Molecular characterization of the plant virus genus Ourmiavirus and evidence of inter-kingdom reassortment of viral genome segments as its possible route of origin.

Ourmia melon virus (OuMV), Epirus cherry virus (EpCV) and Cassava virus C (CsVC) are three species placed in the genus Ourmiavirus. We cloned and sequenced their RNA genomes. The sizes of the three genomic RNAs of OuMV, the type member of the genus, were 2814, 1064 and 974 nt and each had one open reading frame. RNA1 potentially encoded a 97.5 kDa protein carrying the GDD motif typical of RNA-dependent RNA polymerases (RdRps). The putative RdRps of ourmiaviruses are distantly related to known viral RdRps, with the closest similarity and phylogenetic affinity observed with fungal viruses of the genus Narnaviridae. RNA2 encoded a 31.6 kDa protein which, expressed in bacteria as a His-tag fusion protein and in plants through agroinfiltration, reacted specifically with antibodies made against tubular structures found in the cytoplasm. The ORF2 product is significantly similar to movement proteins of the genus Tombusviridae, and phylogenetic analysis supported this evolutionary relationship. The product of OuMV ORF3 is a 23.8 kDa protein. This protein was also expressed in bacteria and plants, and reacted specifically with antisera against the OuMV coat protein. The sequence of the ORF3 protein showed limited but significant similarity to capsid proteins of several plant and animal viruses, although phylogenetic analysis failed to reveal its most likely origin. Taken together, these results indicate that ourmiaviruses comprise a unique group of plant viruses that might have evolved by reassortment of genomic segments of RNA viruses infecting hosts belonging to different eukaryotic kingdoms, in particular, fungi and plants.

Genome of the extremely radiation-resistant bacterium Deinococcus radiodurans viewed from the perspective of comparative genomics.

The bacterium Deinococcus radiodurans shows remarkable resistance to a range of damage caused by ionizing radiation, desiccation, UV radiation, oxidizing agents, and electrophilic mutagens. D. radiodurans is best known for its extreme resistance to ionizing radiation; not only can it grow continuously in the presence of chronic radiation (6 kilorads/h), but also it can survive acute exposures to gamma radiation exceeding 1,500 kilorads without dying or undergoing induced mutation. These characteristics were the impetus for sequencing the genome of D. radiodurans and the ongoing development of its use for bioremediation of radioactive wastes. Although it is known that these multiple resistance phenotypes stem from efficient DNA repair processes, the mechanisms underlying these extraordinary repair capabilities remain poorly understood. In this work we present an extensive comparative sequence analysis of the Deinococcus genome. Deinococcus is the first representative with a completely sequenced genome from a distinct bacterial lineage of extremophiles, the Thermus-Deinococcus group. Phylogenetic tree analysis, combined with the identification of several synapomorphies between Thermus and Deinococcus, supports the hypothesis that it is an ancient group with no clear affinities to any of the other known bacterial lineages. Distinctive features of the Deinococcus genome as well as features shared with other free-living bacteria were revealed by comparison of its proteome to the collection of clusters of orthologous groups of proteins. Analysis of paralogs in Deinococcus has revealed several unique protein families. In addition, specific expansions of several other families including phosphatases, proteases, acyltransferases, and Nudix family pyrophosphohydrolases were detected. Genes that potentially affect DNA repair and recombination and stress responses were investigated in detail. Some proteins appear to have been horizontally transferred from eukaryotes and are not present in other bacteria. For example, three proteins homologous to plant desiccation resistance proteins were identified, and these are particularly interesting because of the correlation between desiccation and radiation resistance. Compared to other bacteria, the D. radiodurans genome is enriched in repetitive sequences, namely, IS-like transposons and small intergenic repeats. In combination, these observations suggest that several different biological mechanisms contribute to the multiple DNA repair-dependent phenotypes of this organism.

Origin of an animal mitochondrial DNA polymerase subunit via lineage-specific acquisition of a glycyl-tRNA synthetase from bacteria of the Thermus-Deinococcus group.

Phylogenetic tree analysis shows that the accessory subunit animal mitochondrial DNA polymerase emerges as a result of horizontal transfer of the gene encoding glycyl-tRNA synthetase from a bacterium of the Thermus-Deinococcus group into the animal nuclear genome. This acquisition by a distinct eukaryotic lineage of a gene encoding a mitochondrial protein from a nonmitochondrial bacterial source underscores the contribution of different types of horizontal transfer event to the evolution of eukaryotes.

Gene conversions in genes encoding outer-membrane proteins in H. pylori and C. pneumoniae.

