The stability of thermophilic proteins: a study based on comprehensive genome comparison

We address the question of the thermal stability of proteins in thermophiles through comprehensive genome comparison, focussing on the occurrence of salt bridges. We compared a set of 12 genomes (from four thermophilic archaeons, one eukaryote, six mesophilic eubacteria, and one thermophilic eubacteria). Our results showed that thermophiles have a greater content of charged residues than mesophiles, both at the overall genomic level and in alpha helices. Furthermore, we found that in thermophiles the charged residues in helices tend to be preferentially arranged with a 1–4 helical spacing and oriented so that intra-helical charge pairs agree with the helix dipole. Collectively, these results imply that intra-helical salt bridges are more prevalent in thermophiles than mesophiles and thus suggest that they are an important factor stabilizing thermophilic proteins. We also found that the proteins in thermophiles appear to be somewhat shorter than those in mesophiles. However, this later observation may have more to do with evolutionary relationships than with physically stabilizing factors. In all our statistics we were careful to controls for various biases. These could have, for instance, arisen due to repetitive or duplicated sequences. In particular, we repeated our calculation using a variety of random and directed sampling schemes. One of these involved making a "stratified sample," a representative cross-section of the genomes derived from a set of 52 orthologous proteins present roughly once in each genome. For another sample, we focused on the subset of the 52 orthologs that had a known 3D structure. This allowed us to determine the frequency of tertiary as well as main-chain salt bridges. Our statistical controls supported our overall conclusion about the prevalence of salt bridges in thermophiles in comparison to mesophiles. Electronic Publication     

Polysaccharide-Degrading Thermophiles Generated by Heterologous Gene Expression in Geobacillus kaustophilus HTA426

Thermophiles have important advantages over mesophiles as host organisms for high-temperature bioprocesses, functional production of thermostable enzymes, and efficient expression of enzymatic activities in vivo. To capitalize on these advantages of thermophiles, we describe here a new inducible gene expression system in the thermophile Geobacillus kaustophilus HTA426. Six promoter regions in the HTA426 genome were identified and analyzed for expression profiles using β-galactosidase reporter assay. This analysis identified a promoter region upstream of a putative amylose-metabolizing gene cluster that directed high-level expression of the reporter gene. The expression was >280-fold that without a promoter and was further enhanced 12-fold by maltose addition. In association with a multicopy plasmid, this promoter region was used to express heterologous genes. Several genes, including a gene whose product was insoluble when expressed in Escherichia coli, were successfully expressed as soluble proteins, with yields of 0.16 to 59 mg/liter, and conferred new functions to G. kaustophilus strains. Remarkably, cellulase and α-amylase genes conferred the ability to degrade cellulose paper and insoluble starch at high temperatures, respectively, generating thermophiles with the potential to degrade plant biomass. Our results demonstrate that this novel expression system expands the potential applications of G. kaustophilus.     

Patterns of temperature adaptation in proteins from Methanococcus and Bacillus

It has long been known that amino acid substitutions in proteins of organisms living at moderate and high temperatures (mesophiles and thermophiles, respectively) are not all symmetrical; for example, more aligned sites have lysine in mesophiles and arginine in thermophiles than have the opposite pattern. This is generally taken to indicate that certain amino acids are favored over others by selection at different temperatures. Previous comparisons of protein sequences from mesophiles and thermophiles have used relatively small numbers of sequences from a diverse array of species, meaning that only the most common amino acid substitutions could be examined and any taxon-specific patterns would be obscured. Here, we compare a large number of proteins between mesophiles and thermophiles in the archaeal genus Methanococcus and the bacterial genus Bacillus. Each genus exhibits dramatically asymmetrical substitution patterns for many pairs of amino acids. There are several pairs of amino acids for which one amino acid is favored in thermophilic Bacillus and the other is favored in thermophilic Methanococcus; this appears to result from the higher G + C content of the DNA of thermophilic Bacillus, a complication not seen in Methanococcus.     

