Analysis of Nanoarchaeum equitans genome and proteome composition: indications for hyperthermophilic and parasitic adaptation

The hyperthermophilic archaeon Nanoarchaeum equitans is characterized by several intriguing features. It is the only known parasitic archaeon and for survival it must be in contact with the crenarchaeon host Ignicoccus. Its genome size is only 490 kb, representing the smallest microbial genome known to date, and yet it has the highest coding density, encoding for 536 genes [1]. Phylogenetic analyses suggested that this microbe is probably a derived, but genomically stable parasite diverged anciently from the archaeal lineage [2].

The genes for several vital metabolic pathways appear to be missing in Nanoarchaeum [1]. This could be due to two plausible reasons. N. equitans might represent an ancient species, and hence possesses a small genome. Alternatively, it might have gone through a process of genome reduction as a strategy of adaptation to the obligatory parasitic lifestyle, as observed in cases of many other parasitic/symbiotic organisms [1]. However, in obligatory intracellular bacteria, many genes involved in DNA recombination and repair along with the biosynthetic and metabolic genes are usually lost [3, 4]. But N. equitans possesses most of the DNA repair enzymes and the complete genetic machinery necessary for transcription, translation and DNA replication. The complexity of its information processing systems and the simplicity of its metabolic apparatus, therefore, suggest the presence of an unanticipated world of organisms yet to be characterized.

Most of the obligatory symbiotic/parasitic bacteria are characterized by the presence of only one/two rRNA operons, a small number of genes for tRNA isoacceptors, slow growth rate and an overall AT-richness [5–7]. It has been proposed that at the beginning of the symbiotic or parasitic integration, the loss of the genes involved in DNA repair favored the bias toward A+T content of such genomes [3, 4, 8]. However, the presence of a full set of archaeal DNA repair and recombination enzymes in N. equitans [1] contradicts the established hypothesis regarding AT-richness of its genome. Evidences for apparently little or no translational selection in synonymous codon usage have been reported for most of the species with reduced genomes examined so far, such as Borrelia burgdorferi [9], Buchnera aphidicola [10], Helicobacter pylori [11], Bartonella [12], Wigglesworthia [13] and Tropheryma whipplei [14] etc. Some of these bacteria exhibit strong base compositional asymmetries between leading and lagging strands of replication. It was, therefore, of interest to investigate whether N. equitans, an archaeon, is also characterized by any of such typical traits of the reduced genomes.

The composition of N. equitans genome and proteome are expected to bear the signatures not only of parasitism, but also of hyperthermophilicity. Comparative analysis of complete genomes of several hyperthermophilic archaea and bacteria revealed that organisms adapted to high temperature require a coordinated set of evolutionary changes towards stability of mRNA, codon-anticodon interactions [15], increased thermostability of encoded proteins by van der Waals interactions [16], larger number of residues in the alpha-helical conformation [17], enhanced secondary structure propensity [18], higher core hydrophobicity [19], additional networks of hydrogen bonds [20], increased ionic interactions [21], increased packing density [22], decreased length of surface loops [23] etc. With increase in growth temperature, microbial organisms tend to acquire base A and lose base C while keeping the contents of bases T and G relatively constant [24], and there is a clear link between a particular pattern of codon usage and the elevated growth temperature [25].

The current report presents an extensive study on the genome and proteome composition of N. equitans, along with a comparative analysis of the compositional characteristics of other mesophiles, hyperthermophiles and obligatory host-associated organisms. Only A+T -rich organisms were selected for analysis, so that the inter-species differences in nucleotide/amino acid usage patterns due to mutational bias could be minimized and any difference in such usage patterns among the mesophilic and hyperthermopilic organisms could be fairly attributed to their adaptation to the growth temperature. The study provides a more detailed view on the genome-wide strategies employed by N. equitans for adaptation to the hyperthermophilic environment and parasitic life style.

The present analysis indicates that the dual adaptation of N. equitans to high temperature and to an obligate parasitism has imposed selective constraints on nucleotide usage at synonymous and nonsynonymous codon positions, modulating thereby its genome/proteome composition. Thermal adaptation involves overrepresentation of purine bases in protein coding sequences, higher GC-content of the structural RNA genes, enhanced usage of positively charged residues, higher frequencies of aromatic residues, decrease in polar uncharged residues in the encoded protein etc., while parasitic adaptation is reflected in the extreme genome reduction, presence of weak translational selection for synonymous codon usage, limited number of tRNAs and rRNAs, large heterogeneity in membrane associated proteins and so on.

