Alternative design of a tRNA core for aminoacylation

The core of Escherichia coli tRNA(Cys) is important for aminoacylation of the tRNA by cysteine-tRNA synthetase. This core differs from the common tRNA core by having a G15:G48, rather than a G15:C48 base-pair. Substitution of G15:G48 with G15:C48 decreases the catalytic efficiency of aminoacylation by two orders of magnitude. This indicates that the design of the core is not compatible with G15:C48. However, the core of E. coli tRNA(Gln), which contains G15:C48, is functional for cysteine-tRNA synthetase. Here, guided by the core of E. coli tRNA(Gln), we sought to test and identify alternative functional design of the tRNA(Cys) core that contains G15:C48. Although analysis of the crystal structure of tRNA(Cys) and tRNA(Gln) implicated long-range tertiary base-pairs above and below G15:G48 as important for a functional core, we showed that this was not the case. The replacement of tertiary interactions involving 9, 21, and 59 in tRNA(Cys) with those in tRNA(Gln) did not construct a functional core that contained G15:C48. In contrast, substitution of nucleotides in the variable loop adjacent to 48 of the 15:48 base-pair created functional cores. Modeling studies of a functional core suggests that the re-constructed core arose from enhanced stacking interactions that compensated for the disruption caused by the G15:C48 base-pair. The repacked tRNA core displayed features that were distinct from those of the wild-type and provided evidence that stacking interactions are alternative means than long-range tertiary base-pairs to a functional core for aminoacylation.     

Efficiencies of different genes and different tree-building methods in recovering a known vertebrate phylogeny

The relative efficiencies of different protein-coding genes of the mitochondrial genome and different tree-building methods in recovering a known vertebrate phylogeny (two whale species, cow, rat, mouse, opossum, chicken, frog, and three bony fish species) was evaluated. The tree-building methods examined were the neighbor joining (NJ), minimum evolution (ME), maximum parsimony (MP), and maximum likelihood (ML), and both nucleotide sequences and deduced amino acid sequences were analyzed. Generally speaking, amino acid sequences were better than nucleotide sequences in obtaining the true tree (topology) or trees close to the true tree. However, when only first and second codon positions data were used, nucleotide sequences produced reasonably good trees. Among the 13 genes examined, Nd5 produced the true tree in all tree-building methods or algorithms for both amino acid and nucleotide sequence data. Genes Cytb and Nd4 also produced the correct tree in most tree-building algorithms when amino acid sequence data were used. By contrast, Co2, Nd1, and Nd41 showed a poor performance. In general, large genes produced better results, and when the entire set of genes was used, all tree-building methods generated the true tree. In each tree-building method, several distance measures or algorithms were used, but all these distance measures or algorithms produced essentially the same results. The ME method, in which many different topologies are examined, was no better than the NJ method, which generates a single final tree. Similarly, an ML method, in which many topologies are examined, was no better than the ML star decomposition algorithm that generates a single final tree. In ML the best substitution model chosen by using the Akaike information criterion produced no better results than simpler substitution models. These results question the utility of the currently used optimization principles in phylogenetic construction. Relatively simple methods such as the NJ and ML star decomposition algorithms seem to produce as good results as those obtained by more sophisticated methods. The efficiencies of the NJ, ME, MP, and ML methods in obtaining the correct tree were nearly the same when amino acid sequence data were used. The most important factor in constructing reliable phylogenetic trees seems to be the number of amino acids or nucleotides used.      

Structure and molecular phylogeny of sasA genes in cyanobacteria: insights into evolution of the prokaryotic circadian system

Cyanobacteria are the simplest organisms known to have a circadian system. In addition to the three well-studied kai genes, kaiA, kaiB, and kaiC, an important element of this system is a two-component sensory transduction histidine kinase sasA. Using publicly available data of complete prokaryotic genomes, we performed structural and phylogenetic analyses of the sasA genes. Results show that this gene has a triple-domain structure, and the domains are under different selective constraints. The sasA gene originated in cyanobacteria probably through the fusion of the ancestral kaiB gene with a double-domain, two-component sensory transduction histidine kinase. The results of the phylogenetic analyses suggest that sasA emerged before the kaiA gene, about 3,000-2,500 MYA, and has evolved in parallel with the evolution of the kaiBC cluster. The observed concordant patterns of the sasA and kaiBC evolution suggest that these genes might compose an ancient KaiBC-SasA-based circadian system, without the kaiA gene, and that such a system still exists in some unicellular cyanobacteria.     

