Eukaryotic signalling domain homologues in archaea and bacteria. Ancient ancestry and horizontal gene transfer.

Phyletic distributions of eukaryotic signalling domains were studied using recently developed sensitive methods for protein sequence analysis, with an emphasis on the detection and accurate enumeration of homologues in bacteria and archaea. A major difference was found between the distributions of enzyme families that are typically found in all three divisions of cellular life and non-enzymatic domain families that are usually eukaryote-specific. Previously undetected bacterial homologues were identified for# plant pathogenesis-related proteins, Pad1, von Willebrand factor type A, src homology 3 and YWTD repeat-containing domains. Comparisons of the domain distributions in eukaryotes and prokaryotes enabled distinctions to be made between the domains originating prior to the last common ancestor of all known life forms and those apparently originating as consequences of horizontal gene transfer events. A number of transfers of signalling domains from eukaryotes to bacteria were confidently identified, in contrast to only a single case of apparent transfer from eukaryotes to archaea.     

Origin and evolution of eukaryotic apoptosis: the bacterial connection

The availability of numerous complete genome sequences of prokaryotes and several eukaryotic genome sequences provides for new insights into the origin of unique functional systems of the eukaryotes. Several key enzymes of the apoptotic machinery, including the paracaspase and metacaspase families of the caspase-like protease superfamily, apoptotic ATPases and NACHT family NTPases, and mitochondrial HtrA-like proteases, have diverse homologs in bacteria, but not in archaea. Phylogenetic analysis strongly suggests a mitochondrial origin for metacaspases and the HtrA-like proteases, whereas acquisition from Actinomycetes appears to be the most likely scenario for AP-ATPases. The homologs of apoptotic proteins are particularly abundant and diverse in bacteria that undergo complex development, such as Actinomycetes, Cyanobacteria and alpha-proteobacteria, the latter being progenitors of the mitochondria. In these bacteria, the apoptosis-related domains typically form multidomain proteins, which are known or inferred to participate in signal transduction and regulation of gene expression. Some of these bacterial multidomain proteins contain fusions between apoptosis-related domains, such as AP-ATPase fused with a metacaspase or a TIR domain. Thus, bacterial homologs of eukaryotic apoptotic machinery components might functionally and physically interact with each other as parts of signaling pathways that remain to be investigated. An emerging scenario of the origin of the eukaryotic apoptotic system involves acquisition of several central apoptotic effectors as a consequence of mitochondrial endosymbiosis and probably also as a result of subsequent, additional horizontal gene transfer events, which was followed by recruitment of newly emerging eukaryotic domains as adaptors.     

Cryo-EM structure of the archaeal 50S ribosomal subunit in complex with initiation factor 6 and implications for ribosome evolution

Translation of mRNA into proteins by the ribosome is universally conserved in all cellular life. The composition and complexity of the translation machinery differ markedly between the three domains of life. Organisms from the domain Archaea show an intermediate level of complexity, sharing several additional components of the translation machinery with eukaryotes that are absent in bacteria. One of these translation factors is initiation factor 6 (IF6), which associates with the large ribosomal subunit. We have reconstructed the 50S ribosomal subunit from the archaeon Methanothermobacter thermautotrophicus in complex with archaeal IF6 at 6.6?? resolution using cryo-electron microscopy (EM). The structure provides detailed architectural insights into the 50S ribosomal subunit from a methanogenic archaeon through identification of the rRNA expansion segments and ribosomal proteins that are shared between this archaeal ribosome and eukaryotic ribosomes but are mostly absent in bacteria and in some archaeal lineages. Furthermore, the structure reveals that, in spite of highly divergent evolutionary trajectories of the ribosomal particle and the acquisition of novel functions of IF6 in eukaryotes, the molecular binding of IF6 on the ribosome is conserved between eukaryotes and archaea. The structure also provides a snapshot of the reductive evolution of the archaeal ribosome and offers new insights into the evolution of the translation system in archaea.     