Genome sequence of Oceanobacillus iheyensis isolated from the Iheya Ridge and its unexpected adaptive capabilities to extreme environments

Oceanobacillus iheyensis HTE831 is an alkaliphilic and extremely halotolerant Bacillus-related species isolated from deep-sea sediment. We present here the complete genome sequence of HTE831 along with analyses of genes required for adaptation to highly alkaline and saline environments. The genome consists of 3.6 Mb, encoding many proteins potentially associated with roles in regulation of intracellular osmotic pressure and pH homeostasis. The candidate genes involved in alkaliphily were determined based on comparative analysis with three Bacillus species and two other Gram-positive species. Comparison with the genomes of other major Gram-positive bacterial species suggests that the backbone of the genus Bacillus is composed of approximately 350 genes. This second genome sequence of an alkaliphilic Bacillus-related species will be useful in understanding life in highly alkaline environments and microbial diversity within the ubiquitous bacilli.     

Using a strategy based on the concept of convergent evolution to identify residue substitutions responsible for thermal adaptation

Factors that are related to thermostability of proteins have been extensively studied in recent years, especially by comparing thermophiles and mesophiles. However, most of them are global characters. It is still not clear how to identify specific residues or fragments which may be more relevant to protein thermostability. Moreover, some of the differences among the thermophiles and mesophiles may be due to phylogenetic differences instead of thermal adaptation. To resolve these problems, I adopted a strategy to identify residue substitutions evolved convergently in thermophiles or mesophiles. These residues may therefore be responsible for thermal adaptation. Four classes of genomes were utilized in this study, including thermophilic archaea, mesophilic archaea, thermophilic bacteria, and mesophilic bacteria. For most clusters of orthologous groups (COGs) with sequences from all of these four classes of genomes, I can identify specific residues or fragments that may potentially be responsible for thermal adaptation. Functional or structural constraints (represented as sequence conservation) were suggested to have higher impact on thermal adaptation than secondary structure or solvent accessibility does. I further compared thermophilic archaea and mesophilic bacteria, and found that the most diverged fragments may not necessarily correspond to the thermostability-determining ones. The usual approach to compare thermophiles and mesophiles without considering phylogenetic relationships may roughly identify sequence features contributing to thermostability; however, to specifically identify residue substitutions responsible for thermal adaptation, one should take sequence evolution into consideration.     

Robust determinants of thermostability highlighted by a codon frequency index capable of discriminating thermophilic from mesophilic genomes

Can genome analysis tell us about the lifestyle of an organism? We ask this question considering a thorough cross comparison of thermophilic and mesophilic genomes, since presently the number of available genomes is enough to ensure statistical significance of the results. We analyze, by means of principal component analysis (PCA), the codon composition of a database comprising 116 genomes, selected so as to include one species for each genus and show that a cross genomic approach can allow the extraction of common determinants of thermostability at the genome level. The results of our analysis indicate that all the known features of thermostability can be found in the 64 component loadings of the second principal axis of PCA. By this, we develop an index of thermostability whose discriminative power between mesophiles and thermophiles scores with 98% accuracy at the genome level and with 95% accuracy at the protein sequence level. We also prove that these results are not due to phylogenetic differences between archaea and bacteria.     

Amino acid coupling patterns in thermophilic proteins

Structural analysis is useful in elucidating structural features responsible for enhanced thermal stability of proteins. However, due to the rapid increase of sequenced genomic data, there are far more protein sequences than the corresponding three-dimensional (3D) structures. The usual sequence-based amino acid composition analysis provides useful but simplified clues about the amino acid types related to thermal stability of proteins. In this work, we developed a statistical approach to identify the significant amino acid coupling sequence patterns in thermophilic proteins. The amino acid coupling sequence pattern is defined as any 2 types of amino acids separated by 1 or more amino acids. Using this approach, we construct the rho profiles for the coupling patterns. The rho value gives a measure of the relative occurrence of a coupling pattern in thermophiles compared with mesophiles. We found that thermophiles and mesophiles exhibit significant bias in their amino acid coupling patterns. We showed that such bias is mainly due to temperature adaptation instead of species or GC content variations. Though no single outstanding coupling pattern can adequately account for protein thermostability, we can use a group of amino acid coupling patterns having strong statistical significance (p values < 10(-7)) to distinguish between thermophilic and mesophilic proteins. We found a good correlation between the optimal growth temperatures of the genomes and the occurrences of the coupling patterns (the correlation coefficient is 0.89). Furthermore, we can separate the thermophilic proteins from their mesophilic orthologs using the amino acid coupling patterns. These results may be useful in the study of the enhanced stability of proteins from thermophiles-especially when structural information is scarce. Proteins 2005. (c) 2005 Wiley-Liss, Inc.     

