Structure, function, and evolution of the family of superoxide dismutase proteins from halophilic archaebacteria.

The protein sequences of seven members of the superoxide dismutase (SOD) family from halophilic archaebacteria have been aligned and compared with each other and with the homologous Mn and Fe SOD sequences from eubacteria and the methanogenic archaebacterium Methanobacterium thermoautotrophicum. Of 199 common residues in the SOD proteins from halophilic archaebacteria, 125 are conserved in all seven sequences, and 64 of these are encoded by single unique triplets. The 74 remaining positions exhibit a high degree of variability, and for almost half of these, the encoding triplets are connected by at least two nonsynonymous nucleotide substitutions. The majority of nucleotide substitutions within the seven genes are nonsynonymous and result in amino acid replacement in the respective protein; silent third-codon-position (synonymous) substitutions are unexpectedly rare. Halophilic SODs contain 30 specific residues that are not found at the corresponding positions of the methanogenic or eubacterial SOD proteins. Seven of these are replacements of highly conserved amino acids in eubacterial SODs that are believed to play an important role in the three-dimensional structure of the protein. Residues implicated in formation of the active site, catalysis, and metal ion binding are conserved in all Mn and Fe SODs. Molecular phylogenies based on parsimony and neighbor-joining methods coherently group the halophile sequences but surprisingly fail to distinguish between the Mn SOD of Escherichia coli and the Fe SOD of M. thermoautotrophicum as the outgroup. These comparisons indicate that as a group, the SODs of halophilic archaebacteria have many unique and characteristic features. At the same time, the patterns of nucleotide substitution and amino acid replacement indicate that these genes and the proteins that they encode continue to be subject to strong and changing selection. This selection may be related to the presence of oxygen radicals and the inter- and intracellular composition and concentration of metal cations.     

Molecular cloning and sequencing of the gene for a halophilic alkaline serine protease (halolysin) from an unidentified halophilic archaea strain (172P1) and expression of the gene in Haloferax volcanii.

The gene of a halophilic alkaline serine protease, halolysin, from an unidentified halophilic archaea (archaebacterium) was cloned and its nucleotide sequence was determined. The deduced amino acid sequence showed that halolysin consists of 411 amino acids, with a molecular weight of 41,963. The highest homology was found with thermitase from Thermoactinomyces vulgaris. Halolysin has a long C-terminal extension of approximately 120 amino acids which has not been found in other extracellular subtilisin type serine proteases. The gene, hly, was expressed in another halophilic archaea, Haloferax volcanii, in a medium containing 18% salts by using a plasmid shuttle vector which has a novobiocin resistance determinant as a selectable marker.     

Specific phosphorylation of the acidic central region of the N-myc protein by casein kinase II.

The central region of the N-myc protein has a characteristic amino acid sequence EDTLSDSDDEDD, which is very similar to those of particular domains of adenovirus E1A, human papilloma virus E7, Simian virus 40 large T, c-myc and L-myc proteins. Domains of these three viral oncoproteins have recently been shown to be specific binding sites for the tumor-suppressor gene retinoblastoma protein. We have noted that the sequence of serine followed by a cluster of acidic amino acids is exactly the same as that of a typical substrate of casein kinase II (CKII). Therefore, we investigated whether these nuclear oncoproteins are phosphorylated by CKII. For this purpose, we fused the beta-galactosidase and N-myc genes including this domain and expressed it in Escherichia coli cells. Several mutant N-myc genes, containing single amino acid substitutions in this domain, were also used to produce fused proteins. Strong phosphorylation by CKII was detected with the fused protein of wild-type N-myc. However, no phosphorylation of beta-galactosidase itself was observed and the phosphorylations of fused mutant proteins were low. Another fused N-myc protein containing most of the C-terminal region downstream of this acidic region was not phosphorylated by CKII. Analysis of phosphorylation sites in synthetic peptides of this acidic region identified the major sites phosphorylated by CKII as Ser261 and Ser263. On two-dimensional tryptic mapping of phosphorylated N-myc proteins, major spots of in vitro-labeled and in-vivo-labeled N-myc proteins were detected in the same positions. These results suggest that two serine residues of the acidic central region of the N-myc protein are phosphorylated by CKII in vivo as well as in vitro. The functional significance of this acidic domain is discussed.     