Probabilistic methods of identifying genes in prokaryotic genomes: connections to the HMM theory

In this paper, we review developments in probabilistic methods of gene recognition in prokaryotic genomes with the emphasis on connections to the general theory of hidden Markov models (HMM). We show that the Bayesian method implemented in GeneMark, a frequently used gene-finding tool, can be augmented and reintroduced as a rigorous forward-backward (FB) algorithm for local posterior decoding described in the HMM theory. Another earlier developed method, prokaryotic GeneMark.hmm, uses a modification of the Viterbi algorithm for HMM with duration to identify the most likely global path through hidden functional states given the DNA sequence. GeneMark and GeneMark.hmm programs are worth using in concert for analysing prokaryotic DNA sequences that arguably do not follow any exact mathematical model. The new extension of GeneMark using the FB algorithm was implemented in the software program GeneMark.fba. Given the DNA sequence, this program determines an a posteriori probability for each nucleotide to belong to coding or non-coding region. Also, for any open reading frame (ORF), it assigns a score defined as a probabilistic measure of all paths through hidden states that traverse the ORF as a coding region. The prediction accuracy of GeneMark.fba determined in our tests was compared favourably to the accuracy of the initial (standard) GeneMark program. Comparison to the prokaryotic GeneMark.hmm has also demonstrated a certain, yet species-specific, degree of improvement in raw gene detection, ie detection of correct reading frame (and stop codon). The accuracy of exact gene prediction, which is concerned about precise prediction of gene start (which in a prokaryotic genome unambiguously defines the reading frame and stop codon, thus, the whole protein product), still remains more accurate in GeneMarkS, which uses more elaborate HMM to specifically address this task.     

Determination of whole prokaryotic phylogeny by the development of a random extraction method

The construction of accurate prokaryotic phylogeny is important not only in the field of evolutionary biology, but also in microbiology and pathology. However, in constructing a phylogenetic tree to trace prokaryotic evolution, the phylogenetic relationship is often changed by the choice of species. For the estimation of the accurate lineage of prokaryotes, a new method, named the "random extraction method", was developed. In this method, 16S rRNA sequence data were randomly extracted 1000 times from each closely-related taxa such as seven phyla of Eubacteria and one domain of Archaea and phylogenetic trees were constructed by the data to clarify the relationship of those groups. Next, the tree topology was counted and the most supported tree topology was found as the most plausible phylogenetic tree. To evaluate the reliability of each node, we developed the "Branching rate" (BR) and calculated for every tree. And also, computational simulation analysis was carried out to confirm these methods. On the assumption that the root of life is between Archaea and Eubacteria, the obtained phylogenetic relationships of phyla are the following. At first, Archaea (Euryarchaeota, Crenarchaeota and Korarchaeota) diverged, and Thermotogales, Cyanobacteria and Chlamydiales diverged in this order, then Firmicutes (Actinobacteria and Bacillus/Clostridium group cluster) and Proteobacteria (alpha and beta/gamma cluster) diverged. In addition, it was shown by the BR that the position of the node of Firmicutes Actinobacteria and Firmicutes Bacillus/Clostridium was changeable for each extraction. Therefore, it was suggested that the differences among the phylogenetic trees of prokaryotes were caused by the influence of these phyla.     

Missing genes in the annotation of prokaryotic genomes

Background  

Protein-coding gene detection in prokaryotic genomes is considered a much simpler problem than in intron-containing eukaryotic genomes. However there have been reports that prokaryotic gene finder programs have problems with small genes (either over-predicting or under-predicting). Therefore the question arises as to whether current genome annotations have systematically missing, small genes.     

Genome phylogenies indicate a meaningful alpha-proteobacterial phylogeny and support a grouping of the mitochondria with the Rickettsiales

Placement of the mitochondrial branch on the tree of life has been problematic. Sparse sampling, the uncertainty of how lateral gene transfer might overwrite phylogenetic signals, and the uncertainty of phylogenetic inference have all contributed to the issue. Here we address this issue using a supertree approach and completed genomic sequences. We first determine that a sensible alpha-proteobacterial phylogenetic tree exists and that it can confidently be inferred using orthologous genes. We show that congruence across these orthologous gene trees is significantly better than might be expected by random chance. There is some evidence of horizontal gene transfer within the alpha-proteobacteria, but it appears to be restricted to a minority of genes ( approximately 23%) most of whom ( approximately 74%) can be categorized as operational. This means that placement of the mitochondrion should not be excessively hampered by interspecies gene transfer. We then show that there is a consistently strong signal for placement of the mitochondrion on this tree and that this placement is relatively insensitive to methodological approach or data set. A concatenated alignment was created consisting of 15 mitochondrion-encoded proteins that are unlikely to have undergone any lateral gene transfer in the timeline under consideration. This alignment infers that the sister group of the mitochondria, for the taxa that have been sampled, is the order Rickettsiales.     

