Biomedical subjects
W Ford Doolittle
Publications and source records attributed to W Ford Doolittle.
Molecular and phylogenetic characterization of syntaxin genes from parasitic protozoa.
Vesicular transport is an integral process in eukaryotic cells and the syntaxins, a member of the SNARE protein superfamily, are a critical piece of the vesicular transport machinery. We have obtained syntaxin homologues from diverse protozoan parasites (including Entamoeba, Giardia, Trichomonas and Trypanosoma), determined the paralogue affinity of the homologues by molecular phylogenetics and compared functionally critical amino acid sites identified in other syntaxins. Surprisingly, three sequences deviate at the signature glutamine residue position, conserved in all previously identified syntaxin homologues. It is known that, despite conserved structure and function of both the syntaxins and the proteins of the regulatory SM superfamily, the various syntaxin paralogues bind their respective SM partners at different regions of the syntaxin molecule. These sites of interactions have been identified down to the individual residues. The pattern of conservation at these residues, in our evolutionarily diverse sampling of syntaxin paralogues, is therefore used to gain further insight into the interaction of these proteins. Phylogenetic analysis confirms and extends previous conclusions that the syntaxin families are present in diverse eukaryotes and that the syntaxin sub-families diverged early in eukaryotic evolution. This result is expanded with the inclusion of new homologues for previously sampled taxa, newly sampled taxa, and newly sampled syntaxin sub-families. Because of their integral role in membrane trafficking, the syntaxin genes represent a valuable potential molecular marker for the experimental study of the endomembrane system of disease-causing protists.
Have archaeal genes contributed to bacterial virulence?
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Phylogenetic reconstruction and lateral gene transfer.
Lateral gene transfer (LGT) is often seen as a form of noise, obscuring the phylogenetic signal with which we might hope to reconstruct the evolution of a group of organisms, or indeed the history of all life (the Tree of Life). Such reconstruction might still be possible if the subset of genes conserved among all genomes in a group (or common to all genomes) comprise a core that is relatively refractory to LGT. Several papers designed to test this notion have recently appeared, and here we re-analyze one, which claims that the core of single-copy orthologs shared by all sequenced genomes of the gammaproteobacteria is essentially free of LGT. This conclusion is unfortunately premature, and it is very hard to determine what fraction of this core has been affected by LGT. We discuss other difficulties with the core concept and suggest that, although the core idea must remain part of our understanding of phylogenetic relationships, it should not be the sole basis for defining such relationships, because these are not exclusively tree-like. We suggest instead a more complex but more natural framework for classification, which we call the Synthesis of Life.
Computing prokaryotic gene ubiquity: rescuing the core from extinction.
The genomic core concept has found several uses in comparative and evolutionary genomics. Defined as the set of all genes common to (ubiquitous among) all genomes in a phylogenetically coherent group, core size decreases as the number and phylogenetic diversity of the relevant group increases. Here, we focus on methods for defining the size and composition of the core of all genes shared by sequenced genomes of prokaryotes (Bacteria and Archaea). There are few (almost certainly less than 50) genes shared by all of the 147 genomes compared, surely insufficient to conduct all essential functions. Sequencing and annotation errors are responsible for the apparent absence of some genes, while very limited but genuine disappearances (from just one or a few genomes) can account for several others. Core size will continue to decrease as more genome sequences appear, unless the requirement for ubiquity is relaxed. Such relaxation seems consistent with any reasonable biological purpose for seeking a core, but it renders the problem of definition more problematic. We propose an alternative approach (the phylogenetically balanced core), which preserves some of the biological utility of the core concept. Cores, however delimited, preferentially contain informational rather than operational genes; we present a new hypothesis for why this might be so.
Origins and evolution of isoprenoid lipid biosynthesis in archaea.
