Exploring Protein Space: From Hydrolase to Ligase by Substitution.

The understanding of how proteins evolve to perform novel functions has long been sought by biologists. In this regard, two homologous bacterial enzymes, PafA and Dop, pose an insightful case study, as both rely on similar mechanistic properties, yet catalyze different reactions. PafA conjugates a small protein tag to target proteins, whereas Dop removes the tag by hydrolysis. Given that both enzymes present a similar fold and high sequence similarity, we sought to identify the differences in the amino acid sequence and folding responsible for each distinct activity. We tackled this question using analysis of sequence-function relationships, and identified a set of uniquely conserved residues in each enzyme. Reciprocal mutagenesis of the hydrolase, Dop, completely abolished the native activity, at the same time yielding a catalytically active ligase. Based on the available Dop and PafA crystal structures, this change of activity required a conformational change of a critical loop at the vicinity of the active site. We identified the conserved positions essential for stabilization of the alternative loop conformation, and tracked alternative mutational pathways that lead to a change in activity. Remarkably, all these pathways were combined in the evolution of PafA and Dop, despite their redundant effect on activity. Overall, we identified the residues and structural elements in PafA and Dop responsible for their activity differences. This analysis delineated, in molecular terms, the changes required for the emergence of a new catalytic function from a preexisting one.

Genomic study of a novel magnetotactic Alphaproteobacteria uncovers the multiple ancestry of magnetotaxis.

Ecological and evolutionary processes involved in magnetotactic bacteria (MTB) adaptation to their environment have been a matter of debate for many years. Ongoing efforts for their characterization are progressively contributing to understand these processes, including the genetic and molecular mechanisms responsible for biomineralization. Despite numerous culture-independent MTB characterizations, essentially within the Proteobacteria phylum, only few species have been isolated in culture because of their complex growth conditions. Here, we report a newly cultivated magnetotactic, microaerophilic and chemoorganoheterotrophic bacterium isolated from the Mediterranean Sea in Marseille, France: Candidatus Terasakiella magnetica strain PR-1 that belongs to an Alphaproteobacteria genus with no magnetotactic relative. By comparing the morphology and the whole genome shotgun sequence of this MTB with those of closer relatives, we brought further evidence that the apparent vertical ancestry of magnetosome genes suggested by previous studies within Alphaproteobacteria hides a more complex evolutionary history involving horizontal gene transfers and/or duplication events before and after the emergence of Magnetospirillum, Magnetovibrio and Magnetospira genera. A genome-scale comparative genomics analysis identified several additional candidate functions and genes that could be specifically associated to MTB lifestyle in this class of bacteria.

leBIBIQBPP: a set of databases and a webtool for automatic phylogenetic analysis of prokaryotic sequences.

BACKGROUND: Estimating the phylogenetic position of bacterial and archaeal organisms by genetic sequence comparisons is considered as the gold-standard in taxonomy. This is also a way to identify the species of origin of the sequence. The quality of the reference database used in such analyses is crucial: the database must reflect the up-to-date bacterial nomenclature and accurately indicate the species of origin of its sequences. DESCRIPTION: leBIBI(QBPP) is a web tool taking as input a series of nucleotide sequences belonging to one of a set of reference markers (e.g., SSU rRNA, rpoB, groEL2) and automatically retrieving closely related sequences, aligning them, and performing phylogenetic reconstruction using an approximate maximum likelihood approach. The system returns a set of quality parameters and, if possible, a suggested taxonomic assigment for the input sequences. The reference databases are extracted from GenBank and present four degrees of stringency, from the "superstringent" degree (one type strain per species) to the loosely parsed degree ("lax" database). A set of one hundred to more than a thousand sequences may be analyzed at a time. The speed of the process has been optimized through careful hardware selection and database design. CONCLUSION: leBIBI(QBPP) is a powerful tool helping biologists to position bacterial or archaeal sequence commonly used markers in a phylogeny. It is a diagnostic tool for clinical, industrial and environmental microbiology laboratory, as well as an exploratory tool for more specialized laboratories. Its main advantages, relatively to comparable systems are: i) the use of a broad set of databases covering diverse markers with various degrees of stringency; ii) the use of an approximate Maximum Likelihood approach for phylogenetic reconstruction; iii) a speed compatible with on-line usage; and iv) providing fully documented results to help the user in decision making.