Helicobacter pylori and Chlamydia pneumoniae are both pathogenic to humans. Their genomes have recently been completed, allowing detailed study of their evolution and organization. Here we describe an evolutionary analysis of the H. pylori and C. pneumoniae genes that encode their outer-membrane proteins. By comparing complete genome sequences of two H. pylori strains and two C. pneumoniae strains, we identify multiple independent conversions among these genes. Such recombination events might provide a selective advantage for these bacterial pathogens.

Improving the accuracy of PSI-BLAST protein database searches with composition-based statistics and other refinements.

PSI-BLAST is an iterative program to search a database for proteins with distant similarity to a query sequence. We investigated over a dozen modifications to the methods used in PSI-BLAST, with the goal of improving accuracy in finding true positive matches. To evaluate performance we used a set of 103 queries for which the true positives in yeast had been annotated by human experts, and a popular measure of retrieval accuracy (ROC) that can be normalized to take on values between 0 (worst) and 1 (best). The modifications we consider novel improve the ROC score from 0.758 +/- 0.005 to 0.895 +/- 0.003. This does not include the benefits from four modifications we included in the 'baseline' version, even though they were not implemented in PSI-BLAST version 2.0. The improvement in accuracy was confirmed on a small second test set. This test involved analyzing three protein families with curated lists of true positives from the non-redundant protein database. The modification that accounts for the majority of the improvement is the use, for each database sequence, of a position-specific scoring system tuned to that sequence's amino acid composition. The use of composition-based statistics is particularly beneficial for large-scale automated applications of PSI-BLAST.

Estimating the number of protein folds and families from complete genome data.

Using the data on proteins encoded in complete genomes, combined with a rigorous theory of the sampling process, we estimate the total number of protein folds and families, as well as the number of folds and families in each genome. The total number of folds in globular, water- soluble proteins is estimated at about 1000, with structural information currently available for about one-third of the number. The sequenced genomes of unicellular organisms encode from approximately 25%, for the minimal genomes of the Mycoplasmas, to 70-80% for larger genomes, such as Escherichia coli and yeast, of the total number of folds. The number of protein families with significant sequence conservation was estimated to be between 4000 and 7000, with structures available for about 20% of these.

Genome trees constructed using five different approaches suggest new major bacterial clades.

BACKGROUND: The availability of multiple complete genome sequences from diverse taxa prompts the development of new phylogenetic approaches, which attempt to incorporate information derived from comparative analysis of complete gene sets or large subsets thereof. Such attempts are particularly relevant because of the major role of horizontal gene transfer and lineage-specific gene loss, at least in the evolution of prokaryotes. RESULTS: Five largely independent approaches were employed to construct trees for completely sequenced bacterial and archaeal genomes: i) presence-absence of genomes in clusters of orthologous genes; ii) conservation of local gene order (gene pairs) among prokaryotic genomes; iii) parameters of identity distribution for probable orthologs; iv) analysis of concatenated alignments of ribosomal proteins; v) comparison of trees constructed for multiple protein families. All constructed trees support the separation of the two primary prokaryotic domains, bacteria and archaea, as well as some terminal bifurcations within the bacterial and archaeal domains. Beyond these obvious groupings, the trees made with different methods appeared to differ substantially in terms of the relative contributions of phylogenetic relationships and similarities in gene repertoires caused by similar life styles and horizontal gene transfer to the tree topology. The trees based on presence-absence of genomes in orthologous clusters and the trees based on conserved gene pairs appear to be strongly affected by gene loss and horizontal gene transfer. The trees based on identity distributions for orthologs and particularly the tree made of concatenated ribosomal protein sequences seemed to carry a stronger phylogenetic signal. The latter tree supported three potential high-level bacterial clades,: i) Chlamydia-Spirochetes, ii) Thermotogales-Aquificales (bacterial hyperthermophiles), and ii) Actinomycetes-Deinococcales-Cyanobacteria. The latter group also appeared to join the low-GC Gram-positive bacteria at a deeper tree node. These new groupings of bacteria were supported by the analysis of alternative topologies in the concatenated ribosomal protein tree using the Kishino-Hasegawa test and by a census of the topologies of 132 individual groups of orthologous proteins. Additionally, the results of this analysis put into question the sister-group relationship between the two major archaeal groups, Euryarchaeota and Crenarchaeota, and suggest instead that Euryarchaeota might be a paraphyletic group with respect to Crenarchaeota. CONCLUSIONS: We conclude that, the extensive horizontal gene flow and lineage-specific gene loss notwithstanding, extension of phylogenetic analysis to the genome scale has the potential of uncovering deep evolutionary relationships between prokaryotic lineages.

