Downloaded from genome.cshlp.org on September 24, 2021 - Published by Cold Spring Harbor Laboratory Press Research Comparative Genomics of the Archaea (Euryarchaeota): Evolution of Conserved Protein Families, the Stable Core, and the Variable Shell Kira S. Makarova,1,2,4 L. Aravind,1,3 Michael Y. Galperin,1 Nick V. Grishin,1 Roman L. Tatusov,1 Yuri I. Wolf,1,4 and Eugene V. Koonin1,5 1National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, Maryland 20894 USA; 2Department of Pathology, F.E. Hebert School of Medicine, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814-4799 USA; 3Department of Biology, Texas A&M University, College Station, Texas 70843 USA Comparative analysis of the protein sequences encoded in the four euryarchaeal species whose genomes have been sequenced completely (Methanococcus jannaschii, Methanobacterium thermoautotrophicum, Archaeoglobus fulgidus, and Pyrococcus horikoshii) revealed 1326 orthologous sets, of which 543 are represented in all four species. The proteins that belong to these conserved euryarchaeal families comprise 31%–35% of the gene complement and may be considered the evolutionarily stable core of the archaeal genomes. The core gene set includes the great majority of genes coding for proteins involved in genome replication and expression, but only a relatively small subset of metabolic functions. For many gene families that are conserved in all euryarchaea, previously undetected orthologs in bacteria and eukaryotes were identified. A number of euryarchaeal synapomorphies (unique shared characters) were identified; these are protein families that possess sequence signatures or domain architectures that are conserved in all euryarchaea but are not found in bacteria or eukaryotes. In addition, euryarchaea-specific expansions of several protein and domain families were detected. In terms of their apparent phylogenetic affinities, the archaeal protein families split into bacterial and eukaryotic families. The majority of the proteins that have only eukaryotic orthologs or show the greatest similarity to their eukaryotic counterparts belong to the core set. The families of euryarchaeal genes that are conserved in only two or three species constitute a relatively mobile component of the genomes whose evolution should have involved multiple events of lineage-specific gene loss and horizontal gene transfer. Frequently these proteins have detectable orthologs only in bacteria or show the greatest similarity to the bacterial homologs, which might suggest a significant role of horizontal gene transfer from bacteria in the evolution of the euryarchaeota. Phylogenetic analysis of rRNA and a set of proteins 1996; Brown and Doolittle 1997; Edgell and Doolittle involved in translation, transcription, and replication 1997). However, it has been aptly noted that archaea has led to the concept of archaea as a third division of have a “eubacterial form and eukaryotic content” life, distinct from either bacteria or eukaryotes (Woese (Keeling et al. 1994). Indeed, beyond the common et al. 1978, 1990; Woese and Gupta 1981; Pace et al. “negative” trait, namely the small cell size and the ab- 1986; Zillig 1991). Furthermore, rooting of paralogous sence of a nucleus, archaea and bacteria share major trees for translation elongation factors and proton aspects of genome organization and expression strat- ATPases suggested that archaea are a sister group of egy. The most important of these common features eukaryotes (Gogarten et al. 1989a,b; Iwabe et al. 1989; include the (typically) single circular chromosome, the Gribaldo and Cammarano 1998). This concept appears absence of introns in protein-coding genes, the oper- to be gaining further support from the generally eu- onic organization of many genes, and the absence of a karyotic layout of the genome expression systems, par- 5Ј-terminal cap and the presence of a ribosomal- ticularly the system of DNA replication whose princi- binding (Shine-Dalgarno) site in archaeal mRNAs pal components are orthologous to the respective rep- (Brown and Doolittle 1997). Furthermore, several op- lication proteins of eukaryotes but apparently do not erons, particularly those encoding ribosomal proteins, have counterparts in bacteria (Mushegian and Koonin are conserved in archaea and bacteria (Brown and Doolittle 1997; Koonin and Galperin 1997). 4Present address: Institute of Cytology and Genetics, Russian Academy of The analysis of the first two completely sequenced Sciences, Novosibirsk 630090, Russia. 