Sequence variation of HIV and bioinformatics

The envelope glycoprotein of human immunodeficiency virus type 1 (HIV-1) interacts with receptors on the target cell and mediates virus entry by fusing the viral and cell membranes. To maintain the viral infectivity, amino acids that interact with receptors are expected to be more conserved than the other sites on the protein surface. In contrast to the functional constraint of amino acids for the receptor binding, some amino acid changes in this protein may produce antigenic variations that enable the virus to escape from recognition of the host immune system. Therefore, both positive selection (higher fitness) and negative selection (lower fitness) against amino acid changes are taking place during evolution of surface proteins of parasites To elucidate the evolutionary mechanisms of the whole HIV-1 gp120 envelope glycoprotein at the single site level, we collected and analyzed all available sequence data for the protein. By analyzing 186 sequences of the HIV-1 gp120 (subtype B), we reevaluated amino acid variability at the single site level, and estimated the numbers of synonymous and nonsynonymous substitutions at each codon position to detect positive and negative selection. We identified 33 amino acid positions which may be under positive selection. Some of these positions may form discontinuous epitopes. We also analyzed amino acid sequences to find amino acid positions responsible for usage of the second receptor. We found that, in addition to the V3 loop, amino acid variation at residue 440 in C4 region is clearly linked with the usage of CXCR 4.     

Think big--giant genes in bacteria

Long genes should be rare in archaea and eubacteria because of the demanding costs of time and resources for protein production. The search in 580 sequenced prokaryotic genomes, however, revealed 0.2% of all genes to be longer than 5 kb (absolute number: 3732 genes). Eighty giant bacterial genes of more than 20 kb in length were identified in 47 taxa that belong to the phyla Thermotogae (1), Chlorobi (3), Planctomycetes (1), Cyanobacteria (2), Firmicutes (7), Actinobacteria (9), Proteobacteria (23) or Euryarchaeota (1) (number of taxa in brackets). Giant genes are strain-specific, differ in their tetranucleotide usage from the bulk genome and occur preferentially in non-pathogenic environmental bacteria. The two longest bacterial genes known to date were detected in the green sulfur bacterium Chlorobium chlorochromatii CaD3 encoding proteins of 36 806 and 20 647 amino acids, being surpassed in length only by the human titin coding sequence. More than 90% of bacterial giant genes either encode a surface protein or a polyketide/non-ribosomal peptide synthetase. Most surface proteins are acidic, threonine-rich, lack cystein and harbour multiple amino acid repeats. Giant proteins increase bacterial fitness by the production of either weapons towards or shields against animate competitors or hostile environments.     

Amino acid signatures of salinity on an environmental scale with a focus on the Dead Sea

The increase of the acidic nature of proteins as an adaptation to hypersalinity has been well documented within halophile isolates. Here we explore the effect of salinity on amino acid preference on an environmental scale. Via pyrosequencing, we have obtained two distinct metagenomic data sets from the Dead Sea, one from a 1992 archaeal bloom and one from the modern Dead Sea. Our data, along with metagenomes from environments representing a range of salinities, show a strong linear correlation (R² = 0.97) between the salinity of an environment and the ratio of acidic to basic amino acids encoded by its inhabitants. Using the amino acid composition of putative protein‐encoding reads and the results of 16S rRNA amplicon sequencing, we differentiate recovered sequences representing microorganisms indigenous to the Dead Sea from lateral gene transfer events and foreign DNA. Our methods demonstrate lateral gene transfer events between a halophilic archaeon and relatives of the thermophilic bacterial genus Thermotoga and suggest the presence of indigenous Dead Sea representatives from 10 traditionally non‐hyperhalophilic bacterial lineages. The work suggests the possibility that amino acid bias of hypersaline environments might be preservable in fossil DNA or fossil amino acids, serving as a proxy for the salinity of an ancient environment. Finally, both the amino acid profile of the 2007 Dead Sea metagenome and the V9 amplicon library support the conclusion that the dominant microorganism inhabiting the Dead Sea is most closely related to a thus far uncultured relative of an alkaliphilic haloarchaeon.     