Elucidation of factors responsible for enhanced thermal stability of proteins: a structural genomics based study

Understanding the molecular basis for the enhanced stability of proteins from thermophiles has been hindered by a lack of structural data for homologous pairs of proteins from thermophiles and mesophiles. To overcome this difficulty, complete genome sequences from 9 thermophilic and 21 mesophilic bacterial genomes were aligned with protein sequences with known structures from the protein data bank. Sequences with high homology to proteins with known structures were chosen for further analysis. High quality models of these chosen sequences were obtained using homology modeling. The current study is based on a data set of models of 900 mesophilic and 300 thermophilic protein single chains and also includes 178 templates of known structure. Structural comparisons of models of homologous proteins allowed several factors responsible for enhanced thermostability to be identified. Several statistically significant, specific amino acid substitutions that occur going from mesophiles to thermophiles are identified. Most of these are at solvent-exposed sites. Salt bridges occur significantly more often in thermophiles. The additional salt bridges in thermophiles are almost exclusively in solvent-exposed regions, and 35% are in the same element of secondary structure. Helices in thermophiles are stabilized by intrahelical salt bridges and by an increase in negative charge at the N-terminus. There is an approximate decrease of 1% in the overall loop content and a corresponding increase in helical content in thermophiles. Previously overlooked cation-pi interactions, estimated to be twice as strong as ion-pairs, are significantly enriched in thermophiles. At buried sites, statistically significant hydrophobic amino acid substitutions are typically consistent with decreased side chain conformational entropy.     

Thermophilic prokaryotes have characteristic patterns of codon usage, amino acid composition and nucleotide content

A number of recent studies have shown that thermophilic prokaryotes have distinguishable patterns of both synonymous codon usage and amino acid composition, indicating the action of natural selection related to thermophily. On the other hand, several other studies of whole genomes have illustrated that nucleotide bias can have dramatic effects on synonymous codon usage and also on the amino acid composition of the encoded proteins. This raises the possibility that the thermophile-specific patterns observed at both the codon and protein levels are merely reflections of a single underlying effect at the level of nucleotide composition. Moreover, such an effect at the nucleotide level might be due entirely to mutational bias. In this study, we have compared the genomes of thermophiles and mesophiles at three levels: nucleotide content, codon usage and amino acid composition. Our results indicate that the genomes of thermophiles are distinguishable from mesophiles at all three levels and that the codon and amino acid frequency differences cannot be explained simply by the patterns of nucleotide composition. At the nucleotide level, we see a consistent tendency for the frequency of adenine to increase at all codon positions within the thermophiles. Thermophiles are also distinguished by their pattern of synonymous codon usage for several amino acids, particularly arginine and isoleucine. At the protein level, the most dramatic effect is a two-fold decrease in the frequency of glutamine residues among thermophiles. These results indicate that adaptation to growth at high temperature requires a coordinated set of evolutionary changes affecting (i) mRNA thermostability, (ii) stability of codon-anticodon interactions and (iii) increased thermostability of the protein products. We conclude that elevated growth temperature imposes selective constraints at all three molecular levels: nucleotide content, codon usage and amino acid composition. In addition to these multiple selective effects, however, the genomes of both thermophiles and mesophiles are often subject to superimposed large changes in composition due to mutational bias.     

