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Yan Boucher

Publications and source records attributed to Yan Boucher.

16 recordsLinked to original sources
PubMed2006

Use of 16S rRNA and rpoB genes as molecular markers for microbial ecology studies.

Several characteristics of the 16S rRNA gene, such as its essential function, ubiquity, and evolutionary properties, have allowed it to become the most commonly used molecular marker in microbial ecology. However, one fact that has been overlooked is that multiple copies of this gene are often present in a given bacterium. These intragenomic copies can differ in sequence, leading to identification of multiple ribotypes for a single organism. To evaluate the impact of such intragenomic heterogeneity on the performance of the 16S rRNA gene as a molecular marker, we compared its phylogenetic and evolutionary characteristics to those of the single-copy gene rpoB. Full-length gene sequences and gene fragments commonly used for denaturing gradient gel electrophoresis were compared at various taxonomic levels. Heterogeneity found between intragenomic 16S rRNA gene copies was concentrated in specific regions of rRNA secondary structure. Such "heterogeneity hot spots" occurred within all gene fragments commonly used in molecular microbial ecology. This intragenomic heterogeneity influenced 16S rRNA gene tree topology, phylogenetic resolution, and operational taxonomic unit estimates at the species level or below. rpoB provided comparable phylogenetic resolution to that of the 16S rRNA gene at all taxonomic levels, except between closely related organisms (species and subspecies levels), for which it provided better resolution. This is particularly relevant in the context of a growing number of studies focusing on subspecies diversity, in which single-copy protein-encoding genes such as rpoB could complement the information provided by the 16S rRNA gene.

Bacteria↗
PubMed2006

Refuting phylogenetic relationships.

BACKGROUND: Phylogenetic methods are philosophically grounded, and so can be philosophically biased in ways that limit explanatory power. This constitutes an important methodologic dimension not often taken into account. Here we address this dimension in the context of concatenation approaches to phylogeny. RESULTS: We discuss some of the limits of a methodology restricted to verificationism, the philosophy on which gene concatenation practices generally rely. As an alternative, we describe a software which identifies and focuses on impossible or refuted relationships, through a simple analysis of bootstrap bipartitions, followed by multivariate statistical analyses. We show how refuting phylogenetic relationships could in principle facilitate systematics. We also apply our method to the study of two complex phylogenies: the phylogeny of the archaea and the phylogeny of the core of genes shared by all life forms. While many groups are rejected, our results left open a possible proximity of N. equitans and the Methanopyrales, of the Archaea and the Cyanobacteria, and as well the possible grouping of the Methanobacteriales/Methanoccocales and Thermosplasmatales, of the Spirochaetes and the Actinobacteria and of the Proteobacteria and firmicutes. CONCLUSION: It is sometimes easier (and preferable) to decide which species do not group together than which ones do. When possible topologies are limited, identifying local relationships that are rejected may be a useful alternative to classical concatenation approaches aiming to find a globally resolved tree on the basis of weak phylogenetic markers. REVIEWERS: This article was reviewed by Mark Ragan, Eugene V Koonin and J Peter Gogarten.

Journal Article↗
PubMed2006

Recovery and evolutionary analysis of complete integron gene cassette arrays from Vibrio.

BACKGROUND: Integrons are genetic elements capable of the acquisition, rearrangement and expression of genes contained in gene cassettes. Gene cassettes generally consist of a promoterless gene associated with a recombination site known as a 59-base element (59-be). Multiple insertion events can lead to the assembly of large integron-associated cassette arrays. The most striking examples are found in Vibrio, where such cassette arrays are widespread and can range from 30 kb to 150 kb. Besides those found in completely sequenced genomes, no such array has yet been recovered in its entirety. We describe an approach to systematically isolate, sequence and annotate large integron gene cassette arrays from bacterial strains. RESULTS: The complete Vibrio sp. DAT722 integron cassette array was determined through the streamlined approach described here. To place it in an evolutionary context, we compare the DAT722 array to known vibrio arrays and performed phylogenetic analyses for all of its components (integrase, 59-be sites, gene cassette encoded genes). It differs extensively in terms of genomic context as well as gene cassette content and organization. The phylogenetic tree of the 59-be sites collectively found in the Vibrio gene cassette pool suggests frequent transfer of cassettes within and between Vibrio species, with slower transfer rates between more phylogenetically distant relatives. We also identify multiple cases where non-integron chromosomal genes seem to have been assembled into gene cassettes and others where cassettes have been inserted into chromosomal locations outside integrons. CONCLUSION: Our systematic approach greatly facilitates the isolation and annotation of large integrons gene cassette arrays. Comparative analysis of the Vibrio sp. DAT722 integron obtained through this approach to those found in other vibrios confirms the role of this genetic element in promoting lateral gene transfer and suggests a high rate of gene gain/loss relative to most other loci on vibrio chromosomes. We identify a relationship between the phylogenetic distance separating two species and the rate at which they exchange gene cassettes, interactions between the non-mobile portion of bacterial genomes and the vibrio gene cassette pool as well as intragenomic translocation events of integrons in vibrios.

