Phylogenetic Affiliation of SSU rRNA Genes Generated by Massively Parallel Sequencing: New Insights into the Freshwater Protist Diversity

International audienceRecent advances in next-generation sequencing (NGS) technologies spur progress in determining the microbial diversity in various ecosystems by highlighting, for example, the rare biosphere. Currently, high-throughput pyrotag sequencing of PCR-amplified SSU rRNA gene regions is mainly used to characterize bacterial and archaeal communities, and rarely to characterize protist communities. In addition, although taxonomic assessment through phylogeny is considered as the most robust approach, similarity and probabilistic approaches remain the most commonly used for taxonomic affiliation. In a first part of this work, a tree-based method was compared with different approaches of taxonomic affiliation (BLAST and RDP) of 18S rRNA gene sequences and was shown to be the most accurate for near full-length sequences and for 400 bp amplicons, with the exception of amplicons covering the V5-V6 region. Secondly, the applicability of this method was tested by running a full scale test using an original pyrosequencing dataset of 18S rRNA genes of small lacustrine protists (0.2-5 mm) from eight freshwater ecosystems. Our results revealed that i) fewer than 5% of the operational taxonomic units (OTUs) identified through clustering and phylogenetic affiliation had been previously detected in lakes, based on comparison to sequence in public databases; ii) the sequencing depth provided by the NGS coupled with a phylogenetic approach allowed to shed light on clades of freshwater protists rarely or never detected with classical molecular ecology approaches; and iii) phylogenetic methods are more robust in describing the structuring of under-studied or highly divergent populations. More precisely, new putative clades belonging to Mamiellophyceae, Foraminifera, Dictyochophyceae and Euglenida were detected. Beyond the study of protists, these results illustrate that the tree-based approach for NGS based diversity characterization allows an in-depth description of microbial communities including taxonomic profiling, community structuring and the description of clades of any microorganisms (protists, Bacteria and Archaea)

Portage de ePANAM sur la grille de calcul

Portage de ePANAM sur la grille de calcu

Temporal Dynamics of Active Prokaryotic Nitrifiers and Archaeal Communities from River to Sea

International audienceTo test if different niches for potential nitrifiers exist in estuarine systems, we assessed by pyrosequencing the diversity of archaeal gene transcript markers for taxonomy (16S ribosomal RNA (rRNA)) during an entire year along a salinity gradient in surface waters of the Charente estuary (Atlantic coast, France). We further investigated the potential for estuarine prokaryotes to oxidize ammonia and hydrolyze urea by quantifying thaumarchaeal amoA and ureC and bacterial amoA transcripts. Our results showed a succession of different nitrifiers from river to sea with bacterial amoA transcripts dominating in the freshwater station while archaeal transcripts were predominant in the marine station. The 16S rRNA sequence analysis revealed that Thaumarchaeota marine group I (MGI) were the most abundant overall but other archaeal groups like Methanosaeta were also potentially active in winter (December–March) and Euryarchaeota marine group II (MGII) were dominant in seawater in summer (April–August). Each station also contained different Thaumarchaeota MGI phylogenetic clusters, and the clusters' microdiversity was associated to specific environmental conditions suggesting the presence of ecotypes adapted to distinct ecological niches. The amoA and ureC transcript dynamics further indicated that some of the Thaumarchaeota MGI sub-clusters were involved in ammonia oxidation through the hy-drolysis of urea. Our findings show that ammonia-oxidizing Archaea and Bacteria were adapted to contrasted conditions and that the Thaumarchaeota MGI diversity probably corresponds to distinct metabolisms or life strategies

Localization and Functional Characterization of the Alternative Oxidase in Naegleria

The Alternative oxidase (AOX) is a protein involved in maintaining the Krebs cycle in instances where the respiratory chain has been inhibited, while allowing for the maintenance of cell growth and necessary metabolic processes for survival. Among eukaryotes, alternative oxidases have disperse distribution and are found in plants, fungi and a few protists, including Naegleria ssp. Naegleria species are free-living unicellular amoeboflagellates, and include the pathogenic species of N. fowleri, the so-called brain eating amoeba. Using a multidisciplinary approach, we aimed to understand the evolution, localization and function of AOX and the role that plays in Naegleria’s biology. Our analyses suggest that the protein was present in last common ancestor of the genus and structure prediction showed that all functional residues are also present in Naegleria species. Using a combination of cellular and biochemical techniques, we also functionally characterize N. gruberi’s AOX in its mitochondria and we demonstrate that its inactivation affects its proliferation. Consequently, we discuss the benefits of the presence of this protein in Naegleria species, along with its potential pathogenicity role in N. fowleri. We predict that our findings will spearhead new explorations to understand the cell biology, metabolism and evolution of Naegleria and other free-living relatives

