Listeria monocytogenes is a model organism for cellular microbiology and host–pathogen interaction studies and an important food-borne pathogen widespread in the environment, thus representing an attractive model to study the evolution of virulence. The phylogenetic structure of L. monocytogenes was determined by sequencing internal portions of seven housekeeping genes (3,288 nucleotides) in 360 representative isolates. Fifty-eight of the 126 disclosed sequence types were grouped into seven well-demarcated clonal complexes (clones) that comprised almost 75% of clinical isolates. Each clone had a unique or dominant serotype (4b for clones 1, 2 and 4, 1/2b for clones 3 and 5, 1/2a for clone 7, and 1/2c for clone 9), with no association of clones with clinical forms of human listeriosis. Homologous recombination was extremely limited (r/m<1 for nucleotides), implying long-term genetic stability of multilocus genotypes over time. Bayesian analysis based on 438 SNPs recovered the three previously defined lineages, plus one unclassified isolate of mixed ancestry. The phylogenetic distribution of serotypes indicated that serotype 4b evolved once from 1/2b, the likely ancestral serotype of lineage I. Serotype 1/2c derived once from 1/2a, with reference strain EGDe (1/2a) likely representing an intermediate evolutionary state. In contrast to housekeeping genes, the virulence factor internalin (InlA) evolved by localized recombination resulting in a mosaic pattern, with convergent evolution indicative of natural selection towards a truncation of InlA protein. This work provides a reference evolutionary framework for future studies on L. monocytogenes epidemiology, ecology, and virulence.

The ability to cross host barriers is an essential virulence determinant of invasive microbial pathogens. Listeria monocytogenes is a model microorganism that crosses human intestinal and placental barriers, and causes severe maternofetal infections by an unknown mechanism. Several studies have helped to characterize the bacterial invasion proteins InlA and InlB. However, their respective species specificity has complicated investigations on their in vivo role. Here we describe two novel and complementary animal models for human listeriosis: the gerbil, a natural host for L. monocytogenes, and a knock-in mouse line ubiquitously expressing humanized E-cadherin. Using these two models, we uncover the essential and interdependent roles of InlA and InlB in fetoplacental listeriosis, and thereby decipher the molecular mechanism underlying the ability of a microbe to target and cross the placental barrier.

Cyanobacteria have played a significant role in the formation of past and modern carbonate deposits at the surface of the Earth using a biomineralization process that has been almost systematically considered induced and extracellular. Recently, a deep-branching cyanobacterial species, Candidatus Gloeomargarita lithophora, was reported to form intracellular amorphous Ca-rich carbonates. However, the significance and diversity of the cyanobacteria in which intracellular biomineralization occurs remain unknown. Here, we searched for intracellular Cacarbonate inclusions in 68 cyanobacterial strains distributed throughout the phylogenetic tree of cyanobacteria. We discovered that diverse unicellular cyanobacterial taxa form intracellular amorphous Ca-carbonates with at least two different distribution patterns, suggesting the existence of at least two distinct mechanisms of biomineralization: (i) one with Ca-carbonate inclusions scattered within the cell cytoplasm such as in Ca. G. lithophora, and (ii) another one observed in strains belonging to the Thermosynechococcus elongatus BP-1 lineage, in which Ca-carbonate inclusions lie at the cell poles. This pattern seems to be linked with the nucleation of the inclusions at the septum of the cells, showing an intricate and original connection between cell division and biomineralization. These findings indicate that intracellular Ca-carbonate biomineralization by cyanobacteria has been overlooked by past studies and open new perspectives on the mechanisms and the evolutionary history of intra-and extracellular Ca-carbonate biomineralization by cyanobacteria.calcification | amorphous calcium carbonate | polyphosphate