DNA, Bacterial↗
PubMed2006

Class 1 integrons potentially predating the association with tn402-like transposition genes are present in a sediment microbial community.

Integrons are genetic elements that contribute to lateral gene transfer in bacteria as a consequence of possessing a site-specific recombination system. This system facilitates the spread of genes when they are part of mobile cassettes. Most integrons are contained within chromosomes and are confined to specific bacterial lineages. However, this is not the case for class 1 integrons, which were the first to be identified and are one of the single biggest contributors to multidrug-resistant nosocomial infections, carrying resistance to many antibiotics in diverse pathogens on a global scale. The rapid spread of class 1 integrons in the last 60 years is partly a result of their association with a specific suite of transposition functions, which has facilitated their recruitment by plasmids and other transposons. The widespread use of antibiotics has acted as a positive selection pressure for bacteria, especially pathogens, which harbor class 1 integrons and their associated antibiotic resistance genes. Here, we have isolated bacteria from soil and sediment in the absence of antibiotic selection. Class 1 integrons were recovered from four different bacterial species not known to be human pathogens or commensals. All four integrons lacked the transposition genes previously considered to be a characteristic of this class. At least two of these integrons were located on a chromosome, and none of them possessed antibiotic resistance genes. We conclude that novel class 1 integrons are present in a sediment environment in various bacteria of the beta-proteobacterial class. These data suggest that the dispersal of this class may have begun before the "antibiotic era."

Bacteria↗
PubMed2005

Improving protein solubility: the use of the Escherichia coli dihydrofolate reductase gene as a fusion reporter.

We have devised a strategy for screening mutant libraries for enzyme variants with enhanced solubility. The method is based on the observation that Escherichia coli can become insensitive to the antibiotic trimethoprim (TMP) if dihydrofolate reductase (DHFR) is expressed at an appropriate level. DHFR is a very soluble protein and can be expressed at levels that exceed normally lethal concentrations of TMP. In our approach, the gene encoding an insoluble target protein is placed in a vector so that the translated protein will be fused to DHFR. The resulting fusion protein will form inclusion bodies and inactivate DHFR-the cells will be susceptible to TMP. Mutations to the target protein that make it more soluble will also make the fusion protein more soluble so that DHFR will be at least partially active-the cells will be resistant to TMP. As the solubility of the target protein increases, the cells will become more resistant to TMP. The system was tested with a putative acetyltransferase (ACE) from a strain of the marine bacterium Vibrio fischerii. The gene encoding this protein was of interest since it is part of a mobile gene cassette within an integron array of the strain in question. After multiple rounds of shuffling and selection, ACE mutants were produced that had significantly improved solubility.

Acetyltransferases↗
PubMed2005

Complex histories of genes encoding 3-hydroxy-3-methylglutaryl-CoenzymeA reductase.

The mevalonate pathway for the synthesis of isoprenoids can be found in organisms from all domains of life. It has been previously demonstrated that the first gene specific to that pathway, which encodes the enzyme 3-hydroxy-3-methylglutaryl-CoenzymeA reductase (HMGR), has been transferred between domains by lateral gene transfer on several occasions. Here we look within the domain Bacteria at lateral acquisition of HMGR, whether as a single gene or as part of a mevalonate pathway cluster. We observe a complex history of multiple transfer events probably reflecting the fact that HMGR could be beneficial in a variety of physiological and genetic contexts. We demonstrate that even in Vibrio species, where HMGR is not clustered with other genes to form an operon or a metabolic cluster, it is under strong purifying selection.

Bacteria↗
PubMed2005

Paradigm change in evolutionary microbiology.

Thomas Kuhn had little to say about scientific change in biological science, and biologists are ambivalent about how applicable his framework is for their disciplines. We apply Kuhn's account of paradigm change to evolutionary microbiology, where key Darwinian tenets are being challenged by two decades of findings from molecular phylogenetics. The chief culprit is lateral gene transfer, which undermines the role of vertical descent and the representation of evolutionary history as a tree of life. To assess Kuhn's relevance to this controversy, we add a social analysis of the scientists involved to the historical and philosophical debates. We conclude that while Kuhn's account may capture aspects of the pattern (or outcome) of an episode of scientific change, he has little to say about how the process of generating new understandings is occurring in evolutionary microbiology. Once Kuhn's application is limited to that of an initial investigative probe into how scientific problem-solving occurs, his disciplinary scope becomes broader.