Fixation of deleterious mutations at critical positions in human proteins

Deleterious mutations associated with human diseases are predominantly found in conserved positions and positions that are essential for the structure and/or function of proteins. However, these mutations are purged from the human population over time and prevented from being fixed. Contrary to this belief, here I show that high proportions of deleterious amino acid changing mutations are fixed at positions critical for the structure and/or function of proteins. Similarly, a high rate of fixation of deleterious mutations was observed in slow-evolving amino acid positions of human proteins. The fraction of deleterious substitutions was found to be two times higher in relatively conserved amino acid positions than in highly variable positions. This study also found fixation of a much higher proportion of radical amino acid changes in primates compared with rodents and artiodactyls in slow-evolving positions. Previous studies observed a higher proportion of nonsynonymous substitutions in humans compared with other mammals, which was taken as indirect evidence for the fixation of deleterious mutations in humans. However, the results of this investigation provide direct evidence for this prediction by suggesting that the excess nonsynonymous mutations fixed in humans are indeed deleterious in nature. Furthermore, these results suggest that studies on disease-associated mutations should consider that a significant fraction of such deleterious mutations has already been fixed in the human genome, and thus, the effects of new mutations at those amino acid positions may not necessarily be deleterious and might even result in reversion to benign phenotypes.     

Unique amino acid composition of proteins in halophilic bacteria

The amino acid compositions of proteins from halophilic archaea were compared with those from non-halophilic mesophiles and thermophiles, in terms of the protein surface and interior, on a genome-wide scale. As we previously reported for proteins from thermophiles, a biased amino acid composition also exists in halophiles, in which an abundance of acidic residues was found on the protein surface as compared to the interior. This general feature did not seem to depend on the individual protein structures, but was applicable to all proteins encoded within the entire genome. Unique protein surface compositions are common in both halophiles and thermophiles. Statistical tests have shown that significant surface compositional differences exist among halophiles, non-halophiles, and thermophiles, while the interior composition within each of the three types of organisms does not significantly differ. Although thermophilic proteins have an almost equal abundance of both acidic and basic residues, a large excess of acidic residues in halophilic proteins seems to be compensated by fewer basic residues. Aspartic acid, lysine, asparagine, alanine, and threonine significantly contributed to the compositional differences of halophiles from meso- and thermophiles. Among them, however, only aspartic acid deviated largely from the expected amount estimated from the dinucleotide composition of the genomic DNA sequence of the halophile, which has an extremely high G+C content (68%). Thus, the other residues with large deviations (Lys, Ala, etc.) from their non-halophilic frequencies could have arisen merely as "dragging effects" caused by the compositional shift of the DNA, which would have changed to increase principally the fraction of aspartic acid alone.     

The rate heterogeneity of nonsynonymous substitutions in mammalian mitochondrial genes

Substitution rates at the three codon positions (r1, r2, and r3) of mammalian mitochondrial genes are in the order of r3 > r1 > r2, and the rate heterogeneity at the three positions, as measured by the shape parameter of the gamma distribution (alpha 1, alpha 2, and alpha 3), is in the order of alpha 3 > alpha 1 > alpha 2. The causes for the rate heterogeneity at the three codon positions remain unclear and, in particular, there has been no satisfactory explanation for the observation of alpha 1 > alpha 2. I attempted to dissect the causes of rate heterogeneity by studying the pattern of nonsynonymous substitutions with respect to codon positions in 10 mitochondrial genes from 19 mammalian species. Nonsynonymous substitutions involve more different amino acid replacements at the second than at the first codon position, which results in r1 > r2. The difference between r1 and r2 increases with the intensity of purifying selection, and so does the rate heterogeneity in nonsynonymous substitutions among sites at the same codon position. All mitochondrial genes appear to have functionally important and unimportant codons, with the latter having all three codon positions prone to nonsynonymous substitutions. Within the functionally important codons, the second codon position is much more conservative than the codon position. This explains why alpha 1 > alpha 2. The result suggests that overweighting of the second codon position in phylogenetic analysis may be a misguided practice.      

