Linking meta-omics and biogeochemistry approaches in soils has remained challenging. This study evaluates the use of an internal RNA extraction standard and its potential for making quantitative estimates of a given microbial community size (biomass) in soil metatranscriptomics. We evaluate commonly used laboratory protocols for RNA processing, metatranscriptomic sequencing and quantitative reverse transcription polymerase chain reaction (qRT-PCR). Metatranscriptomic profiles from soil samples were generated using two library preparation protocols and prepared in triplicates. RNA extracted from pure cultures of Saccharolobus solfataricus was added to the samples as an internal nucleic acid extraction standard (NAEstd). RNA reads originating from NAEstd were identified with a 99.9% accuracy. A remarkable replication consistency between triplicates was seen (average Bray-Curtis dissimilarity 0.03 ± 0.02), in addition to a clear library preparation bias. Nevertheless, the between-sample pattern was not affected by library type. Estimates of 16S rRNA transcript abundance derived from qRT-PCR experiments, NAEstd and a previously published quantification method of metatranscriptomics (hereafter qMeTra) were compared with microbial biomass carbon (MBC) and nitrogen (MBN) extracts. The derived biomass estimates differed by orders of magnitude. While most estimates were significantly correlated with each other, no correlation was observed between NAEstd and MBC extracts. We discuss how simultaneous changes in community size and the soils nucleic acid retention strength might hamper accurate biomass estimation. Adding NAEstd has the potential to shed important light on nucleic acid retention in the substance matrix (e.g., soil) during extraction.

• Klíčová slova
• RNA, biomass estimates, extraction standard, metatranscriptomics, quantitative transcriptomics,
• Publikační typ
• časopisecké články MeSH

In the carnivorous plant genus Genlisea a unique lobster pot trapping mechanism supplements nutrition in nutrient-poor habitats. A wide spectrum of microbes frequently occurs in Genlisea's leaf-derived traps without clear relevance for Genlisea carnivory. We sequenced the metatranscriptomes of subterrestrial traps vs. the aerial chlorophyll-containing leaves of G. nigrocaulis and of G. hispidula. Ribosomal RNA assignment revealed soil-borne microbial diversity in Genlisea traps, with 92 genera of 19 phyla present in more than one sample. Microbes from 16 of these phyla including proteobacteria, green algae, amoebozoa, fungi, ciliates and metazoans, contributed additionally short-lived mRNA to the metatranscriptome. Furthermore, transcripts of 438 members of hydrolases (e.g., proteases, phosphatases, lipases), mainly resembling those of metazoans, ciliates and green algae, were found. Compared to aerial leaves, Genlisea traps displayed a transcriptional up-regulation of endogenous NADH oxidases generating reactive oxygen species as well as of acid phosphatases for prey digestion. A leaf-vs.-trap transcriptome comparison reflects that carnivory provides inorganic P- and different forms of N-compounds (ammonium, nitrate, amino acid, oligopeptides) and implies the need to protect trap cells against oxidative stress. The analysis elucidates a complex food web inside the Genlisea traps, and suggests ecological relationships between this plant genus and its entrapped microbiome.

• Klíčová slova
• Genlisea, RNA-sequencing, algae commensalism, lobster pot trapping, metatranscriptomics, plant carnivory, plant-microbe interaction, whole-genome gene transcription analysis,
• Publikační typ
• časopisecké články MeSH

Cimex lectularius, known as the common bed bug, is a widespread hematophagous human ectoparasite and urban pest that is not known to be a vector of any human infectious disease agents. However, few studies in the era of molecular biology have profiled the microorganisms harbored by field populations of bed bugs. The objective of this study was to examine the viruses present in a large sampling of common bed bugs and related bat bugs (Cimex pipistrelle). RNA sequencing was undertaken on an international sampling of > 500 field-collected bugs, and multiple workflows were used to assemble contigs and query these against reference nucleotide databases to identify viral genomes. Shuangao bed bug virus 2, an uncharacterized rhabdovirus previously discovered in Cimex hemipterus from China, was found in several bed bug pools from the USA and Europe, as well as in C. pipistrelle, suggesting that this virus is common among bed bug populations. In addition, Shuangao bed bug virus 1 was detected in a bed bug pool from China, and sequences matching Enterobacteria phage P7 were found in all bed bug pools, indicating the ubiquitous presence of phage-derived elements in the genome of the bed bug or its enterobacterial symbiont. However, viral diversity was low in bed bugs in our study, as no other viral genomes were detected with significant coverage. These results provide evidence against frequent virus infection in bed bugs. Nonetheless, our investigation had several important limitations, and additional studies should be conducted to better understand the prevalence and composition of viruses in bed bugs. Most notably, our study largely focused on insects from urban areas in industrialized nations, thus likely missing infrequent virus infections and those that could occur in rural or tropical environments or developing nations.

