Phylogeny of prokaryotes: does it exist and why should we care?

Understanding microbial evolution is essential for gathering information on the most ancient events in the history of Life on our planet. Nevertheless, the idea that it is impossible to reconstruct the evolutionary history of prokaryotes because of horizontal gene transfer has become very popular. We review this important debate and how it can be solved.

An archaeal orthologue of the universal protein Kae1 is an iron metalloprotein which exhibits atypical DNA-binding properties and apurinic-endonuclease activity in vitro.

The Kae1 (Kinase-associated endopeptidase 1) protein is a member of the recently identified transcription complex EKC and telomeres maintenance complex KEOPS in yeast. Kae1 homologues are encoded by all sequenced genomes in the three domains of life. Although annotated as putative endopeptidases, the actual functions of these universal proteins are unknown. Here we show that the purified Kae1 protein (Pa-Kae1) from Pyrococcus abyssi is an iron-protein with a novel type of ATP-binding site. Surprisingly, this protein did not exhibit endopeptidase activity in vitro but binds cooperatively to single and double-stranded DNA and induces unusual DNA conformational change. Furthermore, Pa-Kae1 exhibits a class I apurinic (AP)-endonuclease activity (AP-lyase). Both DNA binding and AP-endonuclease activity are inhibited by ATP. Kae1 is thus a novel and atypical universal DNA interacting protein whose importance could rival those of RecA (RadA/Rad51) in the maintenance of genome integrity in all living cells.

Early archives of genetically-restricted proviral DNA in the female genital tract after heterosexual transmission of HIV-1.

OBJECTIVES AND METHOD: In order to characterize human immunodeficiency virus type 1 (HIV-1) variants that are transmitted in women via heterosexual intercourse, the env V1-V3 sequences of HIV-1 provirus (DNA) and free virus (RNA) in paired samples of blood and cervicovaginal secretions of untreated chronically and primary infected African women were compared. RESULTS: Env RNA sequences retrieved from plasma and genital compartments formed a single cluster in primary infection. In contrast, env RNA sequences from these two compartments were distinct in chronically infected women. Analysis of proviral DNA of primary infected women showed that most HIV-1 sequences derived from the genital epithelia form independent clusters from HIV-1 sequences of DNA from peripheral blood mononuclear cells and RNA recovered from plasma and genital secretions. Similarly, the analysis of proviral DNA in the genital compartment of chronically infected women showed the persistence of genetically-restricted cluster of HIV-1. CONCLUSIONS: These observations indicate that a viral subpopulation is archived as proviral DNA in the female genital tract early in primary infection, and suggest that HIV-1 variants from the male donor are selected in the female mucosal site during male to female transmission of HIV-1.

The two tempos of nuclear pore complex evolution: highly adapting proteins in an ancient frozen structure.

BACKGROUND: The origin of the nuclear compartment has been extensively debated, leading to several alternative views on the evolution of the eukaryotic nucleus. Until recently, too little phylogenetic information was available to address this issue by using multiple characters for many lineages. RESULTS: We analyzed 65 proteins integral to or associated with the nuclear pore complex (NPC), including all the identified nucleoporins, the components of their anchoring system and some of their main partners. We used reconstruction of ancestral sequences of these proteins to expand the detection of homologs, and showed that the majority of them, present all over the nuclear pore structure, share homologs in all extant eukaryotic lineages. The anchoring system, by contrast, is analogous between the different eukaryotic lineages and is thus a relatively recent innovation. We also showed the existence of high heterogeneity of evolutionary rates between these proteins, as well as between and within lineages. We show that the ubiquitous genes of the nuclear pore structure are not strongly conserved at the sequence level, and that only their domains are relatively well preserved. CONCLUSION: We propose that an NPC very similar to the extant one was already present in at least the last common ancestor of all extant eukaryotes and it would not have undergone major changes since its early origin. Importantly, we observe that sequences and structures obey two very different tempos of evolution. We suggest that, despite strong constraints that froze the structural evolution of the nuclear pore, the NPC is still highly adaptive, modern, and flexible at the sequence level.

[Luca: the last universal common ancestor]. / Luca: à la recherche du plus proche ancêtre commun universel.