Debaryomyces hansenii is a hemiascomycetous yeast commonly found in natural substrates and in various types of cheese. Pichia guilliermondii is widely distributed in nature and is a common constituent of the normal human microflora. Both species have been described in human infections but are extremely difficult to differentiate phenotypically. Thus, frequent errors in identification occur. The 62 clinical and environmental isolates sent between 2000 and 2007 to the French National Reference Center for Mycoses and Antifungals as D. hansenii or P. guilliermondii were analyzed by using the carbon assimilation pattern, the presence of pseudohyphae, and sequencing of the ITS and D1/D2 regions of the rRNA gene. The objective of this study was to assess using nucleotide sequences whether phenotypic identification was accurate and whether phenotypic characteristics could be used to differentiate the two species when sequencing was not available. We found that 58% of the isolates were misidentified and belong to seven different species: P. guilliermondii , P. caribbica , P. jadinii , D. hansenii , Candida palmioleophila , C. haemulonii type II, and Clavispora lusitaniae . In conclusion, D. hansenii may not be as common a human pathogen as previously thought. Sequencing of either ITS or D1/D2 regions is a good tool for differentiating the species more frequently confused with D. hansenii , keeping in mind that reliable databases should be used.

Cyanobacteria are thought to play a key role in carbonate formation due to their metabolic activity, but other organisms carrying out oxygenic photosynthesis (photosynthetic eukaryotes) or other metabolisms (e.g., anoxygenic photosynthesis, sulfate reduction), may also contribute to carbonate formation. To obtain more quantitative information than that provided by more classical PCR-dependent methods, we studied the microbial diversity of microbialites from the Alchichica crater lake (Mexico) by mining for 16S/18S rRNA genes in metagenomes obtained by direct sequencing of environmental DNA. We studied samples collected at the Western (AL-W) and Northern (AL-N) shores of the lake and, at the latter site, along a depth gradient (1, 5, 10, and 15 m depth). The associated microbial communities were mainly composed of bacteria, most of which seemed heterotrophic, whereas archaea were negligible. Eukaryotes composed a relatively minor fraction dominated by photosynthetic lineages, diatoms in AL-W, influenced by Si-rich seepage waters, and green algae in AL-N samples. Members of the Gammaproteobacteria and Alphaproteobacteria classes of Proteobacteria, Cyanobacteria, and Bacteroidetes were the most abundant bacterial taxa, followed by Planctomycetes, Deltaproteobacteria (Proteobacteria), Verrucomicrobia, Actinobacteria, Firmicutes, and Chloroflexi. Community composition varied among sites and with depth. Although cyanobacteria were the most important bacterial group contributing to the carbonate precipitation potential, photosynthetic eukaryotes, anoxygenic photosynthesizers and sulfate reducers were also very abundant. Cyanobacteria affiliated to Pleurocapsales largely increased with depth. Scanning electron microscopy (SEM) observations showed considerable areas of aragonite-encrusted Pleurocapsa-like cyanobacteria at microscale. Multivariate statistical analyses showed a strong positive correlation of Pleurocapsales and Chroococcales with aragonite formation at macroscale, and suggest a potential causal link. Despite the previous identification of intracellularly calcifying cyanobacteria in Alchichica microbialites, most carbonate precipitation seems extracellular in this system.

