Richard Christen

TreeDyn: towards dynamic graphics and annotations for analyses of trees

BackgroundAnalyses of biomolecules for biodiversity, phylogeny or structure/function studies often use graphical tree representations. Many powerful tree editors are now available, but existing tree visualization tools make little use of meta-information related to the entities under study such as taxonomic descriptions or gene functions that can hardly be encoded within the tree itself (if using popular tree formats). Consequently, a tedious manual analysis and post-processing of the tree graphics are required if one needs to use external information for displaying or investigating trees.ResultsWe have developed TreeDyn, a tool using annotations and dynamic graphical methods for editing and analyzing multiple trees. The main features of TreeDyn are 1) the management of multiple windows and multiple trees per window, 2) the export of graphics to several standard file formats with or without HTML encapsulation and a new format called TGF, which enables saving and restoring graphical analysis, 3) the projection of texts or symbols facing leaf labels or linked to nodes, through manual pasting or by using annotation files, 4) the highlight of graphical elements after querying leaf labels (or annotations) or by selection of graphical elements and information extraction, 5) the highlight of targeted trees according to a source tree browsed by the user, 6) powerful scripts for automating repetitive graphical tasks, 7) a command line interpreter enabling the use of TreeDyn through CGI scripts for online building of trees, 8) the inclusion of a library of packages dedicated to specific research fields involving trees.ConclusionTreeDyn is a tree visualization and annotation tool which includes tools for tree manipulation and annotation and uses meta-information through dynamic graphical operators or scripting to help analyses and annotations of single trees or tree collections.

Marinobacter hydrocarbonoclasticus gen. nov., sp. nov., a new, extremely halotolerant, hydrocarbon-degrading marine bacterium.

On the basis of phenotypical characteristics and analysis of 16S rRNA sequence, a new species belonging to a new genus is described, and the name Marinobacter hydrocarbonoclasticus is proposed. This organism, isolated from Mediterranean seawater near a petroleum refinery, is a gram-negative, aerobic, rod-shaped bacterium. It grows at NaCl concentrations of 0.08 to 3.5 M and uses various hydrocarbons as the sole source of carbon and energy. Its DNA has a guanine-plus-cytosine content of 52.7 mol%. The 16S rRNA analysis shows a clear affiliation between M. hydrocarbonoclasticus and the gamma group of the phylum Proteobacteria. A close phylogenetic relationship appears among the species Marinomonas vaga, Oceanospirillum linum, Halomonas elongata, and Pseudomonas aeruginosa. Because of the impossibility of finding a single most closely related species, we suggest that this bacterium be assigned to a new genus, at least temporarily. The possibility of a revision of this status when new data appear is, however, not excluded. The type strain is M. hydrocarbonoclasticus SP.17 (= ATCC 49840).

Multiple marker parallel tag environmental DNA sequencing reveals a highly complex eukaryotic community in marine anoxic water

Sequencing of ribosomal DNA clone libraries amplified from environmental DNA has revolutionized our understanding of microbial eukaryote diversity and ecology. The results of these analyses have shown that protist groups are far more genetically heterogeneous than their morphological diversity suggests. However, the clone library approach is labour‐intensive, relatively expensive, and methodologically biased. Therefore, even the most intensive rDNA library analyses have recovered only small samples of much larger assemblages, indicating that global environments harbour a vast array of unexplored biodiversity. High‐throughput parallel tag 454 sequencing offers an unprecedented scale of sampling for molecular detection of microbial diversity. Here, we report a 454 protocol for sampling and characterizing assemblages of eukaryote microbes. We use this approach to sequence two SSU rDNA diversity markers—the variable V4 and V9 regions—from 10 L of anoxic Norwegian fjord water. We identified 38 116 V4 and 15 156 V9 unique sequences. Both markers detect a wide range of taxonomic groups but in both cases the diversity detected was dominated by dinoflagellates and close relatives. Long‐tailed rank abundance curves suggest that the 454 sequencing approach provides improved access to rare genotypes. Most tags detected represent genotypes not currently in GenBank, although many are similar to database sequences. We suggest that current understanding of the ecological complexity of protist communities, genetic diversity, and global species richness are severely limited by the sequence data hitherto available, and we discuss the biological significance of this high amplicon diversity.

