Microbial dark matter sequences verification in amplicon sequencing and environmental metagenomics data

Although microorganisms constitute the most diverse and abundant life form on Earth, in many environments, the vast majority of them remain uncultured. As it is based on information gleaned mainly from cultivated microorganisms, our current body of knowledge regarding microbial life is partial and does not reflect actual microbial diversity. That diversity is hidden in the uncultured microbial majority, termed by microbiologists as “microbial dark matter” (MDM), a term borrowed from astrophysics. Metagenomic sequencing analysis techniques (both 16S rRNA gene and shotgun sequencing) compare gene sequences to reference databases, each of which represents only a small fraction of the existing microorganisms. Unaligned sequences lead to groups of “unknown microorganisms” that are usually ignored and rarefied from diversity analysis. To address this knowledge gap, we analyzed the 16S rRNA gene sequences of microbial communities from four different environments—a living organism, a desert environment, a natural aquatic environment, and a membrane bioreactor for wastewater treatment. From those datasets, we chose representative sequences of potentially unknown bacteria for additional examination as “microbial dark matter sequences” (MDMS). Sequence existence was validated by specific amplification and re-sequencing. These sequences were screened against databases and aligned to the Genome Taxonomy Database to build a comprehensive phylogenetic tree for additional sequence classification, revealing potentially new candidate phyla and other lineages. These putative MDMS were also screened against metagenome-assembled genomes from the explored environments for additional validation and for taxonomic and metabolic characterizations. This study shows the immense importance of MDMS in environmental metataxonomic analyses of 16S rRNA gene sequences and provides a simple and readily available methodology for the examination of MDM hidden behind amplicon sequencing results

Identification and rejection of pile-up jets at high pseudorapidity with the ATLAS detector

The rejection of forward jets originating from additional proton–proton interactions (pile-up) is crucial for a variety of physics analyses at the LHC, including Standard Model measurements and searches for physics beyond the Standard Model. The identification of such jets is challenging due to the lack of track and vertex information in the pseudorapidity range |η| > 2.5. This paper presents a novel strategy for forward pile-up jet tagging that exploits jet shapes and topological jet correlations in pile-up interactions. Measurements of the per-jet tagging efficiency are presented using a data set of 3.2 fb−1 of proton–proton collisions at a centre-of-mass energy of 13 TeV collected with the ATLAS detector. The fraction of pile-up jets rejected in the range 2.5 < |η| < 4.5 is estimated in simulated events with an average of 22 interactions per bunch-crossing. It increases with jet transverse momentum and, for jets with transverse momentum between 20 and 50 GeV, it ranges between 49% and 67% with an efficiency of 85% for selecting hard-scatter jets. A case study is performed in Higgs boson production via the vector-boson fusion process, showing that these techniques mitigate the background growth due to additional proton–proton interactions, thus enhancing the reach for such signatures

The Microbiome Structure of the Symbiosis between the Desert Truffle Terfezia boudieri and Its Host Plant Helianthemum sessiliflorum

The desert truffle Terfezia boudieri is an ascomycete fungus that forms ect-endomycorrhiza in the roots of plants belonging to Cistaceae. The fungus forms hypogeous edible fruit bodies, appreciated as gourmet food. Truffles and host plants are colonized by various microbes, which may contribute to their development. However, the diversity and composition of the bacterial community under field conditions in the Negev desert are still unknown. The overall goal of this research was to identify the rhizosphere microbial community supporting the establishment of a symbiotic association between T. boudieri and Helianthemum sessiliflorum. The bacterial community was characterized by fruiting bodies, mycorrhized roots, and rhizosphere soil. Based on next-generation sequencing meta-analyses of the 16S rRNA gene, we discovered diverse bacterial communities of fruit bodies that differed from those found in the roots and rhizosphere. Families of Proteobacteria, Planctomycetes, and Actinobacteria were present in all four samples. Alpha diversity analysis revealed that the rhizosphere and roots contain significantly higher bacterial species numbers compared to the fruit. Additionally, ANOSIM and PCoA provided a comparative analysis of the bacterial taxa associated with fruiting bodies, roots, and rhizosphere. The core microbiome described consists of groups whose biological role triggers important traits supporting plant growth and fruit body development

Microscopic and biomolecular complementary approaches to characterize bioweathering processes at petroglyph sites from the Negev Desert, Israel

