Molecular evolution of the hyperthermophilic archaea of the Pyrococcus genus: analysis of adaptation to different environmental conditions.

BACKGROUND: Prokaryotic microorganisms are able to survive and proliferate in severe environmental conditions. The increasing number of complete sequences of prokaryotic genomes has provided the basis for studying the molecular mechanisms of their adaptation at the genomic level. We apply here a computer-based approach to compare the genomes and proteomes from P. furiosus, P. horikoshii, and P. abyssi to identify features of their molecular evolution related to adaptation strategy to diverse environmental conditions.
RESULTS: Phylogenetic analysis of rRNA genes from 26 Pyrococcus strains suggested that the divergence of P. furiosus, P. horikoshii and P. abyssi might have occurred from ancestral deep-sea organisms. It was demonstrated that the function of genes that have been subject to positive Darwinian selection is closely related to abiotic and biotic conditions to which archaea managed to become adapted. Divergence of the P. furiosus archaea might have been due to loss of some genes involved in cell motility or signal transduction, and/or to evolution under positive selection of the genes for translation machinery. In the course of P. horikoshii divergence, positive selection was found to operate mainly on the transcription machinery; divergence of P. abyssi was related with positive selection for the genes mainly involved in inorganic ion transport. Analysis of radical amino acid replacement rate in evolving P. furiosus, P. horikoshii and P. abyssi showed that the fixation rate was higher for radical substitutions relative to the volume of amino acid side-chain.
CONCLUSIONS: The current results give due credit to the important role of hydrostatic pressure as a cause of variability in the P. furiosus, P. horikoshii and P. abyssi genomes evolving in different habitats. Nevertheless, adaptation to pressure does not appear to be the sole factor ensuring adaptation to environment. For example, at the stage of the divergence of P. horikoshii and P. abyssi, an essential evolutionary role may be assigned to changes in the trophic chain, namely, acquisition of a consumer status at a high (P. horikoshii) or low level (P. abyssi).

Background

It is remarkable how the prokaryotes manage to survive and proliferate in the habitats exposed to an enormous spectrum of conditions despite their simplest organization. It is becoming increasingly apparent that the deep seas are the sources of novel microbial communities and of their so far unclear adaptive abilities. Microbial diversity is not only an academic issue, "it is a treasure house of innovation for the biotechnology industries" [1]. In the postgenomic era, comparative genomics keeps providing powerful tools for unraveling the molecular mechanisms of microbial adaptation. Complete genomic sequences of the prokaryotes give valuable information about the composition of genes, their function, arrangement order in the genome, operon structure, about single nucleotide substitutions in the coding and noncoding parts of the gene as well. High throughputs would allow to uncover the yet unclear molecular mechanisms of the adaptation of prokaryotes to challenging and/or novel environments at the level of genomic organization and protein structure [2].

Here we present the results of comparisons of the genomic sequences of the archaea Pyrococcus performed to elucidate the possible mechanisms of archaeal adaptation to life under different abiotic and biotic environmental conditions. Complete genome sequences are now available for three species of the Pyrococcus genus: P. furiosus [3], P. horikoshii [4], and P. abyssi [5]. The archaea of the genera Pyrococcus belong to the Thermococcales order [6,7]; those of the Thermococcales order are hyperthermophiles, strictly anaerobes and obligate heterotrophs [6,7]. Specific features of the P. furiosus, P. horikoshii, and P. abyssi habitats, certain differences between their genomes are summarized in Table 1. The optimal conditions of temperature, salinity, and pH are similar for their growth. However, their requirements for hydrostatic pressure and habitat depth are different despite the fact that they can proliferate also under pressures close to atmospheric [8]. P. furiosus can exist under pressures not above 20 MPa, it inhabits shallow hydrothermal vents. P. horikoshii inhabits at the depth of about 1400 m (~14 MPa), but it can tolerate pressures as high as 40 MPa. P. abyssi exists at the depth of about 2200 m (~22 MPa) but it can tolerate pressures up to 50 MPa [8]. These organisms differ markedly by the composition of utilized substrates. In contrast to P. furiosus and P. abyssi, P. horikoshii cannot grow on substrates with low content of peptides and amino acids. A probable reason why is the absence of appropriate operons, which renders P. horikoshii unable to synthesize many amino acids (Table 1). Lack of the chemotactic genes is characteristic of P. furiosus [5,9]. From a survey of the features of the habitats of these archaea (Table 1), it may be inferred that the main factors by which they differ are hydrostatic pressure (habitat depth) and specificity of utilized substrate. These archaea may be classified according to these factors as follows: P. furiosus, piezotolerant living in pressures near atmospheric; P. horikoshii, piezotolerant, living in moderate pressures, on amino acid-enriched substrates; P. abyssi, piezotolerant, living predominantly in deep seas. In the course of evolution, the three related species of Pyrococcus became adapted to life in various abiotic and biotic environments. With this in mind, a more thorough analysis of the molecular mechanisms at the genomic level providing this adaptation appears worthwhile [10].