On the lack of good scientific reasons for the growing phylogeny/classification gap

An increasing number of phylogenetic analyses is no longer translated into classifications. The resulting phylogeny/classification gap is undesirable because the precise transmission of phylogenetic insights depends on the frequent revision of Linnaean names. The move away from classifying has numerous correlates. These include: an expanded pool of researchers who are able to produce phylogenetic estimates, a mismatch between the properties of molecular phylogenies and the requirements for verbal Linnaean definitions, the emphasis of statistical representations over the creation and evaluation of scientific terms, and a partial disconnect between the processes of nomenclature and taxonomy. The “taxonomic concept” approach allows systematists to express their varying perspectives with a high precision and can therefore help reduce the aforementioned gap. The phylogeny/classification link must persist in order to ensure community‐wide access to, and continued testing of, the products of systematic research. © The Willi Hennig Society 2005.     

Fossils and ferns in the resolution of land plant phylogeny

Fifty-two taxa of living and extinct vascular plants were evaluated in an unconstrained numerical cladistic analysis using 101 morphological characters to simultaneously resolve the phylogenetic relationships of ferns sensu lato. Included in the analysis were ferns assignable to the Cladoxylales, Stauropteridales, Rhacophytales, Zygopteridales, Ophioglossales, Marattiales, Filicales, and Hydropteridales, as well as a rhyniophyte, a trimerophyte, equisetophytes, lignophytes, and the psilotophytesPsilotum andTmesipteris. The results placed ferns and fernlike plants in three distinct clades, indicating that ferns s.l. are a polyphyletic grade group. Fern clades consist of extinct stauropterids; extinct cladoxylaleans, rhacophytaleans, and zygopteridaleans; and eusporangiates and leptosporangiates with living and extinct species. Psilotophytes occur near the base of the tree rather than nesting with the Filicales, as hypothesized by some. These results place Ophioglossales as the sister group to Marattiales plus the leptosporangiates, supporting the hypothesis that Ophioglossales represent ferns rather than progymnosperms. These analyses are a first attempt, which includes extinct plants, to develop cladistic hypotheses for the overall topology of fern phylogeny and to lay the groundwork for more detailed analyses of relationships among the homosporous leptosporangiates.     

Mitochondrial resolution of a deep branch in the genealogical tree for perching birds.

Animal mitochondrial DNA (mtDNA) is known to contain information about the genealogical relations among closely related species and is shown here to yield information about distant relations as well. Our results also draw attention to the need for caution in using third positions of codons for tree construction. This is evident from comparative studies of the cytochrome b gene in 13 species representing major groups within the order of perching birds (Passeriformes). Sequences of a 924 base-pair segment of this gene were obtained from each of these species via the polymerase chain reaction and a novel set of versatile primers. With a woodpecker sequence as an outgroup, trees that separate songbirds from other perching birds and resolve the ancient branch leading to songbirds were obtained utilizing the conservative first and second positions of codons. Analysis of positions within codons suggests that, for deep branches, the skewed base composition at the fast-changing third positions can result in phylogenetic disinformation, which conflicts with the information retained in the first and second positions. The mitochondrial tree shows broad concordance with that based on hybridization of nuclear DNA; however, parsimony and maximum likelihood methods suggest a close kinship between thrushes and Australian babblers, in agreement with the traditional morphological classification.     

Characterization of the prokaryotic diversity through a stratigraphic permafrost core profile from the Qinghai-Tibet Plateau

Permafrost on the Qinghai-Tibet Plateau is one of the most sensitive regions to climate warming, thus characterizing its microbial diversity and community composition may be important for understanding their potential responses to climate changes. Here, we investigated the prokaryotic diversity in a 10-m-long permafrost core from the Qinghai-Tibet Plateau by restriction fragment length polymorphism analysis targeting the 16S rRNA gene. We detected 191 and 17 bacterial and archaeal phylotypes representing 14 and 2 distinct phyla, respectively. Proteobacteria was the dominant bacterial phylum, while archaeal communities were characterized by a preponderance of Thaumarchaeota. Some of prokaryotic phylotypes were closely related to characterized species involved in carbon and nitrogen cycles, including nitrogen fixation, methane oxidation and nitrification. However, the majority of the phylotypes were only distantly related to known taxa at order or species level, suggesting the potential of novel diversity. Additionally, both bacterial α diversity and community composition changed significantly with sampling depth, where these communities mainly distributed according to core horizons. Arthrobacter-related phylotypes presented at high relative abundance in two active layer soils, while the deeper permafrost soils were dominated by Psychrobacter-related clones. Changes in bacterial community composition were correlated with most measured soil variables, such as carbon and nitrogen contents, pH, and conductivity.     