Evolutionary history of phosphatidylinositol- 3-kinases: ancestral origin in eukaryotes and complex duplication patterns.

BACKGROUND: Phosphatidylinositol-3-kinases (PI3Ks) are a family of eukaryotic enzymes modifying phosphoinositides in phosphatidylinositols-3-phosphate. Located upstream of the AKT/mTOR signalling pathway, PI3Ks activate secondary messengers of extracellular signals. They are involved in many critical cellular processes such as cell survival, angiogenesis and autophagy. PI3K family is divided into three classes, including 14 human homologs. While class II enzymes are composed of a single catalytic subunit, class I and III also contain regulatory subunits. Here we present an in-depth phylogenetic analysis of all PI3K proteins. RESULTS: We confirmed that PI3K catalytic subunits form a monophyletic group, whereas regulatory subunits form three distinct groups. The phylogeny of the catalytic subunits indicates that they underwent two major duplications during their evolutionary history: the most ancient arose in the Last Eukaryotic Common Ancestor (LECA) and led to the emergence of class III and class I/II, while the second - that led to the separation between class I and II - occurred later, in the ancestor of Unikonta (i.e., the clade grouping Amoebozoa, Fungi, and Metazoa). These two major events were followed by many lineage specific duplications in particular in vertebrates, but also in various protist lineages. Major loss events were also detected in Vidiriplantae and Fungi. For the regulatory subunits, we identified homologs of class III in all eukaryotic groups indicating that, for this class, both the catalytic and the regulatory subunits were presents in LECA. In contrast, homologs of the regulatory class I have a more recent origin. CONCLUSIONS: The phylogenetic analysis of the PI3K shed a new light on the evolutionary history of these enzymes. We found that LECA already contained a PI3K class III composed of a catalytic and a regulatory subunit. Absence of class II regulatory subunits and the recent origin of class I regulatory subunits is puzzling given that the class I/II catalytic subunit was present in LECA and has been conserved in most present-day eukaryotic lineages. We also found surprising major loss and duplication events in various eukaryotic lineages. Given the functional specificity of PI3K proteins, this suggests dynamic adaptation during the diversification of eukaryotes.

Molecular and functional evolution of the fungal diterpene synthase genes.

BACKGROUND: Terpenes represent one of the largest and most diversified families of natural compounds and are used in numerous industrial applications. Terpene synthase (TPS) genes originated in bacteria as diterpene synthase (di-TPS) genes. They are also found in plant and fungal genomes. The recent availability of a large number of fungal genomes represents an opportunity to investigate how genes involved in diterpene synthesis were acquired by fungi, and to assess the consequences of this process on the fungal metabolism. RESULTS: In order to investigate the origin of fungal di-TPS, we implemented a search for potential fungal di-TPS genes and identified their presence in several unrelated Ascomycota and Basidiomycota species. The fungal di-TPS phylogenetic tree is function-related but is not associated with the phylogeny based on housekeeping genes. The lack of agreement between fungal and di-TPS-based phylogenies suggests the presence of Horizontal Gene Transfer (HGTs) events. Further evidence for HGT was provided by conservation of synteny of di-TPS and neighbouring genes in distantly related fungi. CONCLUSIONS: The results obtained here suggest that fungal di-TPSs originated from an ancient HGT event of a single di-TPS gene from a plant to a fungus in Ascomycota. In fungi, these di-TPSs allowed for the formation of clusters consisting in di-TPS, GGPPS and P450 genes to create functional clusters that were transferred between fungal species, producing diterpenes acting as hormones or toxins, thus affecting fungal development and pathogenicity.

TPMS: a set of utilities for querying collections of gene trees.

BACKGROUND: The information in large collections of phylogenetic trees is useful for many comparative genomic studies. Therefore, there is a need for flexible tools that allow exploration of such collections in order to retrieve relevant data as quickly as possible. RESULTS: In this paper, we present TPMS (Tree Pattern-Matching Suite), a set of programs for handling and retrieving gene trees according to different criteria. The programs from the suite include utilities for tree collection building, specific tree-pattern search strategies and tree rooting. Use of TPMS is illustrated through three examples: systematic search for incongruencies in a large tree collection, a short study on the Coelomata/Ecdysozoa controversy and an evaluation of the level of support for a recently published Mammal phylogeny. CONCLUSION: TPMS is a powerful suite allowing to quickly retrieve sets of trees matching complex patterns in large collection or to root trees using more rigorous approaches than the classical midpoint method. As it is made of a set of command-line programs, it can be easily integrated in any sequence analysis pipeline for an automated use.