• Klíčová slova
• Arbovirus, Bed bug, Cimex lectularius, Cimex pipistrelle, Metatranscriptomics, RNAseq, Virus,
• MeSH
• infestace ektoparazity * MeSH
• lidé MeSH
• štěnice * genetika   MeSH
• viry * genetika   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Čína MeSH
• Evropa MeSH

Cryoconite holes are small ponds present on the surface of most glaciers filled with meltwater and sediment at the bottom. Although they are characterized by extreme conditions, they host bacterial communities with high taxonomic and functional biodiversity. Despite that evidence for a potential niche for anaerobic microorganisms and anaerobic processes has recently emerged, the composition of the microbial communities of the cryoconite reported so far has not shown the relevant presence of anaerobic taxa. We hypothesize that this is due to the lower growth yield of anaerobes compared to aerobic microorganisms. In this work, we aim at evaluating whether the anaerobic bacterial community represents a relevant fraction of the biodiversity of the cryoconite and at describing its structure and functions. We collected sediment samples from cryoconite holes on the Forni Glacier (Italy) and sequenced both 16S rRNA amplicon genes and 16S rRNA amplicon transcripts at different times of the day along a clear summer day. Results showed that a relevant fraction of taxa has been detected only by 16S rRNA transcripts and was undetectable in 16S rRNA gene amplicons. Furthermore, in the transcript approach, anaerobic taxa were overrepresented compared with DNA sequencing. The metatranscriptomics approach was used also to investigate the expression of the main metabolic functions. Results showed the occurrence of syntrophic and commensalism relationships among fermentative bacteria, hydrogenothrophs, and consumers of fermentation end products, which have never been reported so far in cryoconite. IMPORTANCE Recent evidence disclosed the presence of a potential niche for anaerobic microorganisms and anaerobic processes in supraglacial sediments (cryoconite), but a detailed description of the structure and functions of the anaerobic population is still lacking. This work used rRNA and mRNA sequencing and demonstrated that anaerobes are very active in these environments and represent a relevant albeit neglected part of the ecosystem functions in these environments.

• Klíčová slova
• cryoconite, extremophiles, metatranscriptomics,
• Publikační typ
• časopisecké články MeSH

BACKGROUND: The planetary sulfur cycle is a complex web of chemical reactions that can be microbial-mediated or can occur spontaneously in the environment, depending on the temperature and pH. Inorganic sulfur compounds can serve as energy sources for specialized prokaryotes and are important substrates for microbial growth in general. Here, we investigate dissimilatory sulfur cycling in the brine and sediments of a southwestern Siberian soda lake characterized by an extremely high pH and salinity, combining meta-omics analyses of its uniquely adapted highly diverse prokaryote communities with biogeochemical profiling to identify key microbial players and expand our understanding of sulfur cycling under haloalkaline conditions. RESULTS: Peak microbial activity was found in the top 4 cm of the sediments, a layer with a steep drop in oxygen concentration and redox potential. The majority of sulfur was present as sulfate or iron sulfide. Thiosulfate was readily oxidized by microbes in the presence of oxygen, but oxidation was partially inhibited by light. We obtained 1032 metagenome-assembled genomes, including novel population genomes of characterized colorless sulfur-oxidizing bacteria (SOB), anoxygenic purple sulfur bacteria, heterotrophic SOB, and highly active lithoautotrophic sulfate reducers. Surprisingly, we discovered the potential for nitrogen fixation in a new genus of colorless SOB, carbon fixation in a new species of phototrophic Gemmatimonadetes, and elemental sulfur/sulfite reduction in the "Candidatus Woesearchaeota." Polysulfide/thiosulfate and tetrathionate reductases were actively transcribed by various (facultative) anaerobes. CONCLUSIONS: The recovery of over 200 genomes that encoded enzymes capable of catalyzing key reactions in the inorganic sulfur cycle indicates complete cycling between sulfate and sulfide at moderately hypersaline and extreme alkaline conditions. Our results suggest that more taxonomic groups are involved in sulfur dissimilation than previously assumed.