Phylogenetic analysis of the genera Alteromonas, Shewanella, and Moritella using genes coding for small-subunit rRNA sequences and division of the genus Alteromonas into two genera, Alteromonas (emended) and Pseudoalteromonas gen. nov., and proposal of twelve new species combinations

Small-subunit ribosomal DNA sequences were determined for 17 strains belonging to the genera Alteromonas, Shewanella, Vibrio, and Pseudomonas, and these sequences were analyzed by phylogenetic methods. The resulting data confirmed the existence of the genera Shewanella and Moritella, but suggested that the genus Alteromonas should be split into two genera. We propose that a new genus, the genus Pseudoalteromonas, should be created to accommodate 11 species that were previously Alteromonas species, including Pseudoalteromonas atlantica comb. nov., Pseudoalteromonas aurantia comb. nov., Pseudoalteromonas carrageenovoa comb. nov., Pseudoalteromonas citrea comb. nov., Pseudoalteromonas denitrificans comb. nov., Pseudoalteromonas espejiana comb. nov., Pseudoalteromonas haloplanktis comb. nov. (with two subspecies, Pseudoalteromonas haloplanktis subsp. haloplanktis comb. nov. and Pseudoalteromonas haloplanktis subsp. tetraodonis comb. nov.), Pseudoalteromonas luteoviolacea comb. nov., Pseudoalteromonas nigrifaciens comb. nov., Pseudoalteromonas rubra comb. nov., and Pseudoalteromonas undina comb, nov., and one species that previously was placed in the genus Pseudomonas, Pseudoalteromonas piscicida comb. nov. We propose that P. haloplanktis (type strain, ATCC 14393) should be the type species of the genus Pseudoalteromonas. At this time the emended genus Alteromonas is restricted to a single species, Alteromonas macleodii.

The Protist Ribosomal Reference database (PR2): a catalog of unicellular eukaryote Small Sub-Unit rRNA sequences with curated taxonomy

The interrogation of genetic markers in environmental meta-barcoding studies is currently seriously hindered by the lack of taxonomically curated reference data sets for the targeted genes. The Protist Ribosomal Reference database (PR2, http://ssu-rrna.org/) provides a unique access to eukaryotic small sub-unit (SSU) ribosomal RNA and DNA sequences, with curated taxonomy. The database mainly consists of nuclear-encoded protistan sequences. However, metazoans, land plants, macrosporic fungi and eukaryotic organelles (mitochondrion, plastid and others) are also included because they are useful for the analysis of high-troughput sequencing data sets. Introns and putative chimeric sequences have been also carefully checked. Taxonomic assignation of sequences consists of eight unique taxonomic fields. In total, 136 866 sequences are nuclear encoded, 45 708 (36 501 mitochondrial and 9657 chloroplastic) are from organelles, the remaining being putative chimeric sequences. The website allows the users to download sequences from the entire and partial databases (including representative sequences after clustering at a given level of similarity). Different web tools also allow searches by sequence similarity. The presence of both rRNA and rDNA sequences, taking into account introns (crucial for eukaryotic sequences), a normalized eight terms ranked-taxonomy and updates of new GenBank releases were made possible by a long-term collaboration between experts in taxonomy and computer scientists.

Phylogenetic Analysis and Assessment of the Genera Vibrio, Photobacterium, Aeromonas, and Plesiomonas Deduced from Small-Subunit rRNA Sequences

We sequenced nearly complete small-subunit rRNAs of 54 reference strains belonging to the genera Vibrio, Photobacterium, Aeromonas, and Plesiomonas. We then performed a phylogenetic analysis by comparing the sequences which we obtained with all other known sequences for bacteria belonging to the gamma subgroup of the Proteobacteria (thus providing a data base consisting of 70 sequences for the genera investigated), using methods such as neighbor joining, maximum likelihood, and maximum parsimony, as well as bootstrap, to assess the robustness of each topology. Our results confirmed that the family Vibrionaceae should include only Photobacterium and Vibrio species (but not Vibrio marinus); that Aeromonas species deserve family rank; and that Plesiomonas shigelloides is linked to the family Enterobacteriaceae. The genera Vibrio, Photobacterium, Aeromonas, and Plesiomonas, together with the family Enterobacteriaceae, the family Pasteurellaceae, and probably the genus Alteromonas, form a robust monophyletic unit within the gamma 3 subgroup of the Proteobacteria.

Midgut Microbiota of the Malaria Mosquito Vector Anopheles gambiae and Interactions with Plasmodium falciparum Infection

The susceptibility of Anopheles mosquitoes to Plasmodium infections relies on complex interactions between the insect vector and the malaria parasite. A number of studies have shown that the mosquito innate immune responses play an important role in controlling the malaria infection and that the strength of parasite clearance is under genetic control, but little is known about the influence of environmental factors on the transmission success. We present here evidence that the composition of the vector gut microbiota is one of the major components that determine the outcome of mosquito infections. A. gambiae mosquitoes collected in natural breeding sites from Cameroon were experimentally challenged with a wild P. falciparum isolate, and their gut bacterial content was submitted for pyrosequencing analysis. The meta-taxogenomic approach revealed a broader richness of the midgut bacterial flora than previously described. Unexpectedly, the majority of bacterial species were found in only a small proportion of mosquitoes, and only 20 genera were shared by 80% of individuals. We show that observed differences in gut bacterial flora of adult mosquitoes is a result of breeding in distinct sites, suggesting that the native aquatic source where larvae were grown determines the composition of the midgut microbiota. Importantly, the abundance of Enterobacteriaceae in the mosquito midgut correlates significantly with the Plasmodium infection status. This striking relationship highlights the role of natural gut environment in parasite transmission. Deciphering microbe-pathogen interactions offers new perspectives to control disease transmission.