A comparative categorization of gene flux in diverse microbial species

Microbial genomes harbor genomic islands (GIs), genes presumably acquired via horizontal gene transfer (HGT). We compared GIs of hyperthermophilic, thermophilic, mesophilic, and pathogenic/nonpathogenic species and of small and large genomes. The COG database was used to characterize gene-encoded functions. Putative donors were determined to quantify gene flux between superkingdoms. In hyperthermophiles, more than 10% of the genes were on average acquired across the superkingdom border. For thermophiles and particularly mesophiles, we identified a nearly unidirectional export from bacteria to archaea. Additionally, we analyzed GI composition for Escherichia, and pairs of Listeria, Rhizobiales, Methanosarcinaceae, and Thermus thermophilus/Deinococcus radiodurans. For Escherichia and Listeria, the composition of GIs in pathogenic and nonpathogenic species did not differ significantly with respect to encoded COG classes. The analysis of related genomes showed that the composition of GIs cannot be explained with trends of gene content known to depend on genome size.     

Knowledge-based discovery for designing CRISPR-CAS systems against invading mobilomes in thermophiles

Clustered regularly interspaced short palindromic repeats (CRISPRs) are direct features of the prokaryotic genomes involved in resistance to their bacterial viruses and phages. Herein, we have identified CRISPR loci together with CRISPR-associated sequences (CAS) genes to reveal their immunity against genome invaders in the thermophilic archaea and bacteria. Genomic survey of this study implied that genomic distribution of CRISPR-CAS systems was varied from strain to strain, which was determined by the degree of invading mobiloms. Direct repeats found to be equal in some extent in many thermopiles, but their spacers were differed in each strain. Phylogenetic analyses of CAS superfamily revealed that genes cmr, csh, csx11, HD domain, devR were belonged to the subtypes of cas gene family. The members in cas gene family of thermophiles were functionally diverged within closely related genomes and may contribute to develop several defense strategies. Nevertheless, genome dynamics, geological variation and host defense mechanism were contributed to share their molecular functions across the thermophiles. A thermophilic archaean, Thermococcus gammotolerans and thermophilic bacteria, Petrotoga mobilis and Thermotoga lettingae have shown superoperons-like appearance to cluster cas genes, which were typically evolved for their defense pathways. A cmr operon was identified with a specific promoter in a thermophilic archaean, Caldivirga maquilingensis. Overall, we concluded that knowledge-based genomic survey and phylogeny-based functional assignment have suggested for designing a reliable genetic regulatory circuit naturally from CRISPR-CAS systems, acquired defense pathways, to thermophiles in future synthetic biology.

Electronic supplementary material

The online version of this article (doi:10.1007/s11693-015-9176-8) contains supplementary material, which is available to authorized users.     

Protein thermal stability: insights from atomic displacement parameters (B values)

The factors contributing to the thermal stability of proteins from thermophilic origins are matters of intense debate and investigation. Thermophilic proteins are thought to possess better packed interiors than their mesophilic counterparts, leading to lesser overall flexibility and a corresponding reduction in surface-to-volume ratio. These observations prompted an analysis of B values reported in high-resolution X-ray crystal structures of mesophilic and thermophilic proteins. In this analysis, the following aspects were addressed: (1) frequency distribution of normalized B values (B' factors) over all the proteins and for individual amino acids; (2) amino acid compositions in high B value regions of polypeptide chains; (3) variation in the B values from core to the surface of proteins in terms of their radius of gyration; and (4) degree of dispersion of normalized B values in spheres around the Calpha atoms. The analysis revealed that (1) Ser and Thr have lesser flexibility in thermophiles than in mesophiles, (2) the proportion of Glu and Lys in high B value regions of thermophiles is higher and that of Ser and Thr is lower and (3) the dispersion of B values within spheres at Calpha atoms is similar in mesophiles and thermophiles. These observations reflect plausible differences in the dynamics of thermophilic and mesophilic proteins and suggest amino acid substitutions that are likely to change thermal stability.     