5Corresponding author. archaeal genomes, those of Methanococcus jannaschii E-MAIL [email protected]; FAX (301) 480-9241. (Bult et al. 1996) and Methanobacterium thermoautotro- 608 Genome Research 9:608–628 ©1999 by Cold Spring Harbor Laboratory Press ISSN 1054-9803/99 $5.00; www.genome.org www.genome.org Downloaded from genome.cshlp.org on September 24, 2021 - Published by Cold Spring Harbor Laboratory Press Archaeal Genomics and Evolution phicum (Smith et al. 1997), showed, somewhat unex- thologs that precludes their detection altogether. As a pectedly given the already established archaeal– result, the final step in the construction of the original eukaryotic clade, that the bacterial form of archaea is collection of COGs involved considerable manual cor- complemented by considerable bacterial content. It rection. The distances separating the four archaeal spe- has become clear that the majority of archaeal proteins cies are intermediate between those that are seen show the greatest similarity to their bacterial ho- among close bacterial species such as Escherichia coli mologs, which is likely to indicate bacterial origin, and and Haemophilus influenzae (in the original COG analy- only a minority look “eukaryotic” (Koonin et al. 1997; sis, these species were not considered independently) Smith et al. 1997). In functional terms, there is a clear and those between phylogenetically remote species split between the bacterial and eukaryotic components such as bacteria and eukaryotes. In quantitative terms, of the archaeal genomes—the eukaryotic genes are pri- the mean percent identity of the best hits in all- marily those coding for components of the translation, against-all interspecies comparisons of protein se- transcription, and replication machineries, whereas quences is in the range of 41%–46% for the archaea, the bacterial ones typically encode metabolic enzymes 57% for E. coli versus H. influenzae, and between 30%– and proteins involved in cell division and cell wall bio- 35% for most distant bacterial lineages and bacteria genesis (Koonin et al. 1997; Smith et al. 1997). These versus eukaryotes or archaea (N.V. Grishin, unpubl.; findings raised the issue of possible extensive gene ex- ftp://ncbi.nlm.nih.gov/pub/koonin/gen2gen). It ap- change between bacteria and archaea (Feng et al. 1997; pears that the intermediate level of sequence conser- Koonin et al. 1997; Doolittle and Logsdon 1998). vation seen among the archaea is high enough to pre- Subsequently, the complete genome sequences of vent most, if not all, artificial lumping of COGs attrib- two additional archaeal species, namely Archaeoglobus utable to paralogous families, but low enough for the fulgidus (Klenk et al. 1997) and Pyrococcus horikoshii consistency criterion to be valid and useful. For these (Kawarabayasi et al. 1998a,b), have been reported. All reasons, most of the archaeal COGs delineated by the four available complete archaeal genomes represent automatic procedure were corroborated by subsequent only one of the two (or possibly three) main archaeal case-by-case evaluation. Furthermore, given the typi- subdivisions—the Euryarchaeota (Olsen et al. 1994; cally highly significant similarity between archaeal or- Pace 1997). Nevertheless, they show sufficient diversity thologs, it is most unlikely that any significant number to allow us, for the first time, to embark on a systematic of them have been missed as a result of low sequence comparative analysis of archaeal genomes. We describe conservation. here the results of a detailed comparative analysis of Figure 1 shows the breakdown of the archaeal pro- the four complete euryarchaeal protein sets. Our prin- tein set in terms of their conservation in the four com- cipal approach included the delineation of sets of or- plete genomes. The majority of the proteins in each thologous genes and examination of phylogenetic pat- species—from 58% for P. horikoshii to 71% for M. jan- terns in these families (Tatusov et al. 1997; Koonin et naschii—belong to the archaeal families of likely or- al. 1998). thologs (COGs), and another sizable fraction (from 7% RESULTS AND DISCUSSION Orthologous Families Delineated by Comparison of Four Euryarchaeal Genomes and the Principal Types of Events in Archaeal Evolution The proteins encoded in the genomes of the four eu- ryarchaeal species comprise a very good set for the de- lineation of families of likely orthologs [designated clusters of orthologous groups (of proteins), COGs; Ta- tusov et al. 1997)]. In the original COG analysis, we emphasized that to use consistency between different genomes to support the derivation of COGs, the se- quences of the compared proteins should be maxi- mally independent; therefore, this criterion works best Figure 1 Conserved families and unique proteins encoded in with phylogenetically distant genomes. At large phy- the four complete archaeal genomes. (COGs+) Distant homologs logenetic distances, however,