SURVEY AND SUMMARY: holliday junction resolvases and related nucleases: identification of new families, phyletic distribution and evolutionary trajectories

Holliday junction resolvases (HJRs) are key enzymes of DNA recombination. A detailed computer analysis of the structural and evolutionary relationships of HJRs and related nucleases suggests that the HJR function has evolved independently from at least four distinct structural folds, namely RNase H, endonuclease, endonuclease VII-colicin E and RusA. The endonuclease fold, whose structural prototypes are the phage lambda exonuclease, the very short patch repair nuclease (Vsr) and type II restriction enzymes, is shown to encompass by far a greater diversity of nucleases than previously suspected. This fold unifies archaeal HJRs, repair nucleases such as RecB and Vsr, restriction enzymes and a variety of predicted nucleases whose specific activities remain to be determined. Within the RNase H fold a new family of predicted HJRs, which is nearly ubiquitous in bacteria, was discovered, in addition to the previously characterized RuvC family. The proteins of this family, typified by Escherichia coli YqgF, are likely to function as an alternative to RuvC in most bacteria, but could be the principal HJRs in low-GC Gram-positive bacteria and AQUIFEX: Endonuclease VII of phage T4 is shown to serve as a structural template for many nucleases, including MCR:A and other type II restriction enzymes. Together with colicin E7, endonuclease VII defines a distinct metal-dependent nuclease fold. As a result of this analysis, the principal HJRs are now known or confidently predicted for all bacteria and archaea whose genomes have been completely sequenced, with many species encoding multiple potential HJRs. Horizontal gene transfer, lineage-specific gene loss and gene family expansion, and non-orthologous gene displacement seem to have been major forces in the evolution of HJRs and related nucleases. A remarkable case of displacement is seen in the Lyme disease spirochete Borrelia burgdorferi, which does not possess any of the typical HJRs, but instead encodes, in its chromosome and each of the linear plasmids, members of the lambda exonuclease family predicted to function as HJRs. The diversity of HJRs and related nucleases in bacteria and archaea contrasts with their near absence in eukaryotes. The few detected eukaryotic representatives of the endonuclease fold and the RNase H fold have probably been acquired from bacteria via horizontal gene transfer. The identity of the principal HJR(s) involved in recombination in eukaryotes remains uncertain; this function could be performed by topoisomerase IB or by a novel, so far undetected, class of enzymes. Likely HJRs and related nucleases were identified in the genomes of numerous bacterial and eukaryotic DNA viruses. Gene flow between viral and cellular genomes has probably played a major role in the evolution of this class of enzymes. This analysis resulted in the prediction of numerous previously unnoticed nucleases, some of which are likely to be new restriction enzymes.     

Structure and DNA-binding activity of the Pyrococcus furiosus SMC protein hinge domain

Structural Maintenance of Chromosomes (SMC) proteins are essential for a wide range of processes including chromosome structure and dynamics, gene regulation, and DNA repair. While bacteria and archaea have one SMC protein that forms a homodimer, eukaryotes possess three distinct SMC complexes, consisting of heterodimeric pairs of six different SMC proteins. SMC holocomplexes additionally contain several specific regulatory subunits. The bacterial SMC complex is required for chromosome condensation and segregation. In eukaryotes, this function is carried out by the condensin (SMC2-SMC4) complex. SMC proteins consist of N-terminal and C-terminal domains that fold back onto each other to create an ATPase "head" domain, connected to a central "hinge" domain via a long coiled-coil region. The hinge domain mediates dimerization of SMC proteins and binds DNA. This activity implicates a direct involvement of the hinge domain in the action of SMC proteins on DNA. We studied the SMC hinge domain from the thermophilic archaeon Pyrococcus furiosus. Its crystal structure shows that the SMC hinge domain fold is largely conserved between archaea and bacteria as well as eukarya. Like the eukaryotic condensin hinge domain, the P. furiosus SMC hinge domain preferentially binds single-stranded DNA (ssDNA), but its affinity for DNA is weaker than that of its eukaryotic counterpart, and point mutations reveal that its DNA-binding surface is more confined. The ssDNA-binding activity of its hinge domain might play a role in the DNA-loading process of the prokaryotic SMC complex during replication.     