Two different alanine dehydrogenases from Geobacillus kaustophilus: Their biochemical characteristics and differential expression in vegetative cells and spores

Two putative alanine dehydrogenase (AlaDH) genes (GK2752 and GK3448) were found in the genome of a thermophilic spore-forming bacterium, Geobacillus kaustophilus. The amino acid sequences deduced from the two genes showed mutually high homology (71%), and the phylogenetic tree based on the amino acid sequences of the two putative AlaDHs and the homologous proteins showed that the two putative AlaDH genes (GK2752 and GK3448) belong to different groups. Both of the recombinant gene products exhibited high NAD⁺-dependent AlaDH activity and were purified to homogeneity and characterized in detail. Both enzymes showed high stability against low and high pHs and high temperatures (70 °C). Kinetic analyses showed that the activities of both enzymes proceeded according to the same sequentially ordered Bi-Ter mechanism. X-ray crystallographic analysis showed the two AlaDHs to have similar homohexameric structures. Notably, GK3448-AlaDH was detected in vegetative cells of G. kaustophilus but not spores, while GK2752-AlaDH was present only in the spores. This is the first report showing the presence of two AlaDHs separately expressed in vegetative cells and spores.     

Fatty Acid Distribution in Mesophilic and Thermophilic Strains of the Genus Bacillus

The fatty acid distribution of three mesophilic and three thermophilic strains of the genus Bacillus was determined by gas chromatography of the fatty acid methyl esters. Fatty acid i-15:0 was the most abundant in both the mesophiles (51%) and the thermophiles (41%). The second most abundant fatty acid was a-15:0 in the mesophiles (22%), and i-17:0 in the thermophiles (27%). The fatty acid pair i-15:0, i-17:0 was the most predominant pair in both the mesophiles (61%) and the thermophiles (66%). The fatty acid pair a-15:0, a-17:0 was the second most predominant pair and was much higher in the mesophiles (30%) than in the thermophiles (15%). The average fatty acid chain length was 15.5 for the mesophiles and 16.0 for the thermophiles. The significance of these results for the lipid theory of thermophily is discussed.     

Discrimination of mesophilic and thermophilic proteins using machine learning algorithms

Discriminating thermophilic proteins from their mesophilic counterparts is a challenging task and it would help to design stable proteins. In this work, we have systematically analyzed the amino acid compositions of 3075 mesophilic and 1609 thermophilic proteins belonging to 9 and 15 families, respectively. We found that the charged residues Lys, Arg, and Glu as well as the hydrophobic residues, Val and Ile have higher occurrence in thermophiles than mesophiles. Further, we have analyzed the performance of different methods, based on Bayes rules, logistic functions, neural networks, support vector machines, decision trees and so forth for discriminating mesophilic and thermophilic proteins. We found that most of the machine learning techniques discriminate these classes of proteins with similar accuracy. The neural network-based method could discriminate the thermophiles from mesophiles at the five-fold cross-validation accuracy of 89% in a dataset of 4684 proteins. Moreover, this method is tested with 325 mesophiles in Xylella fastidosa and 382 thermophiles in Aquifex aeolicus and it could successfully discriminate them with the accuracy of 91%. These accuracy levels are better than other methods in the literature and we suggest that this method could be effectively used to discriminate mesophilic and thermophilic proteins.     

Dark matter in archaeal genomes: a rich source of novel mobile elements,defense systems and secretory complexes

Microbial genomes encompass a sizable fraction of poorly characterized, narrowly spread fast-evolving genes. Using sensitive methods for sequences comparison and protein structure prediction, we performed a detailed comparative analysis of clusters of such genes, which we denote “dark matter islands”, in archaeal genomes. The dark matter islands comprise up to 20 % of archaeal genomes and show remarkable heterogeneity and diversity. Nevertheless, three classes of entities are common in these genomic loci: (a) integrated viral genomes and other mobile elements; (b) defense systems, and (c) secretory and other membrane-associated systems. The dark matter islands in the genome of thermophiles and mesophiles show similar general trends of gene content, but thermophiles are substantially enriched in predicted membrane proteins whereas mesophiles have a greater proportion of recognizable mobile elements. Based on this analysis, we predict the existence of several novel groups of viruses and mobile elements, previously unnoticed variants of CRISPR-Cas immune systems, and new secretory systems that might be involved in stress response, intermicrobial conflicts and biogenesis of novel, uncharacterized membrane structures.     