BackgroundThe Chernobyl accident represents a long-term experiment on the effects of exposure to ionizing radiation at the ecosystem level. Though studies of these effects on plants and animals are abundant, the study of how Chernobyl radiation levels affect prokaryotic and eukaryotic microbial communities is practically non-existent, except for a few reports on human pathogens or soil microorganisms. Environments enduring extreme desiccation and UV radiation, such as sunlight exposed biofilms could in principle select for organisms highly resistant to ionizing radiation as well.Methodology/Principal FindingsTo test this hypothesis, we explored the diversity of microorganisms belonging to the three domains of life by cultivation-independent approaches in biofilms developing on concrete walls or pillars in the Chernobyl area exposed to different levels of radiation, and we compared them with a similar biofilm from a non-irradiated site in Northern Ireland. Actinobacteria, Alphaproteobacteria, Bacteroidetes, Acidobacteria and Deinococcales were the most consistently detected bacterial groups, whereas green algae (Chlorophyta) and ascomycete fungi (Ascomycota) dominated within the eukaryotes. Close relatives to the most radio-resistant organisms known, including Rubrobacter species, Deinococcales and melanized ascomycete fungi were always detected. The diversity of bacteria and eukaryotes found in the most highly irradiated samples was comparable to that of less irradiated Chernobyl sites and Northern Ireland. However, the study of mutation frequencies in non-coding ITS regions versus SSU rRNA genes in members of a same actinobacterial operational taxonomic unit (OTU) present in Chernobyl samples and Northern Ireland showed a positive correlation between increased radiation and mutation rates.Conclusions/SignificanceOur results show that biofilm microbial communities in the most irradiated samples are comparable to non-irradiated samples in terms of general diversity patterns, despite increased mutation levels at the single-OTU level. Therefore, biofilm communities growing in sunlight exposed substrates are capable of coping with increased mutation rates and appear pre-adapted to levels of ionizing radiation in Chernobyl due to their natural adaptation to periodical desiccation and ambient UV radiation.

Cyanobacteria are mainly thought to induce carbonate precipitation extracellularly via their photosynthetic activity combined with the nucleation potential of exopolymeric substances. The discovery in microbialites of the alkaline lake Alchichica (Mexico) of Candidatus Gloeomargarita lithophora, a cyanobacterium forming large amounts of intracellular Mg-Ca-Sr-Ba carbonate spherules, showed that intracellular biomineralization in cyanobacteria is also possible. A second cyanobacterium isolated from the same environment, Candidatus Synechococcus calcipolaris G9, has been recently shown to also form intracellular calcium carbonates at the cell poles, a capability shared by all cultured species of the Thermosynechococcus clade, to which it belongs. To explore the diversity of these two distant cyanobacterial lineages representing two different patterns of intracellular calcification, we designed specific primers against their 16S rRNA genes and looked for their occurrence in a wide variety of samples. We identified the presence of members of the Gloeomargarita and Thermosynechococcus/S. calcipolaris lineages in microbialites collected from Lake Alchichica and three other neighboring Mexican lakes. The two clades also occurred in karstic areas and in some thermophilic or hypersaline microbial mats collected in South America and/or Southern Europe. Surprisingly, the within-group diversity in the two clades was low, especially within the S. calcipolaris clade, with all 16S rRNA gene sequences retrieved sharing more than 97% identity. This suggests that these clades are composed of a limited number of operational taxonomic units (OTUs) with cosmopolitan distribution. Moreover, scanning electron microscopy coupled with energy dispersive x-ray spectrometry showed the presence of intracellularly calcifying Gloeomargarita-like cyanobacteria in fresh samples where this clade was relatively abundant, suggesting that these cyanobacteria do precipitate carbonates intracellularly under natural conditions.

Modern phototrophic microbial mats are complex communities often used as analogs of major Precambrian ecosystems. Characterizing biotic, notably metabolic, interactions among different microbial mat members is essential to gain insights into the ecology and biogeochemistry of these systems. We applied 16S/18S rRNA metabarcoding approaches to characterize the structure of archaea, bacteria and protist communities from microbial mats collected along strong physicochemical (oxygen, salinity, temperature, depth) gradients in a shallow pond at the salar de Llamara (Chile). All mats were highly diverse, including members of virtually all known high-rank eukaryotic and prokaryotic taxa but also many novel lineages. Bacterial candidate divisions accounted for almost 50% of sequences in deeper mats, while Archaea represented up to 40% of sequences in some mat layers. Molecular phylogenetic analyses revealed six novel deeply divergent archaeal groups, along abundant and diverse Pacearchaeota and Woesearchaeota. Multivariate statistical analyses showed that local environmental conditions strongly influenced community composition. Co-occurrence network structure was markedly different between surface mats located in the oxygenated zone and mats located in transition and anoxic water layers. We identified potential biotic interactions between various high- and low-rank taxa. Notably, a strong positive correlation was observed between Lokiarchaeota and the poorly known candidate bacterial division TA06.