• Klíčová slova
• Gemmatimonadetes, Haloalkaliphiles, Metagenomics, Metatranscriptomics, Nitrogen fixation, Polysulfide, Soda lake, Tetrathionate, Thiosulfate, Woesearchaeota,
• MeSH
• Archaea klasifikace   genetika   metabolismus   MeSH
• Bacteria klasifikace   genetika   metabolismus   MeSH
• fylogeneze MeSH
• jezera chemie   mikrobiologie   MeSH
• koncentrace vodíkových iontů MeSH
• metagenom MeSH
• oxidace-redukce MeSH
• salinita MeSH
• síra analýza   metabolismus   MeSH
• soli chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Sibiř MeSH
• Názvy látek
• brine MeSH Prohlížeč
• síra MeSH
• soli MeSH

Forests accumulate and store large amounts of carbon (C), and a substantial fraction of this stock is contained in deadwood. This transient pool is subject to decomposition by deadwood-associated organisms, and in this process it contributes to CO2 emissions. Although fungi and bacteria are known to colonize deadwood, little is known about the microbial processes that mediate carbon and nitrogen (N) cycling in deadwood. In this study, using a combination of metagenomics, metatranscriptomics, and nutrient flux measurements, we demonstrate that the decomposition of deadwood reflects the complementary roles played by fungi and bacteria. Fungi were found to dominate the decomposition of deadwood and particularly its recalcitrant fractions, while several bacterial taxa participate in N accumulation in deadwood through N fixation, being dependent on fungal activity with respect to deadwood colonization and C supply. Conversely, bacterial N fixation helps to decrease the constraints of deadwood decomposition for fungi. Both the CO2 efflux and N accumulation that are a result of a joint action of deadwood bacteria and fungi may be significant for nutrient cycling at ecosystem levels. Especially in boreal forests with low N stocks, deadwood retention may help to improve the nutritional status and fertility of soils.IMPORTANCE Wood represents a globally important stock of C, and its mineralization importantly contributes to the global C cycle. Microorganisms play a key role in deadwood decomposition, since they possess enzymatic tools for the degradation of recalcitrant plant polymers. The present paradigm is that fungi accomplish degradation while commensalist bacteria exploit the products of fungal extracellular enzymatic cleavage, but this assumption was never backed by the analysis of microbial roles in deadwood. This study clearly identifies the roles of fungi and bacteria in the microbiome and demonstrates the importance of bacteria and their N fixation for the nutrient balance in deadwood as well as fluxes at the ecosystem level. Deadwood decomposition is shown as a process where fungi and bacteria play defined, complementary roles.

• Klíčová slova
• bacteria, deadwood, decomposition, forest ecosystems, fungi, metatranscriptomics, microbiome, nitrogen fixation, nutrient cycling,
• Publikační typ
• časopisecké články MeSH

Stable isotope probing (SIP) provides the opportunity to label decomposer microorganisms that build their biomass on a specific substrate. In combination with high-throughput sequencing, SIP allows for the identification of microbial community members involved in a particular decomposition process. Further information can be gained (in SIP experiments) through gene-targeted metagenomics and metatranscriptomics, opening the possibility to describe the pool of genes catalyzing specific decomposition reactions in situ and to identify the diversity of genes that are expressed. When combined with gene descriptions of fungal and/or bacterial isolates from the same environment, specific biochemical reactions involved in decomposition can be linked to individual microbial taxa. Here, we describe the use of these methods to explore the decomposer community of fungi and bacteria in forest litter and soil.