Site-directed mutagenesis of the tick-borne encephalitis virus NS3 gene reveals the putative serine protease domain of the NS3 protein.

Several mutations were introduced into the putative serine protease domain of the tick-borne encephalitis virus NS3 protein and into a possible internal cleavage site within the protein. The influence of these mutations on proteolytic activity of NS3 protein and NS3' protein formation was tested in vitro. It was found that NS3' formation was not dependent on the activity of the NS3 N-terminal serine protease. Mutations affecting the Ser-138 residue of the NS3 protein prohibited cleavage between NS2B and NS3 proteins when the NS2B-NS3 part of the viral genome was expressed in vitro, suggesting the key role of Ser-138 in viral serine protease functioning.

A new superfamily of putative NTP-binding domains encoded by genomes of small DNA and RNA viruses.

Statistically significant similarity was revealed between amino acid sequences of NTP-binding pattern-containing domains which are among the most conserved protein segments in dissimilar groups of ss and dsDNA viruses (papova-, parvo-, geminiviruses and P4 bacteriophage), and RNA viruses (picorna-, como- and nepoviruses) with small genomes. Within the aligned domains of 100-120 amino acid residues, three highly conserved sequence segments have been identified, i.e. 'A' and 'B' motifs of the NTP-binding pattern, and a third, C-terminal motif 'C', not described previously. The sequence of the 'B' motif in the proteins of the new superfamily is unusually variable, with substitutions, in some of the members, of the Asp residue conserved in other NTP-binding proteins. The 'C' motif is characterized by an invariant Asn residue preceded by a stretch of hydrophobic residues. As the new superfamily included a well studied DNA and RNA helicase, T antigen of SV40, helicase function could be tentatively assigned also to the other related viral putative NTP-binding proteins. On the other hand, the possibility of different and/or multiple functions for some of these proteins is discussed.

Winnowing sequences from a database search.

In database searches for sequence similarity, matches to a distinct sequence region (e.g., protein domain) are frequently obscured by numerous matches to another region of the same sequence. In order to cope with this problem, algorithms are developed to discard redundant matches. One model for this problem begins with a list of intervals, each with an associated score; each interval gives the range of positions in the query sequence that align to a database sequence, and the score is that of the alignment. If interval I is contained in interval J, and I's score is less than J's, then I is said to be dominated by J. The problem is then to identify each interval that is dominated by at least K other intervals, where K is a given level of "tolerable redundancy." An algorithm is developed to solve the problem in O(N log N) time and O(N*) space, where N is the number of intervals and N* is a precisely defined value that never exceeds N and is frequently much smaller. This criterion for discarding database hits has been implemented in the Blast program, as illustrated herein with examples. Several variations and extensions of this approach are also described.

Short repeats and IS elements in the extremely radiation-resistant bacterium Deinococcus radiodurans and comparison to other bacterial species.

Computer analysis of the complete genome of Deinococcus radiodurans R1 has shown that the number of insertion sequences (ISs) and small noncoding repeats (SNRs) it contains is very high, and comparable with those of Escherichia coli. IS elements and several families of SNRs are described, together with their possible function in the D. radiodurans genome.

The phylogenetic forest and the quest for the elusive tree of life.

Extensive horizontal gene transfer (HGT) among prokaryotes seems to undermine the tree of life (TOL) concept. However, the possibility remains that the TOL can be salvaged as a statistical central trend in the phylogenetic "forest of life" (FOL). A comprehensive comparative analysis of 6901 phylogenetic trees for prokaryotic genes revealed a signal of vertical inheritance that was particularly strong among the 102 nearly universal trees (NUTs), despite the high topological inconsistency among the trees in the FOL, most likely, caused by HGT. The topologies of the NUTs are similar to the topologies of numerous other trees in the FOL; although the NUTs cannot represent the FOL completely, they reflect a significant central trend. Thus, the original TOL concept becomes obsolete but the idea of a "weak" TOL as the dominant trend in the FOL merits further investigation. The totality of gene trees comprising the FOL appears to be a natural representation of the history of life given the inherent tree-like character of the replication process.

From complete genomes to measures of substitution rate variability within and between proteins.

