are so divergent that trees with statistically significant branches cannot be generated except for intein alleles.

The RDP (Ribosomal Database Project).

The Ribosomal Database Project (RDP) is a curated database that offers ribosome-related data, analysis services and associated computer programs. The offerings include phylogenetically ordered alignments of ribosomal RNA (rRNA) sequences, derived phylogenetic trees, rRNA secondary structure diagrams, and various software for handling, analyzing and displaying alignments and trees. The data are available via anonymous FTP (rdp.life.uiuc.edu), electronic mail (server@rdp.life.uiuc.edu), gopher (rdpgopher.life.uiuc.edu) and WWW (http://rdpwww.life.uiuc.edu/ ). The electronic mail and WWW servers provide ribosomal probe checking, approximate phylogenetic placement of user-submitted sequences, screening for possible chimeric rRNA sequences, automated alignment, and a suggested placement of an unknown sequence on an existing phylogenetic tree.