Contributions to a revision of the genus Trichosporon.

The genusTrichosporon was revised using characters of morphology, ultrastructure, physiology, ubiquinone systems, mol% G+C of DNA, DNA/DNA reassociations and 26S ribosomal RNA partial sequences. A total of 101 strains was used, including all available type and authentic cultures of previously described taxa. Nineteen taxa could be distinguished, 15 of which having Q-9 coenzyme systems and 4 having Q-10. Sixteen previously described names were reduced to synonymy. One new species was described.The genus is characterized by the presence of arthroconidia. Few species possess further diagnostic morphological characters, such as the presence of appressoria, macroconidia or meristematic conidiation. The septa of two species were found to be non-perforate, while those of the remaining species contained dolipores at variable degrees of differentiation, with or without vesicular or tubular parenthesomes. All species were able to assimilate a large number of carbon compounds; visible CO2 production was absent. The genus was found to be fairly homogeneous on the basis of a phylogenetic analysis of partial 26S rRNA sequences, with the exception ofT. pullulans which proved to be unrelated.Most taxa were found to occupy well-defined ecological niches. Within the group of taxa isolated from humans, a distinction found be made between those involved in systemic mycoses and those which mainly caused public or non-public white piedras, respectively. One species was consistently associated with animals, while others came mainly from soil or water. One species was mesophilic and another psychrophilic.

Establishment of a Real-Time PCR-Based Approach for Accurate Quantification of Bacterial RNA Targets in Water, Using Salmonella as a Model Organism

ABSTRACT Quantitative PCR (Q-PCR) is a fast and efficient tool to quantify target genes. In eukaryotic cells, quantitative reverse transcription-PCR (Q-RT-PCR) is also used to quantify gene expression, with stably expressed housekeeping genes as standards. In bacteria, such stable expression of housekeeping genes does not occur, and the use of DNA standards leads to a broad underestimation. Therefore, an accurate quantification of RNA is feasible only by using appropriate RNA standards. We established and validated a Q-PCR method which enables the quantification of not only the number of copies of target genes (i.e., the number of bacterial cells) but also the number of RNA copies. The genes coding for InvA and the 16S rRNA of Salmonella enterica serovar Typhimurium were selected for the evaluation of the method. As DNA standards, amplified fragments of the target genes were used, whereas the same DNA standards were transcribed in vitro for the development of appropriate RNA standards. Salmonella cultures and environmental water samples inoculated with bacteria were then employed for the final testing. Both experimental approaches led to a sensitive, accurate, and reproducible quantification of the selected target genes and RNA molecules by Q-PCR and Q-RT-PCR. It is the first time that RNA standards have been successfully used for a precise quantification of the number of RNA molecules in prokaryotes. This demonstrates the potential of this approach for determining the presence and metabolic activity of pathogenic bacteria in environmental samples.

Patterns of Rare and Abundant Marine Microbial Eukaryotes

BACKGROUND Biological communities are normally composed of a few abundant and many rare species. This pattern is particularly prominent in microbial communities, in which most constituent taxa are usually extremely rare. Although abundant and rare subcommunities may present intrinsic characteristics that could be crucial for understanding community dynamics and ecosystem functioning, microbiologists normally do not differentiate between them. Here, we investigate abundant and rare subcommunities of marine microbial eukaryotes, a crucial group of organisms that remains among the least-explored biodiversity components of the biosphere. We surveyed surface waters of six separate coastal locations in Europe, independently considering the picoplankton, nanoplankton, and microplankton/mesoplankton organismal size fractions. RESULTS Deep Illumina sequencing of the 18S rRNA indicated that the abundant regional community was mostly structured by organismal size fraction, whereas the rare regional community was mainly structured by geographic origin. However, some abundant and rare taxa presented similar biogeography, pointing to spatiotemporal structure in the rare microeukaryote biosphere. Abundant and rare subcommunities presented regular proportions across samples, indicating similar species-abundance distributions despite taxonomic compositional variation. Several taxa were abundant in one location and rare in other locations, suggesting large oscillations in abundance. The substantial amount of metabolically active lineages found in the rare biosphere suggests that this subcommunity constitutes a diversity reservoir that can respond rapidly to environmental change. CONCLUSIONS We propose that marine planktonic microeukaryote assemblages incorporate dynamic and metabolically active abundant and rare subcommunities, with contrasting structuring patterns but fairly regular proportions, across space and time.