One of the most important outcomes of modern biology has been the demonstration of the unity of life. All living beings are in fact descendants of a unique ancestor commonly referred to as Luca (the Last universal common ancestor). The discovery - nearly 30 years ago by Carl Woese - that present-day life on our planet can be assigned to only three domains: two of prokaryotic nature (Archaea and Bacteria), and one eukaryoyic (Eucarya), has given birth to a new field of investigation aimed at determining the nature of Luca. Today, thanks to the accumulation of genomic data, we can loop back into the past and infer a few characters of Luca by comparing what present-day organisms have in common. For example, it is now clear that Luca was a cellular organism provided with a cytoplasmic membrane, and that it harboured already a quite sophisticated translation apparatus. However, the inference of other characters of Luca from comparative genomics is less straightforward: for instance, a few key molecular mechanisms for DNA replication are non-homologous across the three domains and their distribution is often puzzling. This evidence has been embraced by proponents of the hypothesis that Luca harboured an RNA genome and that its replacement by DNA and the appearance of the corresponding molecular systems would have occurred independently in the three life domains after their divergence. However, an equally likely scenario would be that of a Luca with a DNA genome and of a subsequent replacement of its DNA-replication systems by non-homologous counterparts either in the bacterial or in the archaeal/eukaroytic branch. Nevertheless, including the viral world into the picture of the tree of life may thus provide us with precious insights into our most distant past since the invention and spread potential of viruses may have played a key role in early evolution.

An emerging phylogenetic core of Archaea: phylogenies of transcription and translation machineries converge following addition of new genome sequences.

BACKGROUND: The concept of a genomic core, defined as the set of genes ubiquitous in all genomes of a monophyletic group, has become crucial in comparative and evolutionary genomics. However, it is still a matter of debate whether lateral gene transfers (LGT) may affect the components of genomic cores, preventing their use to retrace species evolution. We have recently reconstructed the phylogeny of Archaea by using two large concatenated datasets of core proteins involved in translation and transcription, respectively. The resulting trees were largely congruent, showing that informational gene components of the archaeal genomic core belonging to two distinct molecular systems contain a coherent signal for archaeal phylogeny. However, some incongruence remained between the two phylogenies. This may be due either to undetected LGT and/or to a lack of sufficient phylogenetic signal in the datasets. RESULTS: We present evidence strongly favoring of the latter hypothesis. In fact, we have updated our transcription and translation datasets with five new archaeal genomes for a total of 6384 and 2928 amino acid positions, respectively, and 25 taxa. This increase in taxonomic sampling led to the nearly complete convergence of the transcription-based and translation-based trees on a single phylogenetic pattern for archaeal evolution. In fact, only a single incongruence persisted between the two phylogenies. This concerned Methanopyrus kandleri, whose placement remained strongly biased in the transcription tree due to its above average evolutionary rates, and could not be counterbalanced due to the lack of availability of closely related and/or slower-evolving relatives. CONCLUSION: To our knowledge, this is the first report of evidence that the phylogenetic signal harbored by components of the archaeal translation apparatus is confirmed by additional markers belonging to a second molecular system (i.e. transcription). This rules out the risk of circularity when inferring species evolution by small subunit ribosomal RNA and ribosomal protein sequences, since it has been suggested that concerted LGT may affect these markers. Our results strongly support the existence of a core of proteins that has evolved mainly through vertical inheritance in Archaea, and carries a bona fide phylogenetic signal that can be used to retrace the evolutionary history of this domain. The identification and analysis of additional molecular markers not affected by LGT should continue defining the emerging picture of a genuine phylogenetic core for the third domain of life.

Nanoarchaea: representatives of a novel archaeal phylum or a fast-evolving euryarchaeal lineage related to Thermococcales?

BACKGROUND: Cultivable archaeal species are assigned to two phyla -- the Crenarchaeota and the Euryarchaeota -- by a number of important genetic differences, and this ancient split is strongly supported by phylogenetic analysis. The recently described hyperthermophile Nanoarchaeum equitans, harboring the smallest cellular genome ever sequenced (480 kb), has been suggested as the representative of a new phylum -- the Nanoarchaeota -- that would have diverged before the Crenarchaeota/Euryarchaeota split. Confirming the phylogenetic position of N. equitans is thus crucial for deciphering the history of the archaeal domain. RESULTS: We tested the placement of N. equitans in the archaeal phylogeny using a large dataset of concatenated ribosomal proteins from 25 archaeal genomes. We indicate that the placement of N. equitans in archaeal phylogenies on the basis of ribosomal protein concatenation may be strongly biased by the coupled effect of its above-average evolutionary rate and lateral gene transfers. Indeed, we show that different subsets of ribosomal proteins harbor a conflicting phylogenetic signal for the placement of N. equitans. A BLASTP-based survey of the phylogenetic pattern of all open reading frames (ORFs) in the genome of N. equitans revealed a surprisingly high fraction of close hits with Euryarchaeota, notably Thermococcales. Strikingly, a specific affinity of N. equitans and Thermococcales was strongly supported by phylogenies based on a subset of ribosomal proteins, and on a number of unrelated molecular markers. CONCLUSION: We suggest that N. equitans may more probably be the representative of a fast-evolving euryarchaeal lineage (possibly related to Thermococcales) than the representative of a novel and early diverging archaeal phylum.