Table 1

Ecology and genome organization of P. furiosus, P. abyssi, and P. horikoshii (based on [3-5,8,9,15,17,27,70,71])

	P. furiosus	P. abyssi	P. horikoshii
General ecological characteristics
Doubling time (min) [15,70,71]	37	33	32
Pressure optimum (MPa) [8]	11	21	1
Pressure range (MPa) [8]	<0.1-25	<0.1-40	<0.1-35
Temperature optimum (°C) [15,70,71]	100	96	98
Temperature range (°C) [15,70,71]	70-103	67-102	<80--102
Salt concentration optimum (%) [15,70,71]	2	3	2,4
Salt concentration range (%)[15,70,71]	0.5-5	0.7-5	1-5
pH optimum [15,70,71]	7	6.8	7
pH range [15,70,71]	5-9	4-8.5	5-8
Carbohydrate and energy sources
Complex substrates (i.e., yeast extract, peptone, etc.), 20 individual amino acids [15,70,71]	growth	growth	growth
Pyruvate, maltose [15,70,71]	growth	growth	death
Casamino Acids [15,17,70,71]	weak growth	growth	death
β-glucosides (i.e., cellobiose and laminarin) [27]	growth	death	death
Other requirements
S0 [15,70,71]	practically no growth	enhanced growth	enhanced growth
Tryptophan [15,70,71]	not needed	not needed	needed
Genome features
Chromosome size (bp) [3-5]	1,908,256	1,765,118	1,738,505
G + C content (mol %) [3-5]	40.8	44.7	41.9
Number of ORFs [5]	2,208	1,765	2,061
Clusters of long tandem repeats [5]	7	4	6
Insertion sequences [5]	24	1	1
Amino acid biosynthesis (Val, Leu, Ile, Trp), aromatic amino acids biosynthesis, maltose transport, phosphate uptake [5,9]	yes	yes	no
Restriction/modification enzymes (protection from bacteriophages) [5,9]	no	yes	no
Chemotaxis-related genes [5,9]	no	yes	yes
Histidine biosynthesis, riboflavin biosynthesis, trehalose transport, citrate cycle, cobalt transport [5,9]	yes	no	no

In the current study, we analyze genomic variability in three Pyrococcus species with regard to changes in their environmental conditions during evolution. Emphasis is on factors of genome evolution, such as gene loss, radical to conservative amino acid replacement rate ratio. Analysis of radical (large changes in the physico-chemical properties) and conservative (small changes) amino acid fixations yields useful information about modes of protein evolution. An excess of radical over conservative substitutions is a significant indicator of evolution of the proteins under positive natural selection [11,12]. Here, we analyzed the evolution of the protein-coding genes in the archaea of the Pyrococcus genus to detect the gene families evolving under positive selection during adaptation of the Pyrococcus species to different abiotic and biotic conditions. As a result, we demonstrated that the function of the genes evolving under positive selection strictly depends on abiotic and biotic conditions to which P. furiosus, P. abyssi, and P. horikoshii became adapted.