Biological Evolution↗
PubMed2005

Lateral gene transfer and phylogenetic assignment of environmental fosmid clones.

Metagenomic data, especially sequence data from large insert clones, are most useful when reasonable inferences about phylogenetic origins of inserts can be made. Often, clones that bear phylotypic markers (usually ribosomal RNA genes) are sought, but sometimes phylogenetic assignments have been based on the preponderance of blast hits obtained with predicted protein coding sequences (CDSs). Here we use a cloning method which greatly enriches for ribosomal RNA-bearing fosmid clones to ask two questions: (i) how reliably can we judge the phylogenetic origin of a clone (that is, its RNA phylotype) from the sequences of its CDSs? and (ii) how much lateral gene transfer (LGT) do we see, as assessed by CDSs of different phylogenetic origins on the same fosmid? We sequenced 12 rRNA containing fosmid clones, obtained from libraries constructed using DNA isolated from Baltimore harbour sediments. Three of the clones are from bacterial candidate divisions for which no cultured representatives are available, and thus represent the first protein coding sequences from these major bacterial lineages. The amount of LGT was assessed by making phylogenetic trees of all the CDSs in the fosmid clones and comparing the phylogenetic position of the CDS to the rRNA phylotype. We find that the majority of CDSs in each fosmid, 57-96%, agree with their respective rRNA genes. However, we also find that a significant fraction of the CDSs in each fosmid, 7-44%, has been acquired by LGT. In several cases, we can infer co-transfer of functionally related genes, and generate hypotheses about mechanism and ecological significance of transfer.

Anaerobiosis↗
PubMed2005

Higher-level classification of the Archaea: evolution of methanogenesis and methanogens.

We used a phylogenetic approach to analyze the evolution of methanogenesis and methanogens. We show that 23 vertically transmitted ribosomal proteins do not support the monophyly of methanogens, and propose instead that there are two distantly related groups of extant archaea that produce methane, which we have named Class I and Class II. Based on this finding, we subsequently investigated the uniqueness of the origin of methanogenesis by studying both the enzymes of methanogenesis and the proteins that synthesize its specific coenzymes. We conclude that hydrogenotrophic methanogenesis appeared only once during evolution. Genes involved in the seven central steps of the methanogenic reduction of carbon dioxide (CO(2)) are ubiquitous in methanogens and share a common history. This suggests that, although extant methanogens produce methane from various substrates (CO(2), formate, acetate, methylated C-1 compounds), these archaea have a core of conserved enzymes that have undergone little evolutionary change. Furthermore, this core of methanogenesis enzymes seems to originate (as a whole) from the last ancestor of all methanogens and does not appear to have been horizontally transmitted to other organisms or between members of Class I and Class II. The observation of a unique and ancestral form of methanogenesis suggests that it was preserved in two independent lineages, with some instances of specialization or added metabolic flexibility. It was likely lost in the Halobacteriales, Thermoplasmatales and Archaeoglobales. Given that fossil evidence for methanogenesis dates back 2.8 billion years, a unique origin of this process makes the methanogenic archaea a very ancient taxon.

Archaea↗
PubMed2004

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.

Biological Evolution↗
PubMed2004

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.

Alkyl and Aryl Transferases↗
PubMed2004

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.

Base Sequence↗
PubMed2003

Inferring functional constraints and divergence in protein families using 3D mapping of phylogenetic information.

Comparative sequence analysis has been used to study specific questions about the structure and function of proteins for many years. Here we propose a knowledge-based framework in which the maximum likelihood rate of evolution is used to quantify the level of constraint on the identity of a site. We demonstrate that site-rate mapping on 3D structures using datasets of rhodopsin-like G-protein receptors and alpha- and beta-tubulins provides an excellent tool for pinpointing the functional features shared between orthologous and paralogous proteins. In addition, functional divergence within protein families can be inferred by examining the differences in the site rates, the differences in the chemical properties of the side chains or amino acid usage between aligned sites. Two novel analytical methods are introduced to characterize rate- independent functional divergence. These are tested using a dataset of two classes of HMG-CoA reductases for which only one class can perform both the forward and reverse reaction. We show that functionally divergent sites occur in a cluster of sites interacting with the catalytic residues and that this information should facilitate the design of experimental strategies to directly test functional properties of residues.

Animals↗
PubMed2003

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.

Animals↗
PubMed2003

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.

Archaea↗