A characteristic feature of the domain archaea are the lipids forming the hydrophobic core of their cell membrane. These unique lipids are composed of isoprenoid side-chains stereospecifically ether linked to sn-glycerol-1-phosphate. Recently, considerable progress has been made in characterizing the enzymes responsible for the synthesis of archaeal lipids. However, little is known about their evolution. To better understand how this unique biosynthetic apparatus came to be, large-scale database surveys and phylogenetic analyses were performed. All characterized enzymes involved in the biosynthesis of isoprenoid side-chains and the glycerol phosphate backbone along with their assembly in ether lipids were included in these analyses. The sequence data available in public databases was complemented by an in-depth sampling of isoprenoid lipid biosynthesis genes from multiple genera of the archaeal order Halobacteriales, allowing us to look at the evolution of these enzymes on a smaller phylogenetic scale. This investigation of the isoprenoid biosynthesis apparatus of archaea on small and large phylogenetic scales reveals that it evolved through a combination of evolutionary processes, including the co-option of ancestral enzymes, modification of enzymatic specificity, orthologous and non-orthologous gene displacement, integration of components from eukaryotes and bacteria and lateral gene transfer within and between archaeal orders.
Intragenomic heterogeneity and intergenomic recombination among haloarchaeal rRNA genes.
More than one copy of rRNA operons, which code for both the small-subunit (SSU) and large-subunit (LSU) rRNA, are often found in prokaryotes. It is generally assumed that all rRNA operons within a single cell are almost identical. A notable exception is the extremely halophilic archaeal genus Haloarcula, most species of which are known to harbor highly divergent rRNA operons that differ at approximately 5% of the nucleotide positions in the SSU gene and at 1 to 2% of the nucleotide positions in the LSU gene. We report that such intragenomic heterogeneity is not unique to Haloarcula, as high levels of intragenomic sequence variation have been observed for the SSU genes of two other genera of extreme halophiles, Halosimplex and Natrinema. To investigate this in detail, the two rRNA operons of Halosimplex carlsbadense and the four operons of Natrinema sp. strain XA3-1 were cloned and completely sequenced. The SSU and LSU genes of H. carlsbadense show the highest levels of intragenomic heterogeneity observed so far in archaea (6.7 and 2.6%). The operons of Natrinema sp. strain XA3-1 have additional unusual characteristics, such as identical internal transcribed spacers, while one of four SSU genes is 5% divergent and all LSU genes differ from each other by 0.9 to 1.9%. The heterogeneity among the Natrinema sp. strain XA3-1 LSU genes is localized in hot spots, and one of these regions is shown to be the result of a recombination event with a distantly related halophile. This is the first example of interspecies recombination between rRNA genes in archaea, and the recombination occurred over one of the largest phylogenetic distances ever reported for such an event. We suggest that intragenomic heterogeneity of rRNA operons is an ancient and stable trait in several lineages of the Halobacteriales. The impact of this phenomenon on the taxonomy of extremely halophilic archaea is discussed.
Evidence for Golgi bodies in proposed 'Golgi-lacking' lineages.
Golgi bodies are nearly ubiquitous in eukaryotic cells. The apparent lack of such structures in certain eukaryotic lineages might be taken to mean that these protists evolved prior to the acquisition of the Golgi, and it raises questions of how these organisms function in the absence of this crucial organelle. Here, we report gene sequences from five proposed 'Golgi-lacking' organisms (Giardia intestinalis, Spironucleus barkhanus, Entamoeba histolytica, Naegleria gruberi and Mastigamoeba balamuthi). BLAST and phylogenetic analyses show these genes to be homologous to those encoding components of the retromer, coatomer and adaptin complexes, all of which have Golgi-related functions in mammals and yeast. This is, to our knowledge, the first molecular evidence for Golgi bodies in two major eukaryotic lineages (the pelobionts and heteroloboseids). This substantiates the suggestion that there are no extant primitively 'Golgi-lacking' lineages, and that this apparatus was present in the last common eukaryotic ancestor, but has been altered beyond recognition several times.
Active self-splicing group I introns in 23S rRNA genes of hyperthermophilic bacteria, derived from introns in eukaryotic organelles.