The origins of modern proteomes

Distributions of phylogenetically related protein domains (fold superfamilies), or FSFs, among the three Superkingdoms (trichotomy) are assessed. Very nearly 900 of the 1200 FSFs of the trichotomy are shared by two or three Superkingdoms. Parsimony analysis of FSF distributions suggests that the FSF complement of the last common ancestor to the trichotomy was more like that of modern eukaryotes than that of archaea and bacteria. Studies of length distributions among members of orthologous families of proteins present in all three Superkingdoms reveal that such lengths are significantly longer among eukaryotes than among bacteria and archaea. The data also reveal that proteins lengths of eukaryotes are more broadly distributed than they are within archaeal and bacterial members of the same orthologous families. Accordingly, selective pressure for a minimal size is significantly greater for orthologous protein lengths in archaea and bacteria than in eukaryotes. Alignments of orthologous proteins of archaea, bacteria and eukaryotes are characterized by greater sequence variation at their N-terminal and C-terminal domains, than in their central cores. Length variations tend to be localized in the terminal sequences; the conserved sequences of orthologous families are localized in a central core. These data are consistent with the interpretation that the genomes of the last common ancestor (LUCA) encoded a cohort of FSFs not very different from that of modern eukaryotes. Divergence of bacterial and archaeal genomes from that common ancestor may have been accompanied by more intensive reductive evolution of proteomes than that expressed in eukaryotes. Dollo's Law suggests that the evolution of novel FSFs is a very slow process, while laboratory experiments suggests that novel protein genesis from preexisting FSFs can be relatively rapid. Reassortment of FSFs to create novel proteins may have been mediated by genetic recombination before the advent of more efficient splicing mechanisms.     

A thermostable single-strand DNase from Methanococcus jannaschii related to the RecJ recombination and repair exonuclease from Escherichia coli

The RecJ protein of Escherichia coli plays an important role in a number of DNA repair and recombination pathways. RecJ catalyzes processive degradation of single-stranded DNA in a 5'-to-3' direction. Sequences highly related to those encoding RecJ can be found in most of the eubacterial genomes sequenced to date. From alignment of these sequences, seven conserved motifs are apparent. At least five of these motifs are shared among a large family of proteins in eubacteria, eukaryotes, and archaea, including the PPX1 polyphosphatase of yeast and Drosophila Prune. Archaeal genomes are particularly rich in such sequences, but it has not been clear whether any of the encoded proteins play a functional role similar to that of RecJ exonuclease. We have investigated three such proteins from Methanococcus jannaschii with the strongest overall sequence similarity to E. coli RecJ. Two of the genes, MJ0977 and MJ0831, partially complement a recJ mutant phenotype in E. coli. The expression of MJ0977 in E. coli resulted in high levels of a thermostable single-stranded DNase activity with properties similar to those of RecJ exonuclease. Despite overall weak sequence similarity between the MJ0977 product and RecJ, these nucleases are likely to have similar biological functions.     

Evolutionary origin, diversification and specialization of eukaryotic MutS homolog mismatch repair proteins

Most eubacteria, and all eukaryotes examined thus far, encode homologs of the DNA mismatch repair protein MutS. Although eubacteria encode only one or two MutS-like proteins, eukaryotes encode at least six distinct MutS homolog (MSH) proteins, corresponding to conserved (orthologous) gene families. This suggests evolution of individual gene family lines of descent by several duplication/specialization events. Using quantitative phylogenetic analyses (RASA, or relative apparent synapomorphy analysis), we demonstrate that comparison of complete MutS protein sequences, rather than highly conserved C-terminal domains only, maximizes information about evolutionary relationships. We identify a novel, highly conserved middle domain, as well as clearly delineate an N-terminal domain, previously implicated in mismatch recognition, that shows family-specific patterns of aromatic and charged amino acids. Our final analysis, in contrast to previous analyses of MutS-like sequences, yields a stable phylogenetic tree consistent with the known biochemical functions of MutS/MSH proteins, that now assigns all known eukaryotic MSH proteins to a monophyletic group, whose branches correspond to the respective specialized gene families. The rooted phylogenetic tree suggests their derivation from a mitochondrial MSH1-like protein, itself the descendent of the MutS of a symbiont in a primitive eukaryotic precursor.     