Higher-level classification of the Archaea: evolution of methanogenesis and methanogens.

We used a phylogenetic approach to analyze the evolution of methanogenesis and methanogens. We show that 23 vertically transmitted ribosomal proteins do not support the monophyly of methanogens, and propose instead that there are two distantly related groups of extant archaea that produce methane, which we have named Class I and Class II. Based on this finding, we subsequently investigated the uniqueness of the origin of methanogenesis by studying both the enzymes of methanogenesis and the proteins that synthesize its specific coenzymes. We conclude that hydrogenotrophic methanogenesis appeared only once during evolution. Genes involved in the seven central steps of the methanogenic reduction of carbon dioxide (CO(2)) are ubiquitous in methanogens and share a common history. This suggests that, although extant methanogens produce methane from various substrates (CO(2), formate, acetate, methylated C-1 compounds), these archaea have a core of conserved enzymes that have undergone little evolutionary change. Furthermore, this core of methanogenesis enzymes seems to originate (as a whole) from the last ancestor of all methanogens and does not appear to have been horizontally transmitted to other organisms or between members of Class I and Class II. The observation of a unique and ancestral form of methanogenesis suggests that it was preserved in two independent lineages, with some instances of specialization or added metabolic flexibility. It was likely lost in the Halobacteriales, Thermoplasmatales and Archaeoglobales. Given that fossil evidence for methanogenesis dates back 2.8 billion years, a unique origin of this process makes the methanogenic archaea a very ancient taxon.

Horizontal gene transfer and archaeal origin of deoxyhypusine synthase homologous genes in bacteria.

The initiation factor 5A (IF-5A) of archaea and eukaryotes undergoes an unusual post-translational modification consisting of the transformation of a specific conserved lysine residue into the amino acid hypusine. This occurs in a two-step reaction catalysed by the enzymes deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase. Bacteria do not have IF-5A but only a very distant homologue, the elongation factor P (EF-P). Consequently, all bacteria appeared to also lack genes with significant homology to DHS genes. However, we have carried out BLAST searches and found DHS-like genes in a number of bacterial species. The phylogenetic analysis of these sequences strongly suggests that they have been acquired from archaea by horizontal gene transfer (HGT). Our analysis also suggests, although with weaker support, that a single HGT event from archaea, followed by several HGT between bacterial species, accounts for the patchy distribution of DHS-like genes in bacteria. The activity of these genes in bacteria is enigmatic, since we have not found any evidence of interaction between this protein and the bacterial EF-P. Nevertheless, we cannot discard that it exists, since it appears that the interaction between the DHS and its natural substrate, the IF-5A, is rather weak. This is exemplified by the fact that, in archaea, the complex evolutionary history of the DHS is not paralleled by that of the IF-5A, indicating that these proteins do not follow a perfect co-evolution.

Archaeal phylogeny based on proteins of the transcription and translation machineries: tackling the Methanopyrus kandleri paradox.

BACKGROUND: Phylogenetic analysis of the Archaea has been mainly established by 16S rRNA sequence comparison. With the accumulation of completely sequenced genomes, it is now possible to test alternative approaches by using large sequence datasets. We analyzed archaeal phylogeny using two concatenated datasets consisting of 14 proteins involved in transcription and 53 ribosomal proteins (3,275 and 6,377 positions, respectively). RESULTS: Important relationships were confirmed, notably the dichotomy of the archaeal domain as represented by the Crenarchaeota and Euryarchaeota, the sister grouping of Sulfolobales and Aeropyrum pernix, and the monophyly of a large group comprising Thermoplasmatales, Archaeoglobus fulgidus, Methanosarcinales and Halobacteriales, with the latter two orders forming a robust cluster. The main difference concerned the position of Methanopyrus kandleri, which grouped with Methanococcales and Methanobacteriales in the translation tree, whereas it emerged at the base of the euryarchaeotes in the transcription tree. The incongruent placement of M. kandleri is likely to be the result of a reconstruction artifact due to the high evolutionary rates displayed by the components of its transcription apparatus. CONCLUSIONS: We show that two informational systems, transcription and translation, provide a largely congruent signal for archaeal phylogeny. In particular, our analyses support the appearance of methanogenesis after the divergence of the Thermococcales and a late emergence of aerobic respiration from within methanogenic ancestors. We discuss the possible link between the evolutionary acceleration of the transcription machinery in M. kandleri and several unique features of this archaeon, in particular the absence of the elongation transcription factor TFS.

Comparative analysis of a genome fragment of an uncultivated mesopelagic crenarchaeote reveals multiple horizontal gene transfers.