Experimental analysis of diverse actin-like proteins from various magnetotactic bacteria by functional expression in Magnetospirillum gryphiswaldense.

IMPORTANCE: To efficiently navigate within the geomagnetic field, magnetotactic bacteria (MTB) align their magnetosome organelles into chains, which are organized by the actin-like MamK protein. Although MamK is the most highly conserved magnetosome protein common to all MTB, its analysis has been confined to a small subgroup owing to the inaccessibility of most MTB. Our study takes advantage of a genetically tractable host where expression of diverse MamK orthologs together with a resurrected MamK LUCA and uncharacterized actin-like Mad28 proteins from deep-branching MTB resulted in gradual restoration of magnetosome chains in various mutants. Our results further indicate the existence of species-specific MamK interactors and shed light on the evolutionary relationships of one of the key proteins associated with bacterial magnetotaxis.

From conservation to structure, studies of magnetosome associated cation diffusion facilitators (CDF) proteins in Proteobacteria.

Magnetotactic bacteria (MTB) are prokaryotes that sense the geomagnetic field lines to geolocate and navigate in aquatic sediments. They are polyphyletically distributed in several bacterial divisions but are mainly represented in the Proteobacteria. In this phylum, magnetotactic Deltaproteobacteria represent the most ancestral class of MTB. Like all MTB, they synthesize membrane-enclosed magnetic nanoparticles, called magnetosomes, for magnetic sensing. Magnetosome biogenesis is a complex process involving a specific set of genes that are conserved across MTB. Two of the most conserved genes are mamB and mamM, that encode for the magnetosome-associated proteins and are homologous to the cation diffusion facilitator (CDF) protein family. In magnetotactic Alphaproteobacteria MTB species, MamB and MamM proteins have been well characterized and play a central role in iron-transport required for biomineralization. However, their structural conservation and their role in more ancestral groups of MTB like the Deltaproteobacteria have not been established. Here we studied magnetite cluster MamB and MamM cytosolic C-terminal domain (CTD) structures from a phylogenetically distant magnetotactic Deltaproteobacteria species represented by BW-1 strain, which has the unique ability to biomineralize magnetite and greigite. We characterized them in solution, analyzed their crystal structures and compared them to those characterized in Alphaproteobacteria MTB species. We showed that despite the high phylogenetic distance, MamBBW-1 and MamMBW-1 CTDs share high structural similarity with known CDF-CTDs and will probably share a common function with the Alphaproteobacteria MamB and MamM.

Repeated horizontal gene transfers triggered parallel evolution of magnetotaxis in two evolutionary divergent lineages of magnetotactic bacteria.

Under the same selection pressures, two genetically divergent populations may evolve in parallel toward the same adaptive solutions. Here, we hypothesized that magnetotaxis (i.e., magnetically guided chemotaxis) represents a key adaptation to micro-oxic habitats in aquatic sediments and that its parallel evolution homogenized the phenotypes of two evolutionary divergent clusters of freshwater spirilla. All magnetotactic bacteria affiliated to the Magnetospirillum genus (Alphaproteobacteria class) biomineralize the same magnetic particle chains and share highly similar physiological and ultrastructural features. We looked for the processes that could have contributed at shaping such an evolutionary pattern by reconciling species and gene trees using newly sequenced genomes of Magnetospirillum related bacteria. We showed that repeated horizontal gene transfers and homologous recombination of entire operons contributed to the parallel evolution of magnetotaxis. We propose that such processes could represent a more parsimonious and rapid solution for adaptation compared with independent and repeated de novo mutations, especially in the case of traits as complex as magnetotaxis involving tens of interacting proteins. Besides strengthening the idea about the importance of such a function in micro-oxic habitats, these results reinforce previous observations in experimental evolution suggesting that gene flow could alleviate clonal interference and speed up adaptation under some circumstances.

Databases of homologous gene families for comparative genomics.