Potential coexistence of both bacterial and eukaryotic small RNA biogenesis and functional related protein homologs in Archaea

RNA silencing plays crucial roles in both bacteria and eukaryotes, yet its machinery appears to differ in these two kingdoms. A couple of Argonaute protein homologs have been reported in some archaeal species in recent years. As Argonaute protein is the key component of eukaryotic RNA silencing pathways, such findings suggested the possibility of existence of eukaryotic RNA silencing like pathways in Archaea, which present the life forms between prokaryotes and eukaryotes. To further explore such hypothesis, we systematically screened 71 fully sequenced archaeal genomes, and identified some proteins containing homologous regions to the functional domains of eukaryotie RNA silencing pathway key proteins. The phylogenetic relationships of these proteins were analyzed. The conserved time-tional amino acids between archaeal and eukaryotic Piwi domains suggested their functional similarity. Our results provide new clues to the evolution of RNA silencing pathways.     

Prokaryotic structural maintenance of chromosomes (SMC) proteins: distribution, phylogeny, and comparison with MukBs and additional prokaryotic and eukaryotic coiled-coil proteins.

Structural maintenance of chromosomes (SMC) proteins are known to be essential for chromosome segregation in some prokaryotes and in eukaryotes. A systematic search for the distribution of SMC proteins in prokaryotes with fully or partially sequenced genomes showed that they form a larger family than previously anticipated and raised the number of known prokaryotic homologs to 54. Secondary structure predictions revealed that the length of the globular N-terminal and C-terminal domains is extremely well conserved in contrast to the hinge domain and coiled-coil domains which are considerably shorter in several bacterial species. SMC proteins are present in all gram-positive bacteria and in nearly all archaea while they were found in less than half of the gram-negative bacteria. Phylogenetic analyses indicate that the SMC tree roughly resembles the 16S rRNA tree, but that cyanobacteria and Aquifex aeolicus obtained smc genes by lateral transfer from archaea. Fourteen out of 22 smc genes located in fully sequenced genomes seem to be co-transcribed with a second gene out of six different gene families, indicating that the deduced gene products might be involved in similar functions. The SMC proteins were compared with other prokaryotic proteins with long coiled-coil domains. The lengths of different protein domains and signature sequences allowed to differentiate SMCs, MukBs, which were found to be confined to gamma proteobacteria, and two subfamilies of COG 0419 including the SbcC nuclease from E. coli. A phylogenetic analysis was performed including the prokaryotic coiled-coil proteins as well as SMCs and Rad18 proteins from selected eukaryotes.     

Ancestral paralogs and pseudoparalogs and their role in the emergence of the eukaryotic cell

Gene duplication is a crucial mechanism of evolutionary innovation. A substantial fraction of eukaryotic genomes consists of paralogous gene families. We assess the extent of ancestral paralogy, which dates back to the last common ancestor of all eukaryotes, and examine the origins of the ancestral paralogs and their potential roles in the emergence of the eukaryotic cell complexity. A parsimonious reconstruction of ancestral gene repertoires shows that 4137 orthologous gene sets in the last eukaryotic common ancestor (LECA) map back to 2150 orthologous sets in the hypothetical first eukaryotic common ancestor (FECA) [paralogy quotient (PQ) of 1.92]. Analogous reconstructions show significantly lower levels of paralogy in prokaryotes, 1.19 for archaea and 1.25 for bacteria. The only functional class of eukaryotic proteins with a significant excess of paralogous clusters over the mean includes molecular chaperones and proteins with related functions. Almost all genes in this category underwent multiple duplications during early eukaryotic evolution. In structural terms, the most prominent sets of paralogs are superstructure-forming proteins with repetitive domains, such as WD-40 and TPR. In addition to the true ancestral paralogs which evolved via duplication at the onset of eukaryotic evolution, numerous pseudoparalogs were detected, i.e. homologous genes that apparently were acquired by early eukaryotes via different routes, including horizontal gene transfer (HGT) from diverse bacteria. The results of this study demonstrate a major increase in the level of gene paralogy as a hallmark of the early evolution of eukaryotes.     

Archaeal protein translocation crossing membranes in the third domain of life.