Marine planktonic crenarchaeota have escaped all cultivation attempts to date, all crenarchaeota growing in pure culture so far being hyperthermophiles. Here, we present a comparative genomic analysis of a 16S- plus 23S-rDNA-containing fragment of a crenarchaeote retrieved from an environmental genomic library constructed from picoplankton collected at 500 m depth in the Antarctic Polar Front. The clone DeepAnt-EC39 contained an insert of 33.3 kbp, which was completely sequenced. DeepAnt-EC39 appears to represent a lineage specific to deep-sea waters but widespread geographically, as revealed by the analysis of the 16S-23S-rDNA intergenic spacer region. A comparison with previously sequenced marine crenarchaeotal genomic clones also containing an rrn operon (74A4, 4B7 and Cenarchaeum symbiosum strains A and B) revealed a highly variable structure involving gene rearrangements and insertions/deletions. The surroundings of the rrn operon and the contiguous glutamate-1-semialdehyde aminotransferase gene appear hot spots for recombination. Phylogenetic analyses of all individual predicted proteins revealed the existence of several likely cases of horizontal gene transfer both, between the two archaeal kingdoms and between the two prokaryotic domains. The most frequent horizontal transfers appear to involve genes from mesophilic methanogenic euryarchaeota related to Methanosarcinales. We hypothesise that the acquisition of genes from mesophilic bacteria and euryarchaeota has played a major role in the adaptation of Group I crenarchaeota to life at lower temperatures.

Evolution of the Archaea.

Archaea, members of the third domain of life, are bacterial-looking prokaryotes that harbour many unique genotypic and phenotypic properties, testifying for their peculiar evolutionary status. The archaeal ancestor was probably a hyperthermophilic anaerobe. Two archaeal phyla are presently recognized, the Euryarchaeota and the Crenarchaeota. Methanogenesis was the main invention that occurred in the euryarchaeal phylum and is now shared by several archaeal groups. Adaptation to aerobic conditions occurred several times independently in both Euryarchaeota and Crenarchaeota. Recently, many new groups of Archaea that have not yet been cultured have been detected by PCR amplification of 16S ribosomal RNA from environmental samples. The phenotypic and genotypic characterization of these new groups is now a top priority for further studies on archaeal evolution.

Phylogeny: a non-hyperthermophilic ancestor for bacteria.

The first phyla that emerge in the tree of life based on ribosomal RNA (rRNA) sequences are hyperthermophilic, which led to the hypothesis that the universal ancestor, and possibly the original living organism, was hyperthermophilic. Here we reanalyse the bacterial phylogeny based on rRNA using a more reliable approach, and find that hyperthermophilic bacteria (such as Aquificales and Thermotogales) do not emerge first, suggesting that the Bacteria had a non-hyperthermophilic ancestor. It seems that Planctomycetales, a phylum with numerous peculiarities, could be the first emerging bacterial group.

Archaeal phylogeny based on ribosomal proteins.

Until recently, phylogenetic analyses of Archaea have mainly been based on ribosomal RNA (rRNA) sequence comparisons, leading to the distinction of the two major archaeal phyla: the Euryarchaeota and the Crenarchaeota. Here, thanks to the recent sequencing of several archaeal genomes, we have constructed a phylogeny based on the fusion of the sequences of the 53 ribosomal proteins present in most of the archaeal species. This phylogeny was remarkably congruent with the rRNA phylogeny, suggesting that both reflected the actual phylogeny of the domain Archaea even if some nodes remained unresolved. In both cases, the branches leading to hyperthermophilic species were short, suggesting that the evolutionary rate of their genes has been slowed down by structural constraints related to environmental adaptation. In addition, to estimate the impact of lateral gene transfer (LGT) on our tree reconstruction, we used a new method that revealed that 8 genes out of the 53 ribosomal proteins used in our study were likely affected by LGT. This strongly suggested that a core of 45 nontransferred ribosomal protein genes existed in Archaea that can be tentatively used to infer the phylogeny of this domain. Interestingly, the tree obtained using only the eight ribosomal proteins likely affected by LGT was not very different from the consensus tree, indicating that LGT mainly brought random phylogenetic noise. The major difference involves organisms living in similar environments, suggesting that LGTs are mainly directed by the physical proximity of the organisms rather than by their phylogenetic proximity.

Eubacterial phylogeny based on translational apparatus proteins.

Lateral gene transfers are frequent among prokaryotes, although their detection remains difficult. If all genes are equally affected, this questions the very existence of an organismal phylogeny. The complexity hypothesis postulates the existence of a core of genes (those involved in numerous interactions) that are unaffected by transfers. To test the hypothesis, we studied all the proteins involved in translation from 45 eubacterial taxa, and developed a new phylogenetic method to detect transfers. Few of the genes studied show evidence for transfer. The phylogeny based on the genes devoid of transfer is very consistent with the ribosomal RNA tree, suggesting that an eubacterial phylogeny does exist.