Group I introns are common in the 23 rRNA genes of mitochondria and chloroplasts. Often, they encode "homing endonucleases," which target highly conserved gene sequences and drive interorganellar intron mobility, even across species and genus lines. Most bacterial 23S rRNA genes show these same endonuclease-sensitive target sequences. However, only two bacterial 23S rRNA genes are known to contain group I introns: that of Simkania negevensis [Everett, K. D., Kahane, S., Bush, R. M. & Friedman, M. G. (1999) J. Bacteriol. 181, 4734-4740], where the intron is not spliced and probably limits growth, and that of Coxiella burnetii [Seshadri, R., et al. (2003) Proc. Natl. Acad. Sci. USA 100, 5455-5460], where no direct evidence of splicing exists. Both bacteria are intracellular parasites and might have acquired introns from eukaryotic hosts. Here we provide direct evidence for splicing, and evolutionary evidence for mobility, of group I introns in the 23S rRNA genes of several free-living hyperthermophilic bacteria of the genus Thermotoga. These bacteria do not live closely with eukaryotes, but phylogenetic analyses suggest that their introns were also acquired from eukaryotic (probably algal) organelles. In vivo, their introns must be spliced at temperatures approaching 90 degrees C, making them the most thermostable natural ribozymes so far described. We demonstrate that at least some of these introns can also self-splice in vitro.
Phage evolution: new worlds of genomic diversity.
A recent comparative survey of genomes of phages infecting mycobacteria reveals a vast combinatorial network of gene rearrangements and may provide general models for pattern and process in genome evolution.
Diversity of bacteriorhodopsins in different hypersaline waters from a single Spanish saltern.
Haloarchaeal rhodopsins are a diverse group of transmembrane proteins that use light energy to drive several different cellular processes. Two rhodopsins, bacteriorhodopsin and halorhodopsins, are H+ and Cl- ion pumps, respectively, and two rhodopsins, sensory rhodopsin I and II, regulate phototaxis. Bacteriorhodopsin is of special interest as it is a non-chlorophyll-based type of phototrophy (i.e. generation of chemical energy from light energy). However, very little is known about the diversity and distribution of rhodopsin genes in hypersaline environments. Here, we have used environmental PCR and cloning techniques to directly retrieve rhodopsin genes from three different salinity ponds located in a sea salt manufacturing facility near Alicante, Spain. Our survey resulted in the discovery of previously concealed variation including what is hypothesized to be bacteriorhodopsin genes from the uncultivated square morphotype that dominates these environments. In some instances, identical genes were discovered in seemingly different habitats suggesting that some haloarchaea are present over widely varying concentrations of salt.
Targeting clusters of transferred genes in Thermotoga maritima.
We screened a Thermotoga sp. strain RQ2 lambda library for genes present in that strain but absent from the closely related completely sequenced relative Thermotoga maritima strain MSB8, by using probes generated in an earlier genomic subtraction study. Five lambda insert fragments were sequenced, containing, respectively, an archaeal type ATPase operon, rhamnose biosynthetic genes, ORFs with similarity to an arabinosidase, a Thermotoga sp. strain RQ2-specific alcohol dehydrogenase and a novel archaeal Mut-S homologue. All but one of these fragments contained additional Thermotoga sp. strain RQ2-specific sequences not screened for, suggesting that many such strain-specific genes will be found clustered in the genome. Moreover, phylogenetic analyses, phylogenetic distribution and/or G + C content suggests that all the Thermotoga sp. strain RQ2 specific sequences in the sequenced lambda clones have been acquired by lateral gene transfer. We suggest that the use of strain-specific small insert clones obtained by subtractive hybridization to target larger inserts for sequencing is an efficient, economical way to identify environmentally (or clinically) relevant interstrain differences and novel gene clusters, and will be invaluable in comparative genomics.
Comparison of Bayesian and maximum likelihood bootstrap measures of phylogenetic reliability.
Owing to the exponential growth of genome databases, phylogenetic trees are now widely used to test a variety of evolutionary hypotheses. Nevertheless, computation time burden limits the application of methods such as maximum likelihood nonparametric bootstrap to assess reliability of evolutionary trees. As an alternative, the much faster Bayesian inference of phylogeny, which expresses branch support as posterior probabilities, has been introduced. However, marked discrepancies exist between nonparametric bootstrap proportions and Bayesian posterior probabilities, leading to difficulties in the interpretation of sometimes strongly conflicting results. As an attempt to reconcile these two indices of node reliability, we apply the nonparametric bootstrap resampling procedure to the Bayesian approach. The correlation between posterior probabilities, bootstrap maximum likelihood percentages, and bootstrapped posterior probabilities was studied for eight highly diverse empirical data sets and were also investigated using experimental simulation. Our results show that the relation between posterior probabilities and bootstrapped maximum likelihood percentages is highly variable but that very strong correlations always exist when Bayesian node support is estimated on bootstrapped character matrices. Moreover, simulations corroborate empirical observations in suggesting that, being more conservative, the bootstrap approach might be less prone to strongly supporting a false phylogenetic hypothesis. Thus, apparent conflicts in topology recovered by the Bayesian approach were reduced after bootstrapping. Both posterior probabilities and bootstrap supports are of great interest to phylogeny as potential upper and lower bounds of node reliability, but they are surely not interchangeable and cannot be directly compared.