One of the most exciting observations is the significant increase in the usage of positively charged amino acid residues in encoded proteins of N. equitans and other hyperthermophiles compared to that in mesophilic organisms. A strong positive correlation between the optimal growth temperature of the organisms and the percentage of proteins with P/N ratio > 1 in the respective proteomes (Fig. 3b), relatively basic nature of the proteomes of N. equitans and other hyperthermophiles, as depicted by isoelectric point distribution (Fig. 3c) and bias in the replacement of uncharged polar residues of mesophilic proteins by positively charged residues (mainly Lys) in the N. equitans orthologs (Table 3) – all point towards a strong preference for positively charged amino acids in the gene-products of hyperthermophiles. In parallel, there has also been an increase in aromatic residues (especially Tyr) in encoded proteins of N. equitans and other hyperthermophiles (Fig. 3d; Table 3). Greater involvement of positively charged residues at or near protein surfaces may increase the probability of salt bridge formation with negatively charged residues, while simultaneous increase in aromatic residues may strengthen the cation-π interaction. When a cationic side chain comes near an aromatic side chain within a protein, the geometry is known to be biased towards one that would experience a favorable cation-π interaction [41]. It was suggested earlier that both salt-bridge and cation-π interactions may play important roles in thermostability [42]. Tyrosine, by itself, may also contribute to protein thermostability [43]. Selective increase in Lys/Arg and aromatic residues in N. equitans may, therefore, be a strategy of survival at high temperature. A recent study on atomic simulation has revealed that among the charged residues, Lys has much greater number of accessible rotamers than Arg and may entropically stabilize the folded states of proteins [44].

Marked reduction in the frequencies of uncharged polar residues may also contribute to thermostability by avoiding the deamination and backbone cleavages involving Asn and Gln, which can be catalyzed by Ser and Thr [45, 46]. According to the Sweet and Eisenberg scale [34], there is a significant increase in average hydrophobicity in the encoded proteins of N. equitans, as compared to their mesophilic orthologs. This may also be a part of the measures taken for environmental adaptation. The Kyte-Doolittle scale [47], however, did not indicate any significant difference in average hydrophobicity between these two groups of proteins. It was suggested earlier by Haney et al. [32] that some of the established hydrophobicity scales are strongly correlated to the differences between the proteins of mesophiles and thermophiles, whereas others are not. Replacement of the uncharged polar residues of mesophilic proteins by more hydrophobic residues in N. equitans orthologs may lead to an increase in the extent of the hydrophobic core and hence to a decrease in the solvent accessible surface area of the protein. Therefore the stability of N. equitans proteins in extremely high temperatures is apparently provided by significant modifications in their sequences toward enrichment of certain residues.

It is interesting to note that unlike mesophilic organisms, in N. equitans and other hyperthermophiles, there is a markedly differential selection for nucleotide usage in protein coding and structural RNA sequences. Both nonsynonymous and synonymous codon positions of their coding sequences are purine rich, and this has two probable consequences. Firstly, due to purine richness of protein-coding sequences, the organisms may minimize unnecessary RNA-RNA interactions and prevent the double-stranded RNA formation within the molecule [30]. Secondly, the higher purine content in nonsynonymous codon position has good correlation with the increased frequencies of certain residues in the encoded proteins. These may help the organism to adapt in high temperature. In contrast, the structural RNA sequences (tRNAs and rRNAs) of these organisms are characterized by significantly higher GC-content and hence by an increased number of hydrogen bonds, which may facilitate intramolecular stabilization at elevated temperature [29, 48]. Thus, in N. equitans, selection for the prevalence of purine bases in ORFs and the GC- richness of non-coding RNA sequences may also be the consequence of its hyperthermophilic adaptation.