A dynamic, ring-forming MucB / RseB-like protein influences spore shape in Bacillus subtilis

How organisms develop into specific shapes is a central question in biology. The maintenance of bacterial shape is connected to the assembly and remodelling of the cell envelope. In endospore-forming bacteria, the pre-spore compartment (the forespore) undergoes morphological changes that result in a spore of defined shape, with a complex, multi-layered cell envelope. However, the mechanisms that govern spore shape remain poorly understood. Here, using a combination of fluorescence microscopy, quantitative image analysis, molecular genetics and transmission electron microscopy, we show that SsdC (formerly YdcC), a poorly-characterized new member of the MucB / RseB family of proteins that bind lipopolysaccharide in diderm bacteria, influences spore shape in the monoderm Bacillus subtilis. Sporulating cells lacking SsdC fail to adopt the typical oblong shape of wild-type forespores and are instead rounder. 2D and 3D-fluorescence microscopy suggest that SsdC forms a discontinuous, dynamic ring-like structure in the peripheral membrane of the mother cell, near the mother cell proximal pole of the forespore. A synthetic sporulation screen identified genetic relationships between ssdC and genes involved in the assembly of the spore coat. Phenotypic characterization of these mutants revealed that spore shape, and SsdC localization, depend on the coat basement layer proteins SpoVM and SpoIVA, the encasement protein SpoVID and the inner coat protein SafA. Importantly, we found that the ΔssdC mutant produces spores with an abnormal-looking cortex, and abolishing cortex synthesis in the mutant largely suppresses its shape defects. Thus, SsdC appears to play a role in the proper assembly of the spore cortex, through connections to the spore coat. Collectively, our data suggest functional diversification of the MucB / RseB protein domain between diderm and monoderm bacteria and identify SsdC as an important factor in spore shape development

Genetic screens identify additional genes implicated in envelope remodeling during the engulfment stage of Bacillus subtilis sporulation

During bacterial endospore formation, the developing spore is internalized into the mother cell through a phagocytic-like process called engulfment, which involves synthesis and hydrolysis of peptidoglycan. Engulfment peptidoglycan hydrolysis requires the widely conserved and well-characterized DMP complex, composed of SpoIID, SpoIIM, and SpoIIP. In contrast, although peptidoglycan synthesis has been implicated in engulfment, the protein players involved are less well defined. The widely conserved SpoIIIAH-SpoIIQ interaction is also required for engulfment efficiency, functioning like a ratchet to promote membrane migration around the forespore. Here, we screened for additional factors required for engulfment using transposon sequencing in Bacillus subtilis mutants with mild engulfment defects. We discovered that YrvJ, a peptidoglycan hydrolase, and the MurA paralog MurAB, involved in peptidoglycan precursor synthesis, are required for efficient engulfment. Cytological analyses suggest that both factors are important for engulfment when the DMP complex is compromised and that MurAB is additionally required when the SpoIIIAH-SpoIIQ ratchet is abolished. Interestingly, despite the importance of MurAB for sporulation in B. subtilis, phylogenetic analyses of MurA paralogs indicate that there is no correlation between sporulation and the number of MurA paralogs and further reveal the existence of a third MurA paralog, MurAC, within the Firmicutes. Collectively, our studies identify two new factors that are required for efficient envelop remodeling during sporulation and highlight the importance of peptidoglycan precursor synthesis for efficient engulfment in B. subtilis and likely other endospore-forming bacteria