Proper cell function relies on correct protein localization. As a first step in the delivery of extracytoplasmic proteins to their ultimate destinations, the hydrophobic barrier presented by lipid-based membranes must be overcome. In contrast to the well-defined bacterial and eukaryotic protein translocation systems, little is known about how proteins cross the membranes of archaea, the third and most recently described domain of life. In bacteria and eukaryotes, protein translocation occurs at proteinaceous sites comprised of evolutionarily conserved core components acting in concert with other, domain-specific elements. Examination of available archaeal genomes as well as cloning of individual genes from other archaeal strains reveals the presence of homologues to selected elements of the bacterial or eukaryotic translocation machines. Archaeal genomic searches, however, also reveal an apparent absence of other, important components of these two systems. Archaeal translocation may therefore represent a hybrid of the bacterial and eukaryotic models yet may also rely on components or themes particular to this domain of life. Indeed, considering the unique chemical composition of the archaeal membrane as well as the extreme conditions in which archaea thrive, the involvement of archaeal-specific translocation elements could be expected. Thus, understanding archaeal protein translocation could reveal the universal nature of certain features of protein translocation which, in some cases, may not be readily obvious from current comparisons of bacterial and eukaryotic systems. Alternatively, elucidation of archaeal translocation could uncover facets of the translocation process either not yet identified in bacteria or eukaryotes, or which are unique to archaea. In the following, the current status of our understanding of protein translocation in archaea is reviewed.     

The prokaryotic V4R domain is the likely ancestor of a key component of the eukaryotic vesicle transport system

   

Intracellular vesicle traffic that enables delivery of proteins between the endoplasmic reticulum, Golgi and various endosomal subcompartments is one of the hallmarks of the eukaryotic cell. Its evolutionary history is not well understood but the process itself and the core vesicle traffic machinery are believed to be ancient. We show here that the 4-vinyl reductase (V4R) protein domain present in bacteria and archaea is homologous to the Bet3 subunit of the TRAPP1 vesicle-tethering complex that is conserved in all eukaryotes. This suggests, for the first time, a prokaryotic origin for one of the key eukaryotic trafficking proteins.     

Evolutionary Comparisons of RecA-Like Proteins Across All Major Kingdoms of Living Organisms

Protein sequences with similarities to Escherichia coli RecA were compared across the major kingdoms of eubacteria, archaebacteria, and eukaryotes. The archaeal sequences branch monophyletically and are most closely related to the eukaryotic paralogous Rad51 and Dmc1 groups. A multiple alignment of the sequences suggests a modular structure of RecA-like proteins consisting of distinct segments, some of which are conserved only within subgroups of sequences. The eukaryotic and archaeal sequences share an N-terminal domain which may play a role in interactions with other factors and nucleic acids. Several positions in the alignment blocks are highly conserved within the eubacteria as one group and within the eukaryotes and archaebacteria as a second group, but compared between the groups these positions display nonconservative amino acid substitutions. Conservation within the RecA-like core domain identifies possible key residues involved in ATP-induced conformational changes. We propose that RecA-like proteins derive evolutionarily from an assortment of independent domains and that the functional homologs of RecA in noneubacteria comprise an array of RecA-like proteins acting in series or cooperatively. Received: 25 October 1996 / Accepted: 31 December 1996     

GGDEF domain is homologous to adenylyl cyclase

The GGDEF domain is detected in many prokaryotic proteins, most of which are of unknown function. Several bacteria carry 12-22 different GGDEF homologues in their genomes. Conducting extensive profile-based searches, we detect statistically supported sequence similarity between GGDEF domain and adenylyl cyclase catalytic domain. From this homology, we deduce that the prokaryotic GGDEF domain is a regulatory enzyme involved in nucleotide cyclization, with the fold similar to that of the eukaryotic cyclase catalytic domain. This prediction correlates with the functional information available on two GGDEF-containing proteins, namely diguanylate cyclase and phosphodiesterase A of Acetobacter xylinum, both of which regulate the turnover of cyclic diguanosine monophosphate. Domain architecture analysis shows that GGDEF is typically present in multidomain proteins containing regulatory domains of signaling pathways or protein-protein interaction modules. Evolutionary tree analysis indicates that GGDEF/cyclase superfamily forms a large diversified cluster of orthologous proteins present in bacteria, archaea, and eukaryotes. Proteins 2001;42:210-216.     