Lateral gene transfer and the origins of prokaryotic groups.
Lateral gene transfer (LGT) is now known to be a major force in the evolution of prokaryotic genomes. To date, most analyses have focused on either (a) verifying phylogenies of individual genes thought to have been transferred, or (b) estimating the fraction of individual genomes likely to have been introduced by transfer. Neither approach does justice to the ability of LGT to effect massive and complex transformations in basic biology. In some cases, such transformation will be manifested as the patchy distribution of a seemingly fundamental property (such as aerobiosis or nitrogen fixation) among the members of a group classically defined by the sharing of other properties (metabolic, morphological, or molecular, such as small subunit ribosomal RNA sequence). In other cases, the lineage of recipients so transformed may be seen to comprise a new group of high taxonomic rank ("class" or even "phylum"). Here we review evidence for an important role of LGT in the evolution of photosynthesis, aerobic respiration, nitrogen fixation, sulfate reduction, methylotrophy, isoprenoid biosynthesis, quorum sensing, flotation (gas vesicles), thermophily, and halophily. Sometimes transfer of complex gene clusters may have been involved, whereas other times separate exchanges of many genes must be invoked.
Can commercial protection be good for research?
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Lateral transfer of an EF-1alpha gene: origin and evolution of the large subunit of ATP sulfurylase in eubacteria.
It is generally accepted that new genes arise via duplication and functional divergence of existing genes, in accordance with Ohno's model, now called "Mutation During Redundancy," or MDR. In this model, one of the two gene copies is free to acquire novel (although likely related) activities through mutation, since only one copy is required for its original function. However, duplication within a genome is not the only process that might give rise to this situation: acquisition of a functionally redundant gene by lateral gene transfer (LGT) could also initiate the MDR process. Here we describe a probable instance, involving LGT of an archaeal or eukaryotic elongation factor 1alpha (EF-1alpha) gene. The large subunit of ATP sulfurylase (CysN or the N-terminal portion of NodQ), found mainly in proteobacteria, is clearly related to translation elongation factors. However, our analyses show that cysN arose from an EF-1alpha gene initially acquired by LGT, not from a within-genome duplication of the resident EF-Tu gene. To our knowledge, this is the first unequivocal case of LGT followed by functional modification to be described; this mechanism could be a potentially important force in establishing genes with novel functions in genomes.
Novel syntaxin gene sequences from Giardia, Trypanosoma and algae: implications for the ancient evolution of the eukaryotic endomembrane system.
SNAP receptors or SNARES are crucial components of the intracellular membrane system of eukaryotes. The syntaxin family of SNAREs have been shown to have roles in neurotransmission, vesicular transport, membrane fusion and even internal membrane compartment reconstruction. While syntaxins and SNAREs in general have been well characterized in mammalian and yeast models, little is known about their overall distribution across eukaryotic diversity or about the evolution of the syntaxin gene family. By combining bioinformatic, molecular biological and phylogenetic approaches, we demonstrate that various syntaxin homologs are not only present in 'eukaryotic crown taxa' but across a wide range of eukaryotic lineages. The alignment of evolutionarily diverse syntaxin paralogs shows that an isoleucine residue critical to nSec1-syntaxin complex formation and the characteristic syntaxin glutamine residue are nearly universally conserved, implying a general functional importance for these residues. Other identified functional residues involved in botulism toxicity and calcium-binding-protein interactions are also compared. The presence of Golgi-related syntaxins in the intestinal parasite Giardia intestinalis provides further evidence for a cryptic Golgi in this 'adictyosomal' taxon, and another likely case of secondary reduction in this parasite. The phylogeny of syntaxins shows a number of nested duplications, including a case of parallel evolution in the plasma membrane-associated syntaxins, and ancestral duplications in the other syntaxin paralogs. These speak to ancient events in the evolution of the syntaxin system and emphasize the universal role of the syntaxins in the eukaryotic intracellular compartment system.