Single-Stranded DNA-Binding Proteins in the Archaea

International audienceSingle-stranded (ss) DNA-binding proteins are found in all three domains of life where they play vital roles in nearly all aspects of DNA metabolism by binding to and stabilizing exposed ssDNA and acting as platforms onto which DNA-processing activities can assemble. The ssDNA-binding factors SSB and RPA are extremely well conserved across bacteria and eukaryotes, respectively, and comprise one or more OB-fold ssDNA-binding domains. In the third domain of life, the archaea, multiple types of ssDNA-binding protein are found with a variety of domain architectures and subunit compositions, with OB-fold ssDNA-binding domains being a characteristic of most, but not all. This chapter summarizes current knowledge of the distribution, structure, and biological function of the archaeal ssDNA-binding factors, highlighting key features shared between clades and those that distinguish the proteins of different clades from one another. The likely cellular functions of the proteins are discussed and gaps in current knowledge identified

Deciphering biodiversity and interactions between bacteria and microeukaryotes within epilithic biofilms from the Loue River, France

Epilithic river biofilms are complex matrix-enclosed communities harboring a great diversity of prokaryotic and eukaryotic microorganisms. Interactions between these communities and the relative impacts of environmental factors on their compositions are poorly understood. In this study, we assessed the spatio-temporal variation in the diversity and composition of bacterial and microeukaryotic communities within biofilms in a French river. Significant changes were found in the composition of these microbial communities over the sampling period and between the upstream and downstream stations. In addition, the beta diversity of the bacterial community tended to decrease along the river, mostly as a result of turnover. These changes could be caused by the different water temperatures and geological and hydrological river contexts at the sampling sites (from karst landscape to river plain). Finally, our network analysis showed multiple correlations among dominant OTUs. Among them, negative correlations between Rhodobacteraceae and two other dominant groups of photosynthetic microorganisms (cyanobacteria and diatoms) were particularly interesting, which raises the question of what environmental factors trigger the changes occurring in benthic microbial photosynthetic communities

Diversification of division mechanisms in endospore-forming bacteria revealed by analyses of peptidoglycan synthesis in Clostridioides difficile

Abstract The bacterial enzymes FtsW and FtsI, encoded in the highly conserved dcw gene cluster, are considered to be universally essential for the synthesis of septal peptidoglycan (PG) during cell division. Here, we show that the pathogen Clostridioides difficile lacks a canonical FtsW/FtsI pair, and its dcw-encoded PG synthases have undergone a specialization to fulfill sporulation-specific roles, including synthesizing septal PG during the sporulation-specific mode of cell division. Although these enzymes are directly regulated by canonical divisome components during this process, dcw-encoded PG synthases and their divisome regulators are dispensable for cell division during normal growth. Instead, C. difficile uses a bifunctional class A penicillin-binding protein as the core divisome PG synthase, revealing a previously unreported role for this class of enzymes. Our findings support that the emergence of endosporulation in the Firmicutes phylum facilitated the functional repurposing of cell division factors. Moreover, they indicate that C. difficile, and likely other clostridia, assemble a distinct divisome that therefore may represent a unique target for therapeutic interventions

Comparative genomic analysis of Methanimicrococcus blatticola provides insights into host adaptation in archaea and the evolution of methanogenesis

International audienceAbstract Other than the Methanobacteriales and Methanomassiliicoccales, the characteristics of archaea that inhabit the animal microbiome are largely unknown. Methanimicrococcus blatticola , a member of the Methanosarcinales, currently reunites two unique features within this order: it is a colonizer of the animal digestive tract and can only reduce methyl compounds with H 2 for methanogenesis, a increasingly recognized metabolism in the archaea and whose origin remains debated. To understand the origin of these characteristics, we have carried out a large-scale comparative genomic analysis. We infer the loss of more than a thousand genes in M. blatticola , by far the largest genome reduction across all Methanosarcinales. These include numerous elements for sensing the environment and adapting to more stable gut conditions, as well as a significant remodeling of the cell surface components likely involved in host and gut microbiota interactions. Several of these modifications parallel those previously observed in phylogenetically distant archaea and bacteria from the animal microbiome, suggesting large-scale convergent mechanisms of adaptation to the gut. Strikingly, M. blatticola has lost almost all genes coding for the H 4 MPT methyl branch of the Wood–Ljungdahl pathway (to the exception of mer ), a phenomenon never reported before in any member of Class I or Class II methanogens. The loss of this pathway illustrates one of the evolutionary processes that may have led to the emergence of methyl-reducing hydrogenotrophic methanogens, possibly linked to the colonization of organic-rich environments (including the animal gut) where both methyl compounds and hydrogen are abundant