As known, protein structure alters under the changes in pressure [13]. In fact, pairwise comparison of the shallow-water P. furiosus with the deep-water P. abyssi demonstrated that amino acid substitution is asymmetrical: arginine, serine, glycine, valine, and aspartic acid were more frequently, while tyrosine and glutamine were less frequently used in P. abyssi [14]. The inference was that the more polar amino acids are more piezophilic than the heavy ones [14]. In this work, we also analyzed the proteome evolution of the three species of the Pyrococcus genus in terms of tendencies toward fixation of amino acids with particular physicochemical properties. We disclosed that the changes in protein structure of Pyrococcus might have been related with optimization of hydrophobic core volume in the vast majority of proteins, but not with adaptive evolution of proteins with specific function.

Thus, pressure might not have been the sole agent that brought about adaptation of the P. furiosus, P. horikoshii, and P. abyssi genomes. For example, at the stage of the divergence of P. horikoshii and P. abyssi, change in the trophic level might have been a no less important evolutionary factor. P. horikoshii might have acquired a consumer status at a high level, whereas P. abyssi at a low one.

Results and Discussion

Pyrococcus phylogeny

A phylogenetic tree of archaea of the Pyrococcus genus derived from comparisons of 16S rRNA gene sequences is given in Figure 1. Organisms whose genomes have been completely sequenced are underlined. To get an idea of how these microorganisms are distributed according to depth, habitat depth is given to the right of each species name. Most archaea of this genus inhabit at the depth of 1300 - 2600 m. They also include inhabitants of shallow water (P. furiosus [15] and P. woesi [16]), and the obligate piezophile Pyrococcus CH1 [8]. Pyrococcus CH1 has been sampled at the depth of 4700 m; experimental data indicate that they die at pressures below 15 MPa, 50-60 MPa being optimal for their growth.

Figure 1

Phylogenetic relationships among Pyrococcus strains (from [3-5,8,16,70-82]) based on the 16S rRNA gene sequences. Bayesian posterior probabilities of nodes are shown.

The data on phylogeny and habitat conditions allowed us to assume that the common ancestor of the P. furiosus, P. horikoshii, and P. abyssi might have inhabited at the sea depth of ~2000 m under high pressure (~20 MPa). Evolving from the common Pyrococcus ancestor, the branch with the P. furiosus ancestor separated. These organisms might have coped with life in shallow water and probably lost some of the functions providing survival under high pressure. The ancestor of P. horikoshii and P. abyssi might have remained tolerant to high pressure. However, P. horikoshii was less tolerant to high pressure and it became adapted to existence in an amino acid enriched environment (Table 1). P. abyssi remained in deep sea habitat where P. horikoshii and P. abyssi common ancestor occurred.

Gene loss events in the Pyrococcus proteomes

We have analyzed a sample of 164 groups of orthologous genes P. furiosus, P. horikoshii, and P. abyssi, of which one of the three species underwent gene loss (Additional data file 1). To define the functional classes of genes most susceptible to changes and the organisms, which as a rule lose genes with these functions, we compared the deviation of the number of lost genes with the 1:1:1 uniform distribution in different functional groups for the three genomes. The χ2 test was used to determine the significance of the deviation. The results are set out in Table 2. The data for gene loss in 164 groups of orthologous genes are in agreement with those reported elsewhere for comparisons of the complete Pyrococcus genomes (compare with Table 1). Table 2 clearly shows that gene loss according to function and organism are nonuniform. P. furiosus lost the chemotaxis and cell motility genes in the course of evolution; according to our classification, these are the genes referred to the functional group "signal transduction mechanisms" and "cell motility", respectively. Gene loss was dramatic in P. horikoshii. It concerned the genes for metabolism, those for energy production and conversion, transport and metabolism of carbohydrates and lipids. Taken together with the experimental data (Table 1), this allowed us to assign archaea of the P. horikoshii species to the group inhabiting enriched microbiological communities of hydrothermal vents, habitats saturated with amino acids, i.e. with a status of a high level consumer. In contrast, the number of gene losses during evolution is minimal in P. abyssi. Thanks to it, P. abyssi is able to exist in deficient aggressive environments [17] it is, probably, a consumer of a low level in microbiological communities of hydrothermal vents.