Pattern of Nucleotide Substitutions in Growth Hormone-Prolactin Gene Family: A Paradigm for Evolution by Gene Duplication

The growth hormone-prolactin gene family in mammals is an interesting example of evolution by gene duplication. Divergence among members of duplicated gene families and among species was examined by using reported gene sequences of growth hormone, prolactin and their receptors. Sequence divergence among species was found to show a general tendency in which a generation-time effect is pronounced for synonymous substitutions but not so for nonsynonymous substitutions. Divergence among duplicated genes is characterized by the relatively high rate of nonsynonymous substitutions, i.e., the rate is close to that of synonymous ones. In view of the stage- and tissue-specific expression of duplicated genes, some of the amino acid substitutions among duplicated genes is likely to be caused by positive Darwinian selection.     

Rates of Transition and Transversion in Coding Sequences since the Human-Rodent Divergence

Protein-coding sequences of 337 human genes were compared with those of homologous genes from rodent (mouse or rat). A composite alignment containing 477,189 nucleotide positions was constructed, and 21,570 amino acid replacements were inferred. The rates of transitional and transversional silent substitutions in fourfold degenerate sites are estimated as 1.71 × 10^-9 and 1.22 × 10^-9 site^-1 year^-1, respectively. Rates of substitutions in replacement sites, subject to selective constraints mediated by the genetic code, are lower, but also reflect a transitional bias. The amino acid exchange rejected least often during evolution is Asp/Glu, which is fixed at 30% the rate of transversions in silent sites. The most mutable amino acids in this survey are threonine and serine; serine coded by AGY is more mutable than serine coded by TCN. A scoring matrix for evaluating amino acid similarity was derived from this study.     

Two variable active site residues modulate response regulator phosphoryl group stability

Many signal transduction networks control their output by switching regulatory elements on or off. To synchronize biological response with environmental stimulus, switching kinetics must be faster than changes in input. Two-component regulatory systems (used for signal transduction by bacteria, archaea and eukaryotes) switch via phosphorylation or dephosphorylation of the receiver domain in response regulator proteins. Although receiver domains share conserved active site residues and similar three-dimensional structures, rates of self-catalysed dephosphorylation span a >or= 40,000-fold range in response regulators that control diverse biological processes. For example, autodephosphorylation of the chemotaxis response regulator CheY is 640-fold faster than Spo0F, which controls sporulation. Here we demonstrate that substitutions at two variable active site positions decreased CheY autodephosphorylation up to 40-fold and increased the Spo0F rate up to 110-fold. Particular amino acids had qualitatively similar effects in different response regulators. However, mutant proteins matched to other response regulators at the two key variable positions did not always exhibit similar autodephosphorylation kinetics. Therefore, unknown factors also influence absolute rates. Understanding the effects that particular active site amino acid compositions have on autodephosphorylation rate may allow manipulation of phosphoryl group stability for useful purposes, as well as prediction of signal transduction kinetics from amino acid sequence.     

Further Examples of Evolution by Gene Duplication Revealed through DNA Sequence Comparisons

To test the theory that evolution by gene duplication occurs as a result of positive Darwinian selection that accompanies the acceleration of mutant substitutions, DNA sequences of recent duplication were analyzed by estimating the numbers of synonymous and nonsynonymous substitutions. For the troponin C family, at the period of differentiation of the fast and slow isoforms, amino acid substitutions were shown to have been accelerated relative to synonymous substitutions. Comparison of the first exon of α-actin genes revealed that amino acid substitutions were accelerated when the smooth muscle, skeletal and cardiac isoforms differentiated. Analysis of members of the heat shock protein 70 gene family of mammals indicates that heat shock responsive genes including duplicated copies are evolving rapidly, contrary to the cognitive genes which have been evolutionarily conservative. For the α(1)-antitrypsin reactive center, the acceleration of amino acid substitution has been found for gene pairs of recent duplication.     