Birds in a bush: five genes indicate explosive evolution of avian orders

All recent studies of bird phylogeny have produced poorly resolved relationships among the orders of Neoaves, the lineage that includes most modern birds. This "bush" result suggests the possibility of an explosive and potentially unresolvable evolutionary radiation. However, simultaneous radiations of multiple lineages are thought to be rare or nonexistent in nature and difficult to corroborate empirically because lack of phylogenetic resolution can also be caused by analytical artifacts. Here we examine the predictions of the explosive radiation hypothesis for five independent genetic datasets for Neoaves. We propose a methodology for testing for polytomies of evolutionary lineages, perform likelihood-ratio tests to compare trees with zero-length branches to more resolved trees, compare topologies between independent gene trees, and propose a power test for the SOWH test. The evidence of (1) extremely short (in some cases zero-length) branches for interordinal relationships across independent gene trees and (2) topological incongruence among gene trees suggests that the bird tree includes essentially simultaneous radiation of multiple lineages. This result explains why a robust phylogeny of birds has not been produced despite much effort on the part of avian systematists.     

Contribution of Archaea to total prokaryotic production in the deep Atlantic Ocean

Fluorescence in situ hybridization (FISH) in combination with polynucleotide probes revealed that the two major groups of planktonic Archaea (Crenarchaeota and Euryarchaeota) exhibit a different distribution pattern in the water column of the Pacific subtropical gyre and in the Antarctic Circumpolar Current system. While Euryarchaeota were found to be more dominant in nearsurface waters, Crenarchaeota were relatively more abundant in the mesopelagic and bathypelagic waters. We determined the abundance of archaea in the mesopelagic and bathypelagic North Atlantic along a south-north transect of more than 4,000 km. Using an improved catalyzed reporter deposition-FISH (CARD-FISH) method and specific oligonucleotide probes, we found that archaea were consistently more abundant than bacteria below a 100-m depth. Combining microautoradiography with CARD-FISH revealed a high fraction of metabolically active cells in the deep ocean. Even at a 3,000-m depth, about 16% of the bacteria were taking up leucine. The percentage of Euryarchaeota and Crenarchaeaota taking up leucine did not follow a specific trend, with depths ranging from 6 to 35% and 3 to 18%, respectively. The fraction of Crenarchaeota taking up inorganic carbon increased with depth, while Euryarchaeota taking up inorganic carbon decreased from 200 m to 3,000 m in depth. The ability of archaea to take up inorganic carbon was used as a proxy to estimate archaeal cell production and to compare this archaeal production with total prokaryotic production measured via leucine incorporation. We estimate that archaeal production in the mesopelagic and bathypelagic North Atlantic contributes between 13 to 27% to the total prokaryotic production in the oxygen minimum layer and 41 to 84% in the Labrador Sea Water, declining to 10 to 20% in the North Atlantic Deep Water. Thus, planktonic archaea are actively growing in the dark ocean although at lower growth rates than bacteria and might play a significant role in the oceanic carbon cycle.     

Rubisco genes indicate a close phylogenetic relation between the plastids of Chromophyta and Rhodophyta

The genes for both subunits of Rubisco (rbcL, rbcS) are located on the plastome of the brown alga Ectocarpus siliculosus (Chromophyta, Phaeophyceae). The organization of these genes in the form of an operon was similar to that found in rhodoplasts, cyanobacteria and the plastids of Cryptomonas . Sequence analysis of the complete operon revealed a high degree of homology and great structural similarities to corresponding genes from two red algae. In contrast, sequence homology to Rubisco genes from chloroplasts and cyanobacteria was much lower. This clearly indicated a close phylogenetic relationship between the plastids of Rhodophyta and Chromophyta which seem to have evolved independently from the chloroplasts (polyphyletic origin). Our data suggest that the plastids of Chromophyta and Cryptophyta have originated from endosymbiotic unicellular red algae. Surprisingly, red and brown algal Rubiscos show a significantly higher degree of homology to that from a hydrogen bacterium than to those from cyanobacteria.     