Proteome Analysis Database: online application of InterPro and CluSTr for the functional classification of proteins in whole genomes

The SWISS-PROT group at EBI has developed the Proteome Analysis Database utilising existing resources and providing comparative analysis of the predicted protein coding sequences of the complete genomes of bacteria, archaea and eukaryotes (http://www.ebi.ac. uk/proteome/). The two main projects used, InterPro and CluSTr, give a new perspective on families, domains and sites and cover 31-67% (InterPro statistics) of the proteins from each of the complete genomes. CluSTr covers the three complete eukaryotic genomes and the incomplete human genome data. The Proteome Analysis Database is accompanied by a program that has been designed to carry out InterPro proteome comparisons for any one proteome against any other one or more of the proteomes in the database.     

Molecular Evolution of Translin Superfamily Proteins Within the Genomes of Eubacteria, Archaea and Eukaryotes

Translin and its interacting partner protein, TRAX, are members of the translin superfamily. These proteins are involved in mRNA regulation and in promoting RISC activity by removing siRNA passenger strand cleavage products, and have been proposed to play roles in DNA repair and recombination. Both homomeric translin and heteromeric translin-TRAX complex bind to ssDNA and RNA; however, the heteromeric complex is a key activator in siRNA-mediated silencing in human and drosophila. The residues critical for RNase activity of the complex reside in TRAX sequence. Both translin and TRAX are well conserved in eukaryotes. In present work, a single translin superfamily protein is detected in Chloroflexi eubacteria, in the known phyla of archaea and in some unicellular eukaryotes. The prokaryotic proteins essentially share unique sequence motifs with eukaryotic TRAX, while the proteins possessing both the unique sequences and conserved indels of TRAX or translin can be identified from protists. Intriguingly, TRAX protein in all the known genomes of extant Chloroflexi share high sequence similarity and conserved indels with the archaeal protein, suggesting occurrence of TRAX at least at the time of Chloroflexi divergence as well as evolutionary relationship between Chloroflexi and archaea. The mirror phylogeny in phylogenetic tree, constructed using diverse translin and TRAX sequences, indicates gene duplication event leading to evolution of translin in unicellular eukaryotes, prior to divergence of multicellular eukayrotes. Since Chloroflexi has been debated to be near the last universal common ancestor, the present analysis indicates that TRAX may be useful to understand the tree of life.     

The rhomboids: a nearly ubiquitous family of intramembrane serine proteases that probably evolved by multiple ancient horizontal gene transfers

Background

The rhomboid family of polytopic membrane proteins shows a level of evolutionary conservation unique among membrane proteins. They are present in nearly all the sequenced genomes of archaea, bacteria and eukaryotes, with the exception of several species with small genomes. On the basis of experimental studies with the developmental regulator rhomboid from Drosophila and the AarA protein from the bacterium Providencia stuartii, the rhomboids are thought to be intramembrane serine proteases whose signaling function is conserved in eukaryotes and prokaryotes.

Results

Phylogenetic tree analysis carried out using several independent methods for tree constructions and the corresponding statistical tests suggests that, despite its broad distribution in all three superkingdoms, the rhomboid family was not present in the last universal common ancestor of extant life forms. Instead, we propose that rhomboids evolved in bacteria and have been acquired by archaea and eukaryotes through several independent horizontal gene transfers. In eukaryotes, two distinct, ancient acquisitions apparently gave rise to the two major subfamilies, typified by rhomboid and PARL (presenilins-associated rhomboid-like protein), respectively. Subsequent evolution of the rhomboid family in eukaryotes proceeded by multiple duplications and functional diversification through the addition of extra transmembrane helices and other domains in different orientations relative to the conserved core that harbors the protease activity.

Conclusions

Although the near-universal presence of the rhomboid family in bacteria, archaea and eukaryotes appears to suggest that this protein is part of the heritage of the last universal common ancestor, phylogenetic tree analysis indicates a likely bacterial origin with subsequent dissemination by horizontal gene transfer. This emphasizes the importance of explicit phylogenetic analysis for the reconstruction of ancestral life forms. A hypothetical scenario for the origin of intracellular membrane proteases from membrane transporters is proposed.