乌勇布拉克盐湖嗜盐古菌絮凝效果筛选及活性检测

【目的】从嗜盐古菌中筛选可产生生物絮凝剂的菌株,对发酵液、上清液、菌悬液、胞外聚合物的絮凝作用进行检测,筛选能够适应高盐废水处理,且具有广谱盐度及pH作用范围的微生物絮凝剂。【方法】以新疆乌勇布拉克干盐湖沉积物为研究对象,利用纯培养方法对嗜盐古菌进行分离,对絮凝菌株进行初筛及16S rRNA基因测序,构建系统进化树,初步判断菌株分类地位;复筛检测不同生物材料的絮凝效果;选择絮凝效果较好的生物材料,检测其盐度、pH的絮凝效果稳定性。【结果】采用纯培养方法共分离到28株嗜盐古菌,絮凝初筛共筛选出16株嗜盐古菌,分布于碱线菌属(Natrinema)、盐缓长菌属(Halopiger)和盐土生菌属(Haloterrigena)。菌株发酵液、上清液、菌悬液、胞外聚合物具有不同程度的絮凝效果。菌株A279-1、A133、RP33、NGA0064、RM-152、A389的发酵液、上清液的絮凝效果较好,其中菌株A389的发酵液絮凝率为61.06%,上清液为67.92%。所有菌株菌悬液的絮凝率达到80%以上。菌株所产胞外聚合物表现出较好的絮凝效果,菌株RM-152所产胞外聚合物的絮凝率最高,达89.86%,其次是A389 (81.53%)。菌株A389所产胞外聚合物的产量最大,达12.53 g/L,具有广泛的盐度和pH适应性。【结论】乌勇布拉克干盐湖沉积物中蕴含丰富的可产生微生物絮凝剂的嗜盐古菌资源。嗜盐古菌菌株发酵液、上清液、菌悬液及胞外聚合物均具有良好的絮凝作用,尤其是胞外聚合物表现出较好的絮凝效果,具有广谱的盐度和pH耐受性。嗜盐古菌所产生物絮凝剂的发现对于后续高盐废水功能材料开发具有重要应用价值。     

Molecular evolution of cadherin-related neuronal receptor/protocadherin(alpha) (CNR/Pcdh(alpha)) gene cluster in Mus musculus subspecies

The mouse cadherin-related neuronal receptor/protocadherin (CNR/Pcdh) gene clusters are located on chromosome 18. We sequenced single-nucleotide polymorphisms (SNPs) of the CNR/Pcdh(alpha)-coding region among 12 wild-derived and four laboratory strains; these included the four major subspecies groups of Mus musculus: domesticus, musculus, castaneus, and bactrianus. We detected 883 coding SNPs (cSNPs) in the CNR/Pcdh(alpha) variable exons and three in the constant exons. Among all the cSNPs, 586 synonymous (silent) and 297 nonsynonymous (amino acid exchanged) substitutions were found; therefore, the K(a)/K(s) ratio (nonsynonymous substitutions per synonymous substitution) was 0.51. The synonymous cSNPs were relatively concentrated in the first and fifth extracellular cadherin domain-encoding regions (ECs) of CNR/Pcdh(alpha). These regions have high nucleotide homology among the CNR/Pcdh(alpha) paralogs, suggesting that gene conversion events in synonymous and homologous regions of the CNR/Pcdh(alpha) cluster are related to the generation of cSNPs. A phylogenetic analysis revealed gene conversion events in the EC1 and EC5 regions. Assuming that the common sequences between rat and mouse are ancestral, the GC content of the third codon position has increased in the EC1 and EC5 regions, although biased substitutions from GC to AT were detected in all the codon positions. In addition, nonsynonymous substitutions were extremely high (11 of 13, K(a)/K(s) ratio 5.5) in the laboratory mouse strains. The artificial environment of laboratory mice may allow positive selection for nonsynonymous amino acid variations in CNR/Pcdh(alpha) during inbreeding. In this study, we analyzed the direction of cSNP generation, and concluded that subspecies-specific nucleotide substitutions and region-restricted gene conversion events may have contributed to the generation of genetic variations in the CNR/Pcdh genes within and between species.     