Distribution of genes for lysine biosynthesis through the aminoadipate pathway among prokaryotic genomes

Deinococcus radioduranshas homologous genes to the genes which from the Thermus: thermophilus gene cluster for lysine biosynthesis. Interestingly, those genes are clustered in Thermus, nevertheless they are scattered in Deinococcus. A similar gene cluster has only been found in Pyrococcus However, the phylogenetic analyses indicated that the deduced gene products from Deinococcus were the most closely related to the proteins encoded in the Thermus gene cluster for lysine biosynthesis. Therefore, those genes had not been transferred horizontally between Pyrococcus and Thermus. It is strongly suggested that a common ancestor of Deinococcus and Thermus possessed the genes for lysine biosynthesis through the aminoadipate pathway. These had been clustered through the evolution of Thermus or had been scattered from the gene cluster through the evolution of Deinococcus. In addition, I showed that LysW and its homologues were specialized proteins for the prokaryotic lysine biosynthesis through the aminoadipate pathway.     

HGT-DB: a database of putative horizontally transferred genes in prokaryotic complete genomes

The Horizontal Gene Transfer DataBase (HGT-DB) is a genomic database that includes statistical parameters such as G+C content, codon and amino-acid usage, as well as information about which genes deviate in these parameters for prokaryotic complete genomes. Under the hypothesis that genes from distantly related species have different nucleotide compositions, these deviated genes may have been acquired by horizontal gene transfer. The current version of the database contains 88 bacterial and archaeal complete genomes, including multiple chromosomes and strains. For each genome, the database provides statistical parameters for all the genes, as well as averages and standard deviations of G+C content, codon usage, relative synonymous codon usage and amino-acid content. It also provides information about correspondence analyses of the codon usage, plus lists of extraneous group of genes in terms of G+C content and lists of putatively acquired genes. With this information, researchers can explore the G+C content and codon usage of a gene when they find incongruities in sequence-based phylogenetic trees. A search engine that allows searches for gene names or keywords for a specific organism is also available. HGT-DB is freely accessible at http://www.fut.es/~debb/HGT.     

Utility of 17 chloroplast genes for inferring the phylogeny of the basal angiosperms

Sequences from 14 slowly evolving chloroplast genes (including three highly conserved introns) were obtained for representative basal angiosperm and seed-plant taxa, using novel primers described here. These data were combined with published sequences from atpB, rbcL, and newly obtained sequences from ndhF. Combined data from these 17 genes permit sturdy, well-resolved inference of major aspects of basal angiosperm relationships, demonstrating that the new primers are valuable tools for sorting out the deepest events in flowering plant phylogeny. Sequences from the inverted repeat (IR) proved to be particularly reliable (low homoplasy, high retention index). Representatives of Cabomba and Illicium were the first two successive branches of the angiosperms in an initial sampling of 19 exemplar taxa. This result was strongly supported by bootstrap analysis and by two small insertion/deletion events in the slowly evolving introns. Several paleoherb groups (representatives of Piperales) formed a strongly supported clade with taxa representing core woody magnoliids (Laurales, Magnoliales, and Winteraceae). The monophyly of the sampled eudicots and monocots was also well supported. Analyses of three major partitions of the data showed many of the same clades and supported the rooting seen with all the data combined. While Amborella trichopoda was supported as the sister group of the remaining angiosperms when we added Amborella and Nymphaea odorata to the analysis, a strongly conflicting rooting was observed when Amborella alone was added.     

Alternative designs for construction of the class II transfer RNA tertiary core

The structural requirements for assembly of functional class II transfer RNA core regions have been examined by sequence analysis and tested by reconstruction of alternative folds into the tertiary domain of Escherichia coli tRNA(2)Gln. At least four distinct designs have been identified that permit stable folding and efficient synthetase recognition, as assessed by thermal melting profiles and glutaminylation kinetics. Although most large variable-arm tRNAs found in nature possess an enlarged D-loop, lack of this feature can be compensated for by insertion of nucleotides either 3' to the variable loop or within the short acceptor/D-stem connector region. Rare pyrimidines at nt 9 in the core region can be accommodated in the class II framework, but only if specific nucleotides are present either in the D-loop or 3' to the variable arm. Glutaminyl-tRNA synthetase requires one or two unpaired uridines 3' to the variable arm to efficiently aminoacylate several of the class II frameworks. Because there are no specific enzyme contacts in the tRNAGln core region, these data suggest that tRNA discrimination by GlnRS depends in part on indirect readout of RNA sequence information.