Throughout the Negev Desert highlands, thousands of ancient petroglyphs sites are susceptible to deterioration processes that may result in the loss of this unique rock art. Therefore, the overarching goal of the current study was to characterize the composition, diversity and effects of microbial colonization of the rocks to find ways of protecting these unique treasures. The spatial organization of the microbial colonizers and their relationships with the lithic substrate were analysed using scanning electron microscopy. This approach revealed extensive epilithic and endolithic colonization and close microbial–mineral interactions. Shotgun sequencing analysis revealed various taxa from the archaea, bacteria and some eukaryotes. Metagenomic coding sequences (CDS) of these microbial lithobionts exhibited specific metabolic pathways involved in the rock elements' cycles and uptake processes. Thus, our results provide evidence for the potential participation of the microorganisms colonizing these rocks during different solubilization and mineralization processes. These damaging actions may contribute to the deterioration of this extraordinary rock art and thus threaten this valuable heritage. Shotgun metagenomic sequencing, in conjunction with the in situ scanning electron microscopy study, can thus be considered an effective strategy to understand the complexity of the weathering processes occurring at petroglyph sites and other cultural heritage assets.The microscopy study was supported by program TOP-HERITAGE S2018/NMT-4372 (Comunidad de Madrid, FSE, FEDER).Peer reviewe

Microscopic and biomolecular complementary approaches to characterize bioweathering processes at petroglyph sites from the Negev Desert, Israel

Throughout the Negev Desert highlands, thousands of ancient petroglyphs sites are susceptible to deterioration processes that may result in the loss of this unique rock art. Therefore, the overarching goal of the current study was to characterize the composition, diversity and effects of microbial colonization of the rocks to find ways of protecting these unique treasures. The spatial organization of the microbial colonizers and their relationships with the lithic substrate were analysed using scanning electron microscopy. This approach revealed extensive epilithic and endolithic colonization and close microbial–mineral interactions. Shotgun sequencing analysis revealed various taxa from the archaea, bacteria and some eukaryotes. Metagenomic coding sequences (CDS) of these microbial lithobionts exhibited specific metabolic pathways involved in the rock elements' cycles and uptake processes. Thus, our results provide evidence for the potential participation of the microorganisms colonizing these rocks during different solubilization and mineralization processes. These damaging actions may contribute to the deterioration of this extraordinary rock art and thus threaten this valuable heritage. Shotgun metagenomic sequencing, in conjunction with the in situ scanning electron microscopy study, can thus be considered an effective strategy to understand the complexity of the weathering processes occurring at petroglyph sites and other cultural heritage assets.The microscopy study was supported by program TOP-HERITAGE S2018/NMT-4372 (Comunidad de Madrid, FSE, FEDER).Peer reviewe

Diversity of Bacterial Biota in Capnodis tenebrionis (Coleoptera: Buprestidae) Larvae

The bacterial biota in larvae of Capnodis tenebrionis, a serious pest of cultivated stone-fruit trees in the West Palearctic, was revealed for the first time using the MiSeq platform. The core bacterial community remained the same in neonates whether upon hatching or grown on peach plants or an artificial diet, suggesting that C. tenebrionis larvae acquire much of their bacterial biome from the parent adult. Reads affiliated with class levels Gammaproteobacteria and Alphaproteobacteria (phylum Proteobacteria ca. 86%), and Actinobacteria (ca. 14%) were highly abundant. Most diverse reads belong to the families Xanthomonadaceae (50%), Methylobacteriaceae (20%), Hyphomicrobiaceae (9%), Micrococcaceae (7%) and Geodermatophilaceae (4.5%). About two-thirds of the reads are affiliated with the genera Lysobacter, Microvirga, Methylobacterium, and Arthrobacter, which encompass species displaying cellulolytic and lipolytic activities. This study provides a foundation for future studies to elucidate the roles of bacterial biota in C. tenebrionis

Performance of the ATLAS track reconstruction algorithms in dense environments in LHC Run 2

With the increase in energy of the Large Hadron Collider to a centre-of-mass energy of 13 TeV for Run 2, events with dense environments, such as in the cores of highenergy jets, became a focus for new physics searches as well as measurements of the Standard Model. These environments are characterized by charged-particle separations of the order of the tracking detectors sensor granularity. Basic track quantities are compared between 3.2 fb(-1) of data collected by the ATLAS experiment and simulation of protonproton collisions producing high-transverse-momentum jets at a centre-of-mass energy of 13 TeV. The impact of chargedparticle separations and multiplicities on the track reconstruction performance is discussed. The track reconstruction efficiency in the cores of jets with transverse momenta between 200 and 1600 GeV is quantified using a novel, datadriven, method. The method uses the energy loss, dE/ dx, to identify pixel clusters originating from two charged particles. Of the charged particles creating these clusters, themeasured fraction that fail to be reconstructed is 0.061 +/- 0.006 (stat.) +/- 0.014 (syst.) and 0.093 +/- 0.017 (stat.) +/- 0.021 (syst.) for jet transverse momenta of 200-400GeV and 1400-1600GeV, respectively