Correlated asymmetry of sequence and functional divergence between duplicate proteins of Saccharomyces cerevisiae

The role of sequence divergence in functional divergence of duplicate genes is a topic of great interest. In this study, we compare the numbers of amino acid substitutions in each sequence since two yeast duplicates diverged, using a preduplication ancestral outgroup. Using this strategy, we explored the relationship between sequence divergence and functional divergence between duplicate partners. We show that the degree of relative functional asymmetry between duplicate proteins is proportional to the relative sequence divergence between them. Furthermore, of the two duplicates, the copy closer to their ancestral sequence (fewer number of amino acid substitutions) interacts with more proteins and affects fitness more severely when deleted. Therefore, asymmetric sequence divergence between duplicates is correlated with asymmetric functional divergence and may underlie the duplicate's role in genetic robustness against mutations. Among the functional traits considered, protein abundance appears to have the strongest correlation with the nonsynonymous divergence between duplicates. Taken together with the results from whole-genome analyses, our results indicate that within-species duplicates are subject to the same evolutionary force that acts on interspecific sequence and functional divergence. In particular, we detect signs of purifying selection on the more slowly evolving duplicate.     

Mitochondrial ATPase subunit 6 and cytochrome B gene polymorphisms in young obese adults.

We hypothesized that the mutational strand asymmetry is more strongly exerted upon the mitochondrial cytochrome b (Cytb) gene, which is distant from the origin of the light-strand replication (Ori(L)), than upon the ATPase subunit 6 (ATP6) gene, which is close to the Ori(L). To test this hypothesis, we determined the sequences of these two genes in 96 Japanese young obese adults. The frequency of G-->A transitions was significantly higher than that of C-->T transitions in the Cytb gene, whereas the frequencies of G-->A and C-->T transitions were not significantly different in the ATP6 gene. The marked mutational strand asymmetry in the Cytb gene can be explained by the deamination of C to uracil in the long single-stranded state of the heavy strand during replication. The ratio of the nonsynonymous substitutions at the second codon positions to those at the first codon positions was significantly lower in the Cytb gene than in the ATP6 gene. The physicochemical differences between the standard and the replaced amino acid residues were significantly smaller in the Cytb gene than in ATP6 one. The present study indicates that amino acid sequences are less variable for Cytb than for ATP6 in spite of the strong mutational strand asymmetry for the Cytb gene.     

Haloadaptation: insights from comparative modeling studies of halophilic archaeal DHFRs

Proteins of halophilic archaea function in high-salt concentrations that inactivate or precipitate homologous proteins from non-halophilic species. Haloadaptation and the mechanism behind the phenomenon are not yet fully understood. In order to obtain useful information, homology modeling studies of dihydrofolate reductases (DHFRs) from halophilic archaea were performed that led to the construction of structural models. These models were subjected to energy minimization, structural evaluation and analysis. Complementary approaches concerning calculations of the amino acid composition and visual inspection of the surfaces and cores of the models, as well as calculations of electrostatic surface potentials, in comparison to non-halophilic DHFRs were also performed. The results provide evidence that sheds some light on the phenomenon of haloadaptation: DHFRs from halophilic archaea may maintain their fold, in high-salt concentrations, by sharing highly negatively charged surfaces and weak hydrophobic cores.     

What Amino Acid Properties Affect Protein Evolution?

We studied 10 protein-coding mitochondrial genes from 19 mammalian species to evaluate the effects of 10 amino acid properties on the evolution of the genetic code, the amino acid composition of proteins, and the pattern of nonsynonymous substitutions. The 10 amino acid properties studied are the chemical composition of the side chain, two polarity measures, hydropathy, isoelectric point, volume, aromaticity, aliphaticity, hydrogenation, and hydroxythiolation. The genetic code appears to have evolved toward minimizing polarity and hydropathy but not the other seven properties. This can be explained by our finding that the presumably primitive amino acids differed much only in polarity and hydropathy, but little in the other properties. Only the chemical composition (C) and isoelectric point (IE) appear to have affected the amino acid composition of the proteins studied, that is, these proteins tend to have more amino acids with typical C and IE values, so that nonsynonymous mutations tend to result in small differences in C and IE. All properties, except for hydroxythiolation, affect the rate of nonsynonymous substitution, with the observed amino acid changes having only small differences in these properties, relative to the spectrum of all possible nonsynonymous mutations. Received: 2 January 1998 / Accepted: 25 April 1998