BACKGROUND: Comparative genomics is a central step in many sequence analysis studies, from gene annotation and the identification of new functional regions in genomes, to the study of evolutionary processes at the molecular level (speciation, single gene or whole genome duplications, etc.) and phylogenetics. In that context, databases providing users high quality homologous families and sequence alignments as well as phylogenetic trees based on state of the art algorithms are becoming indispensable. METHODS: We developed an automated procedure allowing massive all-against-all similarity searches, gene clustering, multiple alignments computation, and phylogenetic trees construction and reconciliation. The application of this procedure to a very large set of sequences is possible through parallel computing on a large computer cluster. RESULTS: Three databases were developed using this procedure: HOVERGEN, HOGENOM and HOMOLENS. These databases share the same architecture but differ in their content. HOVERGEN contains sequences from vertebrates, HOGENOM is mainly devoted to completely sequenced microbial organisms, and HOMOLENS is devoted to metazoan genomes from Ensembl. Access to the databases is provided through Web query forms, a general retrieval system and a client-server graphical interface. The later can be used to perform tree-pattern based searches allowing, among other uses, to retrieve sets of orthologous genes. The three databases, as well as the software required to build and query them, can be used or downloaded from the PBIL (Pôle Bioinformatique Lyonnais) site at http://pbil.univ-lyon1.fr/.

Automatic identification of large collections of protein-coding or rRNA sequences.

The number of available genomic sequences is growing very fast, due to the development of massive sequencing techniques. Sequence identification is needed and contributes to the assessment of gene and species evolutionary relationships. Automated bioinformatics tools are thus necessary to carry out these identification operations in an accurate and fast way. We developed HoSeqI (Homologous Sequence Identification), a software environment allowing this kind of automated sequence identification using homologous gene family databases. HoSeqI is accessible through a Web interface (http://pbil.univ-lyon1.fr/software/HoSeqI/) allowing to identify one or several sequences and to visualize resulting alignments and phylogenetic trees. We also implemented another application, MultiHoSeqI, to quickly add a large set of sequences to a family database in order to identify them, to update the database, or to help automatic genome annotation. Lately, we developed an application, ChiSeqI (Chimeric Sequence Identification), to automate the processes of identification of bacterial 16S ribosomal RNA sequences and of detection of chimeric sequences.

Integrating transcription factor binding site information with gene expression datasets.

MOTIVATION: Microarrays are widely used to measure gene expression differences between sets of biological samples. Many of these differences will be due to differences in the activities of transcription factors. In principle, these differences can be detected by associating motifs in promoters with differences in gene expression levels between the groups. In practice, this is hard to do. RESULTS: We combine correspondence analysis, between group analysis and co-inertia analysis to determine which motifs, from a database of promoter motifs, are strongly associated with differences in gene expression levels. Given a database of motifs and gene expression levels from a set of arrays, the method produces a ranked list of motifs associated with any specified split in the arrays. We give an example using the Gene Atlas compendium of gene expression levels for human tissues where we search for motifs that are associated with expression in central nervous system (CNS) or muscle tissues. Most of the motifs that we find are known from previous work to be strongly associated with expression in CNS or muscle. We give a second example using a published prostate cancer dataset where we can simply and clearly find which transcriptional pathways are associated with differences between benign and metastatic samples. AVAILABILITY: The source code is freely available upon request from the authors.

HoSeqI: automated homologous sequence identification in gene family databases.

UNLABELLED: We present a web service allowing to automatically assign sequences to homologous gene families from a set of databases. After identification of the most similar gene family to the query sequence, this sequence is added to the whole alignment and the phylogenetic tree of the family is rebuilt. Thus, the phylogenetic position of the query sequence in its gene family can be easily identified. AVAILABILITY: http://pbil.univ-lyon1.fr/software/HoSeqI/.

IsoSel: Protein Isoform Selector for phylogenetic reconstructions.

The reliability of molecular phylogenies is strongly dependent on the quality of the assembled datasets. In the case of eukaryotes, the selection of only one protein isoform per genomic locus is mandatory to avoid biases linked to redundancy. Here, we present IsoSel, a tool devoted to the selection of alternative isoforms in the context of phylogenetic reconstruction. It provides a better alternative to the widely used approach consisting in the selection of the longest isoforms and it performs better than Guidance, its only available counterpart. IsoSel is publicly available at http://doua.prabi.fr/software/isosel.

Use and misuse of correspondence analysis in codon usage studies.