Recent studies on several microbial genomes indicate a close connection between synonymous codon usage bias, tRNA abundance, number of rRNA operons, optimal generation time and genome size [6, 7, 49]. Most of the species with reduced genomes are host-associated microorganisms, characterized by the presence of only one or two rRNA operons, a small number of tRNA genes, long generation time and overall AT-richness. Evidences for apparently little translational selection have been reported in most of these organisms. N. equitans, which is an archaeal parasite with the smallest genome known so far, encodes only a limited number of tRNAs (38 identified tRNAs) and single copies of 5S, 16S and 23S rRNA. Although N. equitans is a hyperthermophilic archaeon, evidence for a relatively poor translational selection for synonymous codon usage is consistent with the earlier observations on several bacteria adapted to strictly host-associated lifestyle. Furthermore, the synonymous codon usage pattern of N. equitans forms a subset of the patterns observed typically in hyperthermophilic microbial species and is quite distinct from the patterns of the mesophilic organisms (Fig. 5a). Synonymous codon usage in Nanoarchaeal genes, therefore, reveals a dual adaptation to obligatory parasitism and hyperthermophilicity.

As demonstrated by the COA on amino acid usage, probable membrane associated proteins exhibit two different clusters (Fig. 4a). The amino acid usage profile and the predicted secondary structures of the members of these two clusters are quite distinct from one another (Table 5). Most of the variations in cell-surface proteins may be potentially important for N. equitans to interact with the Ignicoccus host and probably evolved during the course of parasitic and/or thermal adaptation. It is also important to note that in spite of its obligatory parasitic lifestyle, there is a tendency to follow the cost-minimization hypothesis at lower level as proteins encoded by highly expressed genes are preferentially constructed with some smaller and energetically less expensive amino acids. Existence of cost minimization effect in host-associated organisms might be due to a genome-level adaptation to utilize less expensive and small residues from the host in the highly expressed genes [12, 14, 50]. This might have an evolutionary advantage to minimize host energy exhaustion for maintaining continued association and the chance of elimination by the host.

Hyperthermophilic organisms, in general, have comparatively smaller genome than the mesophilic organisms. It might be advantageous in hyperthermophiles to maintain multiple copies of chromosomes per cell due to a probable need of a reserve supply of intact chromosome to compensate for the greater chance of DNA double strand breaks at high temperature [51, 52]. Furthermore, the faster replication of small genome is likely to be more favorable in the environment having temperatures near to or above 100°C [52]. Many microbial organisms living in close association with other organisms in an obligate symbiotic or parasitic relationship also experienced a reduction in genome size with respect to their free-living ancestors. The N. equitans genome lacks the genes for central metabolism, primary biosynthesis and bioenergetic apparatus [1], which are expected to be present in the common archaeal ancestor. In contrast to mesophilic organisms, it possesses the simplest functional protein folding system – the genome contains only single copies of homologues of prefoldin α- and β-subunits, Hsp60 and sHsp [52]. Unlike other obligate symbiotic/parasitic organisms, N. equitans has well-organized DNA repair mechanism with a full set of archaeal DNA repair and recombination enzymes [1]. Furthermore, despite the small genome size, it devotes a large amount of coding capacity for surface-associated proteins, suggesting that the interaction with its host may play a major part in the parasitic adaptation of the organism. Hence, it can be inferred that the unusual genome reduction and genome composition in N. equitans are the consequences of both hyperthermophilic and parasitic adaptation and during the coevolutionary process with Ignicoccus host, N. equitans may have experienced a dramatic decrease of genome size, retaining only the essential genes for its thermo-parasitic lifestyle.

Comprehensive analysis on the N. equitans genome along with its comparison to other mesophiles, hyperthermophiles and host-associated organisms allowed us to understand how the dual adaptation of N. equitans to high temperature and to an obligate parasitism can influence the nucleotide usage at synonymous and nonsynonymous codon positions, modulating thereby its genome/proteome composition. Thermal adaptation involves overrepresentation of purine bases in protein coding sequences, higher GC-content of the structural RNAs, enhanced usage of positively charged residues and aromatic residues, decrease in polar uncharged residues in the encoded protein and so on, while the parasitic adaptation is reflected in the extreme genome reduction, presence of weak translational selection for synonymous codon usage, large heterogeneity in membrane associated proteins etc. Our findings not only offer an insight into the mechanisms of genomic adaptation of N. equitans to high temperature and parasitism, but also evaluate the generality of such mechanisms in the microbial world.