The COG database: new developments in phylogenetic classification of proteins from complete genomes

INTRODUCTION

The database of Clusters of Orthologous Groups of proteins (COGs) has been incepted as a phylogenetic classification of proteins from complete genomes (1). Each COG includes proteins that are thought to be orthologous, i.e. connected through vertical evolutionary descent (2). Orthology may involve not only one-to-one, but also, in cases of lineage-specific gene duplications, one-to-many and many-to-many relationships (hence Orthologous Groups of proteins). The purpose of the COGs database is to serve as a platform for functional annotation of newly sequenced genomes and for studies on genome evolution. To facilitate functional studies, the COGs have been classified into 17 broad functional categories, including a class for which only a general functional prediction, usually that of biochemical activity, was feasible and a class of uncharacterized COGs. Additionally, some of the COGs with known functions are organized to represent specific cellular systems and biochemical pathways. The database is accompanied by the COGNITOR program, which assigns new proteins, typically from newly sequenced genomes, to pre-existing COGs. Here we describe the new developments in the COGs database in the year 2000, which included both the quantitative update through addition of new genomes and development of new functionalities associated with the database.

THE CURRENT STATUS OF THE COGS—NEW GENOMES

Since the second release of the COG database in January 2000 (3), nine new genomes have been added to the database using the COGNITOR program with subsequent manual validation to identify new members of pre-existing COGs and previously described procedures for the construction of new COGs. The additions included the first sequenced genome of a crenarchaeon (representative of the second major division of the archaea), Aeropyrum pernix; a fifth representative of the Euryarchaea, Pyrococcus abyssi; and seven bacterial genomes, including those from unusual organisms such as the extremely radio­resistant Deinococcus radiodurans (Table 1). The previously described trend held with the new genomes in that 60–80% of the proteins from each of the prokaryotic genomes could be included in COGs (Table 1).

The genome of the crenarchaeon A.pernix (4), which was of particular interest because this major evolutionary lineage had not been previously represented among completely sequenced genomes, was investigated in detail as a benchmark for annotation of newly sequenced genomes using the COG system (5). The COG analysis resulted in an ∼50% increase in confident functional prediction for A.pernix genes compared to the original annotations. On the other hand, a significant fraction of open reading frames (ORFs), originally annotated as genes, did not show detectable similarity to any proteins in current databases, but overlapped with proteins included in the COGs, strongly suggesting that these ORFs were not real genes (Table 2). Thus the analysis of the genome of an organism that had no close relatives among other organisms with sequenced genomes appears to corroborate the effectiveness of the COG system as a genome annotation tool.

Given the accumulation of multiple, complete genome sequences, we were interested in the growth dynamics of the COG set with the increased number of included genomes. The growth curve was constructed by imitating the COG formation for each of the 10⁶ random orders of genome inclusion (Fig. 1). For each number of species, the maximum, the minimum and the average number of COGs was determined. The minimal and the maximal curves define the area containing all possible growth curves (Fig. 1). The average curve approximates the expected dynamics of the COG growth. Given that the number of completely sequenced genomes is still relatively small and that some of them are closely related, it remains uncertain whether or not the number of COGs is starting to approach saturation, and if it is, what is the asymptotic value.

ADDING PROTEINS FROM MULTICELLULAR EUKARYOTES TO PROKARYOTIC COGs

The current COG collection includes multiple bacterial and archaeal genomes and only one eukaryotic species, the yeast Saccharomyces cerevisiae. Incorporating the larger genomes of multicellular eukaryotes into the COG system is a challenging task due to the preponderance of multidomain proteins in these organisms. As a first step toward this goal, we sought to identify eukaryotic proteins that fit into already existing COGs, in other words, those eukaryotic proteins that have orthologs in at least two prokaryotic species. To this end, 19 895 protein sequences from the (nearly) complete genome of the nematode Caenorhabditis elegans (6) and 14 100 sequences from the genome of the fruit fly Drosophila melanogaster (7) were analyzed using the COGNITOR program, which assigns proteins to COGs on the basis of multiple genome-specific best hits and splits multi­domain protein into individual domains if these show affinity with different COGs. After manual validation of the results, 20% of the D.melanogaster proteins and 14% of the C.elegans proteins were assigned to COGs; a significant number of proteins from each of the multicellular eukaryotes were included in COGs of each functional category, with the notable exception of ‘Cell division and chromosome partitioning’ and ‘Cell motility and secretion’, which consist primarily of prokaryote-specific proteins (Table 3). The COG analysis of the worm and fly proteins yielded numerous functional predictions, which have not been described previously (I.V.Garkavtsev and E.V.Koonin, unpublished observations). Eukaryotic proteins that have orthologs in prokaryotes belong to two major categories: (i) ancient proteins inherited from the last common ancestor of all extant life forms or at least the common ancestor of archaea and eukaryotes; (ii) proteins encoded by genes that have been horizontally transferred from organelles to the eukaryotic nucleus or otherwise acquired by eukaryotes from bacteria (8). Analysis of the phylogenetic patterns in the COGs may help distinguish between these two categories.

After three distant eukaryotic genomes were included in the prokaryotic COGs, it was of interest to analyze their co-occurrence. As expected, the majority of COGs with eukaryotic members include all three genomes; at the same time, a considerable number of COGs include all possible pairs of eukaryotic genomes and each of the individual species (Table 4). These observations, which will be analyzed in detail elsewhere, support the major role of lineage-specific gene loss and horizontal gene transfer in eukaryotic evolution.

DETECTING MISSED GENES

One of the features associated with the COG database is the analysis of phylogenetic patterns, i.e. the patterns of species that are represented or not represented in each of the COGs. Unexpected phylogenetic patterns, for example, those that contain all but one bacterial species or those that include only one of a pair of closely related species, may be due to omission of genes in genome annotations submitted to GenBank or to unusual evolutionary phenomena such as non-orthologous displacement of a nearly ubiquitous gene. Before considering the second hypothesis, the first one should be tested, and we undertook a systematic analysis of COGs with unexpected phylogenetic patterns in search of missing members (9). The nucleotide sequence of the genome in question was searched using the TNBLASTN program (10) and the sequences of members of the respective COGs as queries. As a result, missing genes coding for members of 48 COGs were identified (Table 5); most of the predicted new proteins are small, which explains why they have escaped the original genome annotations. Thus the COG system is instrumental in improving genome annotation not only with respect to functional predictions, but also for gene identification per se.

NEW FEATURES ASSOCIATED WITH THE COGS

THE COG WORLDWIDE WEB SITE

The COG database is accessible at http://www.ncbi.nlm.nih.gov/COG. The site includes the following main features: complete list of all COGs hyperlinked to individual COG pages; COGs organized by functional category; COGs organized by functional complexes and pathways; an interactive matrix of co-occurrence of genomes in COGs; a phylogenetic pattern search tool; a principal component classification tool; COGNITOR; a COG Help page. Each of the individual COG pages is hyperlinked to: (i) pictorial representations of BLAST search outputs for each member of the COG, which also include links to the respective GenBank and Entrez-Genomes entries, (ii) a multiple alignment of the COG members produced automatically by using the ClustalW program, (iii) a COG-Info page (reached by clicking on the COG number). The supplement to the COGs, which shows proteins from C.elegans and D.melanogaster assigned to each COG is accessible at http://www.ncbi.nlm.nih.gov/COG/euk. The COG data set is also available by anonymous ftp at ftp://ncbi.nlm.nih.gov/pub/COG.