Table 2

Events of gene losses in the P. furiosus, P. horikoshii and P. abyssi genomes

Functional group	Number of genes lost
	P. horiko-shii	P. abyssi	P. furiosus	p* (χ2)
INFORMATION STORAGE AND PROCESSING	9	2	10	0.066
Translation; ribosomal structure and biogenesis	1	1	3	0.449
Transcription	5	1	4	0.273
Replication; recombination and repair	3	0	3	0.223
CELLULAR PROCESSES AND SIGNALING	4	4	25	0.000002
Cell cycle control; cell division; chromosome partitioning	1	0	0	0.368
Defense mechanisms	1	1	5	0.102
Signal transduction mechanisms	1	1	11	0.0004
Cell wall/membrane/envelope biogenesis	1	0	4	0.074
Cell motility	0	0	3	0.05
Intracellular trafficking; secretion; vesicular transport	0	1	1	0.607
Posttranslational modification; protein turnover; chaperones	0	1	1	0.607
METABOLISM	34	9	21	0.0007
Energy production and conversion	11	1	5	0.011
Carbohydrate transport and metabolism	8	0	4	0.018
Amino acid transport and metabolism	3	1	0	0.174
Nucleotide transport and metabolism	5	2	4	0.529
Coenzyme transport and metabolism	3	4	2	0.717
Lipid transport and metabolism	3	0	0	0.05
Inorganic ion transport and metabolism	1	1	5	0.102
Secondary metabolites biosynthesis; transport and catabolism	0	0	1	0.368
FUNCTION UNKNOWN	23	13	10	0.049

It has been shown that the archaeal genes of the Pyrococcus genus are prone to horizontal transfer [7,18,19]. Six regions extremely variable because of multiple gene transfers from related species/strains have been distinguished in P. furiosus [19]. Horizontal gene transfer may affect statistical estimates of gene loss significance. This prompted us to perform additional studies to estimate the frequency of these events in the analyzed 164 gene clusters and to judge how this may possibly affect the statistical relation between gene functional class and gene loss event (Additional data file 1). To identify the cluster with the horizontally transferred genes, we used the HGT-DB database [20,21]. This database contains the genes from the genomes of various prokaryotes, which they might have been acquired through horizontal transfer. Of the 164 clusters with ongoing gene loss, 9 (~5%) only according to the HGT-DB data potentially contained the horizontally transferred Pyrococcus genes. Of these 9, 6 were members of the group with unknown functions, the other 3 we annotated as performing functions of "nucleotide transport and metabolism", "posttranslational modification, protein turnover, chaperones", and "cell motility". Of the three functional groups according to the data in Table 2, the "cell motility" group only shows statistically significant deviation from 1:1:1, with all the lost genes in the group belonging to P. furiosus. The gene with the "cell motility" function, in which horizontal transfer might have occurred (GenBank GI 14590358), belongs to the P. horikoshii species. It is of interest that the horizontally transferred genes in the "nucleotide transport and metabolism" (GenBank GI 14590010) "posttranslational modification, protein turnover, chaperones" (GenBank GI 14590433) groups belong to P. horikoshii, too. Thus, the events of horizontal gene transfer for the genomes we analyzed could not have significantly affected the identification of functional classes dramatically undergoing gene loss.

Positive selection of the Pyrococcus protein-coding genes

Search of genes evolving under Darwinian positive selection was performed for 911 orthologous clusters (Additional data file 2). For each cluster, there were 4 homologous sequences of genes encoding proteins of the three Pyrococcus species (P. furiosus, P. horikoshii, and P. abyssi) and one T. onnurineus species as outgroup in each cluster (see Methods). The phylogenetic tree for these four microorganisms built from concatenated protein alignments from the 911 clusters is shown in Figure 2. Our further analysis proceeded from this tree. We were mostly interested in the evolutionary events associated with change in environmental conditions for P. abyssi, P. horikoshii, and P. furiosus species as they diverged from common ancestors on the internal nodes of the tree. The tree branches representing the divergence are designated as a, b, c, respectively (Figure 2). The internal tree branch representing evolution from the common ancestor of P. furiosus and T. onnurineus to the recent common ancestor of P. abyssi and P. horikoshii is designated as d.