Correspondence analysis has frequently been used for codon usage studies but this method is often misused. Because amino acid composition exerts constraints on codon usage, it is common to use tables containing relative codon frequencies (or ratios of frequencies) instead of simple codon counts to get rid of these amino acid biases. The problem is that some important properties of correspondence analysis, such as rows weighting, are lost in the process. Moreover, the use of relative measures sometimes introduces other biases and often diminishes the quantity of information to analyse, occasionally resulting in interpretation errors. For instance, in the case of an organism such as Borrelia burgdorferi, the use of relative measures led to the conclusion that there was no translational selection, while analyses based on codon counts show that there is a possibility of a selective effect at that level. In this paper, we expose these problems and we propose alternative strategies to correspondence analysis for studying codon usage biases when amino acid composition effects must be removed.

RTKdb: database of Receptor Tyrosine Kinase.

Receptor Tyrosine Kinases (RTK) are transmembrane receptors specifically found in metazoans. They represent an excellent model for studying evolution of cellular processes in metazoans because they encompass large families of modular proteins and belong to a major family of contingency generating molecules in eukaryotic cells: the protein kinases. Because tyrosine kinases have been under close scrutiny for many years in various species, they are associated with a wealth of information, mainly in mammals. Presently, most categories of RTK were identified in mammals, but in a near future other model species will be sequenced, and will bring us RTKs from other metazoan clades. Thus, collecting RTK sequences would provide a good starting point as a new model for comparative and evolutionary studies applying to multigene families. In this context, we are developing the Receptor Tyrosine Kinase database (RTKdb), which is the only database on tyrosine kinase receptors presently available. In this database, protein sequences from eight model metazoan species are organized under the format previously used for the HOVERGEN, HOBACGEN and NUREBASE systems. RTKdb can be accessed through the PBIL (Pôle Bioinformatique Lyonnais) World Wide Web server at http://pbil.univ-lyon1.fr/RTKdb/, or through the FamFetch graphical user interface available at the same address.

The European ribosomal RNA database.

The European ribosomal RNA database aims to compile all complete or nearly complete ribosomal RNA sequences from both the small (SSU) and large (LSU) ribosomal subunits. All sequences are available in aligned format. Sequence alignment is based on the secondary structure of the molecules, as determined by comparative sequence analysis. Additional information about the sequences, such as taxonomic classification of the organism from which they have been obtained, and literature references are also provided. In order to identify the closest relatives to newly determined sequences, BLAST searches can be performed, after which the best matching sequences are aligned and a phylogenetic tree is inferred. As of 2003, the European ribosomal RNA database is maintained at Ghent University (Belgium). The database can be consulted at http://www.psb.ugent.be/rRNA/.

NUREBASE: database of nuclear hormone receptors.

Nuclear hormone receptors are an abundant class of ligand activated transcriptional regulators, found in varying numbers in all animals. Based on our experience of managing the official nomenclature of nuclear receptors, we have developed NUREBASE, a database containing protein and DNA sequences, reviewed protein alignments and phylogenies, taxonomy and annotations for all nuclear receptors. The reviewed NUREBASE is completed by NUREBASE_DAILY, automatically updated every 24 h. Both databases are organized under a client/server architecture, with a client written in Java which runs on any platform. This client, named FamFetch, integrates a graphical interface allowing selection of families, and manipulation of phylogenies and alignments. NUREBASE sequence data is also accessible through a World Wide Web server, allowing complex queries. All information on accessing and installing NUREBASE may be found at http://www.ens-lyon.fr/LBMC/laudet/nurebase.html.

Update of NUREBASE: nuclear hormone receptor functional genomics.

Nuclear hormone receptors are an abundant class of ligand-activated transcriptional regulators, found in varying numbers in all animals. Based on our experience of managing the official nomenclature of nuclear receptors, we have developed NUREBASE, a database containing protein and DNA sequences, reviewed protein alignments and phylogenies, taxonomy and annotations for all nuclear receptors. New developments in NUREBASE include explicit declaration of alternative transcripts of each gene, and expression data for human and mouse nuclear receptors. The core of NUREBASE is reviewed, and it is completed by NUREBASE_DAILY, automatically updated every 24 h. All information on accessing and installing NUREBASE may be found at http://www. ens-lyon.fr/LBMC/laudet/nurebase/nurebase.html.