Accumulation of complete genome sequences of diverse organisms creates new possibilities for evolutionary inferences from whole-genome comparisons. In the present study, we analyze the distributions of substitution rates among proteins encoded in 19 complete genomes (the interprotein rate distribution). To estimate these rates, it is necessary to employ another fundamental distribution, that of the substitution rates among sites in proteins (the intraprotein distribution). Using two independent approaches, we show that intraprotein substitution rate variability appears to be significantly greater than generally accepted. This yields more realistic estimates of evolutionary distances from amino-acid sequences, which is critical for evolutionary-tree construction. We demonstrate that the interprotein rate distributions inferred from the genome-to-genome comparisons are similar to each other and can be approximated by a single distribution with a long exponential shoulder. This suggests that a generalized version of the molecular clock hypothesis may be valid on genome scale. We also use the scaling parameter of the obtained interprotein rate distribution to construct a rooted whole-genome phylogeny. The topology of the resulting tree is largely compatible with those of global rRNA-based trees and trees produced by other approaches to genome-wide comparison.

Interkingdom gene fusions.

BACKGROUND: Genome comparisons have revealed major lateral gene transfer between the three primary kingdoms of life - Bacteria, Archaea, and Eukarya. Another important evolutionary phenomenon involves the evolutionary mobility of protein domains that form versatile multidomain architectures. We were interested in investigating the possibility of a combination of these phenomena, with an invading gene merging with a pre-existing gene in the recipient genome. RESULTS: Complete genomes of fifteen bacteria, four archaea and one eukaryote were searched for interkingdom gene fusions (IKFs); that is, genes coding for proteins that apparently consist of domains originating from different primary kingdoms. Phylogenetic analysis supported 37 cases of IKF, each of which includes a 'native' domain and a horizontally acquired 'alien' domain. IKFs could have evolved via lateral transfer of a gene coding for the alien domain (or a larger protein containing this domain) followed by recombination with a native gene. For several IKFs, this scenario is supported by the presence of a gene coding for a second, stand-alone version of the alien domain in the recipient genome. Among the genomes investigated, the greatest number of IKFs has been detected in Mycobacterium tuberculosis, where they are almost always accompanied by a stand-alone alien domain. For most of the IKF cases detected in other genomes, the stand-alone counterpart is missing. CONCLUSIONS: The results of comparative genome analysis show that IKF formation is a real, but relatively rare, evolutionary phenomenon. We hypothesize that IKFs are formed primarily via the proposed two-stage mechanism, but other than in the Actinomycetes, in which IKF generation seems to be an active, ongoing process, most of the stand-alone intermediates have been eliminated, perhaps because of functional redundancy.

Prediction of the archaeal exosome and its connections with the proteasome and the translation and transcription machineries by a comparative-genomic approach.

By comparing the gene order in the completely sequenced archaeal genomes complemented by sequence profile analysis, we predict the existence and protein composition of the archaeal counterpart of the eukaryotic exosome, a complex of RNAses, RNA-binding proteins, and helicases that mediates processing and 3'->5' degradation of a variety of RNA species. The majority of the predicted archaeal exosome subunits are encoded in what appears to be a previously undetected superoperon. In Methanobacterium thermoautotrophicum, this predicted superoperon consists of 15 genes; in the Crenarchaea, Sulfolobus solfataricus and Aeropyrum pernix, one and two of the genes from the superoperon, respectively, are relocated in the genome, whereas in other Euryarchaeota, the superoperon is split into a variable number of predicted operons and solitary genes. Methanococcus jannaschii partially retains the superoperon, but lacks the three core exosome subunits, and in Halobacterium sp., the superoperon is divided into two predicted operons, with the same three exosome subunits missing. This suggests concerted gene loss and an alteration of the structure and function of the predicted exosome in the Methanococcus and Halobacterium lineages. Additional potential components of the exosome are encoded by partially conserved predicted small operons. Along with the orthologs of eukaryotic exosome subunits, namely an RNase PH and two RNA-binding proteins, the predicted archaeal exosomal superoperon also encodes orthologs of two protein subunits of RNase P. This suggests a functional and possibly a physical interaction between RNase P and the postulated archaeal exosome, a connection that has not been reported in eukaryotes. In a pattern of apparent gene loss complementary to that seen in Methanococcus and Halobacterium, Thermoplasma acidophilum lacks the RNase P subunits. Unexpectedly, the identified exosomal superoperon, in addition to the predicted exosome components, encodes the catalytic subunits of the archaeal proteasome, two ribosomal proteins and a DNA-directed RNA polymerase subunit. These observations suggest that in archaea, a tight functional coupling exists between translation, RNA processing and degradation, (apparently mediated by the predicted exosome) and protein degradation (mediated by the proteasome), and may have implications for cross-talk between these processes in eukaryotes.