Patterns of temperature adaptation in proteins from the bacteria Deinococcus radiodurans and Thermus thermophilus

Asymmetrical patterns of amino acid substitution in proteins of organisms living at moderate and high temperatures (mesophiles and thermophiles, respectively) are generally taken to indicate selection favoring different amino acids at different temperatures due to their biochemical properties. If that were the case, comparisons of different pairs of mesophilic and thermophilic taxa would exhibit similar patterns of substitutional asymmetry. A previous comparison of mesophilic versus thermophilic Methanococcus with mesophilic versus thermophilic Bacillus revealed several pairs of amino acids for which one amino acid was favored in thermophilic Bacillus and the other was favored in thermophilic Methanococcus. Most of this could be explained by the higher G+C content of the DNA of thermophilic Bacillus, a phenomenon not seen in the Methanococcus comparison. Here, I compared the mesophilic bacterium Deinococcus radiodurans and its thermophilic relative Thermus thermophilus, which are similar in G+C content. Of the 190 pairs of amino acids, 83 exhibited significant substitutional asymmetry, consistent with the pervasive effects of selection. Most of these significantly asymmetrical pairs of amino acids were asymmetrical in the direction predicted from the Methanococcus data, consistent with thermal adaptation resulting from universal biochemical properties of the amino acids. However, 12 pairs of amino acids exhibited asymmetry significantly different from and in the opposite direction of that found in the Methanococcus comparison, and 21 pairs of amino acids exhibited asymmetry that was significantly different from that found in the Bacillus comparison and could not be explained by the greater G+C content in thermophilic Bacillus. This suggests that selection due to universal biochemical properties of the amino acids and differences in G+C content are not the only causes of substitutional asymmetry between mesophiles and thermophiles. Instead, selection on taxon-specific properties of amino acids, such as their metabolic cost, may play a role in causing asymmetrical patterns of substitution.     

Conjugative plasmid transfer from <Emphasis Type="Italic">Escherichia coli</Emphasis> is a versatile approach for genetic transformation of thermophilic <Emphasis Type="Italic">Bacillus</Emphasis> and <Emphasis Type="Italic">Geobacillus</Emphasis> species

We previously demonstrated efficient transformation of the thermophile Geobacillus kaustophilus HTA426 using conjugative plasmid transfer from Escherichia coli BR408. To evaluate the versatility of this approach to thermophile transformation, this study examined genetic transformation of various thermophilic Bacillus and Geobacillus spp. using conjugative plasmid transfer from E. coli strains. E. coli BR408 successfully transferred the E. coli–Geobacillus shuttle plasmid pUCG18T to 16 of 18 thermophiles with transformation efficiencies between 4.1 × 10^?7 and 3.8 × 10^?2/recipient. Other E. coli strains that are different from E. coli BR408 in intracellular DNA methylation also generated transformants from 9 to 15 of the 18 thermophiles, including one that E. coli BR408 could not transform, although the transformation efficiencies of these strains were generally lower than those of E. coli BR408. The conjugation was performed by simple incubation of an E. coli donor and a thermophile recipient without optimization of experimental conditions. Moreover, thermophile transformants were distinguished from abundant E. coli donor only by high temperature incubation. These observations suggest that conjugative plasmid transfer, particularly using E. coli BR408, is a facile and versatile approach for plasmid introduction into thermophilic Bacillus and Geobacillus spp., and potentially a variety of other thermophiles.     

Genome-wide patterns of nucleotide substitution reveal stringent functional constraints on the protein sequences of thermophiles

To test the hypothesis that the proteins of thermophilic prokaryotes are subject to unusually stringent functional constraints, we estimated the numbers of synonymous and nonsynonymous nucleotide substitutions per site between 17,957 pairs of orthologous genes from 22 pairs of closely related species of Archaea and Bacteria. The average ratio of nonsynonymous to synonymous substitutions was significantly lower in thermophiles than in nonthermophiles, and this effect was observed in both Archaea and Bacteria. There was no evidence that this difference could be explained by factors such as nucleotide content bias. Rather, the results support the hypothesis that proteins of thermophiles are subject to unusually strong purifying selection, leading to a reduced overall level of amino acid evolution per mutational event. The results show that genome-wide patterns of sequence evolution can be influenced by natural selection exerted by a species' environment and shed light on a previous observation that relatively few of the mutations arising in a thermophilic archaeon were nucleotide substitutions in contrast to indels.