Figure 2

Phylogenetic relationships among the P. abyssi, P. horikoshii, P. furiosus, and T. onnurineus genomes based on the concatenated amino acid sequences. Bayesian posterior probabilities of nodes are shown. Ingroup branches are labeled a through d.

To analyze the evolution mode of the genes, we used the sequences reconstructed for the inner tree nodes (Figure 2). We detected positive selection modes by calculation of the radical over conservative replacement ratio for genes and of the pairwise γ-distances for proteins (see Methods section).

As a result, we identified a number of genes that underwent positive selection at various stages of the evolution of the Pyrococcus genus. Of these, the genes in 154 clusters underwent positive selection for the branch a (Figure 2); in 208 clusters for the branch b; in 131 clusters for the branch c; in 168 clusters for the branch d (Additional data file 3). Positive selection was not identified on one of the branches leading to the Pyrococcus species in 422 clusters. A part of the genes underwent positive selection on one of the branches might have also underwent positive selection on its other branches. However, we also detected genes, which underwent selection pressure on a single branch of the tree. We designated such clusters as "unique". The relative proportion of the total number of "unique" clusters was 48.7% (75 clusters) for the branch a, 49.5% (103 clusters) for the branch b, 51.9% (68 clusters) for the branch c, and 57.1% (96 clusters) for the branch d (Additional data file 3).

Further analysis was aimed at detection of functional classes whose genes mainly underwent positive selection on the indicated branches (Additional data file 3). We determined significance of associations between the evolution under positive selection and the functional class of genes using the permutation statistical test (see Methods). The test was applied to all the genes undergoing pressure of positive selection and to the genes undergoing it on a certain branch (the "unique" clusters). The results for each of the examined branches are presented in Table 3.

Table 3

Functional groups enrichment with clusters under different evolution modes

Functional group	Total clusters in group	All positively selected clusters on the Pyrococcus tree branches (as in Figure 2) †				Unique positively selected clusters on the Pyrococcus tree branches (as in Figure 2) †				Clusters not under positive selection †
		a	b	c	d	a	b	c	d
Translation; ribosomal structure and biogenesis	168	32	45	48 ****	37	13	16	26 ****	19	55****
Transcription	62	13	25 ***	13	15	3	10	5	5	20**
Replication; recombination and repair	51	10	10	4*	6	6	6	3	5	26
Cell cycle control; cell division; chromosome partitioning	13	4	1*	1	1	2	0	0	1	8
Defense mechanisms	12	2	3	2	3	0	1	0	1	7
Signal transduction mechanisms	10	0	3	1	1	0	3	1	1	5
Cell wall/membrane/envelope biogenesis	19	2	1**	1*	2	2	1	1	2	13 *
Cell motility	11	3	2	3	2	1	2	1	1	4
Intracellular trafficking; secretion; vesicular transport	11	2	3	1	5 *	1	1	0	3	3*
Posttranslational modification; protein turnover; chaperones	32	6	8	2*	6	2	4	1	4	16
Energy production and conversion	78	13	14	10	12	5	9	8	5	40
Carbohydrate transport and metabolism	49	4*	10	6	8	2	6	2	6	28
Amino acid transport and metabolism	61	13	18	2***	10	7	9	2*	4	29
Nucleotide transport and metabolism	38	4	10	8	13 *	2	6	3	10 **	10**
Coenzyme transport and metabolism	58	12	17	8	11	5	9	4	5	23
Lipid transport and metabolism	15	1	2	0	3	0	2	0	2	10
Inorganic ion transport and metabolism	46	9	4**	4	4*	8 *	3	4	4	26
Secondary metabolites biosynthesis; transport and catabolism	7	2	1	0	2	2	0	0	1	3
Function unknown	170	22*	31*	17*	27	14	15	7*	17	96 **
Total	911	154	208	131	168	75	103	68	96	422

† - Bold denotes cases with excess of clusters (*0.01

† - Bold denotes cases with excess of clusters (*0.01