• Klíčová slova
• Metagenomics, Microbial communities, Organic matter decomposition, Soil ecology, Stable isotope probing, metatranscriptomics,
• MeSH
• Bacteria metabolismus   MeSH
• biomasa MeSH
• houby metabolismus   MeSH
• lesy MeSH
• mykobiom * MeSH
• půda chemie   MeSH
• půdní mikrobiologie MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• půda MeSH

Despite their ecological importance, nothing is known about the diversity and abundance of RNA viruses in termites (Termitoidae). We used a metatranscriptomics approach to determine the RNA virome structure of 50 diverse species of termite that differ in both phylogenetic position and colony composition. From these samples, we identified 67 novel RNA viruses, characterized their genomes, quantified their abundance and inferred their evolutionary history. These viruses were found within or similar to those from the Togaviridae, Iflaviridae, Polycipiviridae, Flaviviridae, Leviviridae, Narnaviridae, Mitoviridae, Lispivirdae, Phasmaviridae, Picobirnaviridae and Partitiviridae. However, all viruses identified were novel and divergent, exhibiting only 20% to 45% amino acid identity to previously identified viruses. Our analysis suggested that 17 of the viruses identified were termite-infecting, with the remainder likely associated with the termite microbiome or diet. Unclassified sobemo-like and bunya-like viruses dominated termite viromes, while most of the phylogenetic diversity was provided by the picobirna- and mitovirus-like viruses. Of note was the identification of a novel flavi-like virus most closely related to those found in marine vertebrates and invertebrates. Notably, the sampling procedure had the strongest association with virome composition, with greater RNA virome diversity in libraries prepared from whole termite bodies than those that only sampled heads.

• Klíčová slova
• RNA sequencing, RNA viruses, ecology, evolution, metatranscriptomics, termites,
• MeSH
• genetická variace MeSH
• genom virový genetika   MeSH
• Isoptera virologie   MeSH
• RNA virová genetika   MeSH
• RNA-viry klasifikace   genetika   izolace a purifikace   MeSH
• virom genetika   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• RNA virová MeSH

The productivity of the ocean is largely dependent on iron availability, and marine phytoplankton have evolved sophisticated mechanisms to cope with chronically low iron levels in vast regions of the open ocean. By analyzing the metabarcoding data generated from the Tara Oceans expedition, we determined how the global distribution of the model marine chlorarachniophyte Bigelowiella natans varies across regions with different iron concentrations. We performed a comprehensive proteomics analysis of the molecular mechanisms underpinning the adaptation of B. natans to iron scarcity and report on the temporal response of cells to iron enrichment. Our results highlight the role of phytotransferrin in iron homeostasis and indicate the involvement of CREG1 protein in the response to iron availability. Analysis of the Tara Oceans metagenomes and metatranscriptomes also points to a similar role for CREG1, which is found to be widely distributed among marine plankton but to show a strong bias in gene and transcript abundance toward iron-deficient regions. Our analyses allowed us to define a new subfamily of the CobW domain-containing COG0523 putative metal chaperones which are involved in iron metabolism and are restricted to only a few phytoplankton lineages in addition to B. natans At the physiological level, we elucidated the mechanisms allowing a fast recovery of PSII photochemistry after resupply of iron. Collectively, our study demonstrates that B. natans is well adapted to dynamically respond to a changing iron environment and suggests that CREG1 and COG0523 are important components of iron homeostasis in B. natans and other phytoplankton.IMPORTANCE Despite low iron availability in the ocean, marine phytoplankton require considerable amounts of iron for their growth and proliferation. While there is a constantly growing knowledge of iron uptake and its role in the cellular processes of the most abundant marine photosynthetic groups, there are still largely overlooked branches of the eukaryotic tree of life, such as the chlorarachniophytes. In the present work, we focused on the model chlorarachniophyte Bigelowiella natans, integrating physiological and proteomic analyses in culture conditions with the mining of omics data generated by the Tara Oceans expedition. We provide unique insight into the complex responses of B. natans to iron availability, including novel links to iron metabolism conserved in other phytoplankton lineages.

• Klíčová slova
• Bigelowiella natans, iron, metagenomics, metatranscriptomics, photosynthesis, phytoplankton, proteomics,
• Publikační typ
• časopisecké články MeSH

Stable isotope probing (SIP) provides the opportunity to label decomposer microorganisms that build their biomass on a specific substrate. In combination with high-throughput sequencing, SIP allows for the identification of fungal community members involved in a particular decomposition process. Further information can be gained through gene-targeted metagenomics and metatranscriptomics, opening the possibility to describe the pool of genes catalyzing specific decomposition reactions in situ and to identify the diversity of genes that are expressed. When combined with gene descriptions of fungal isolates from the same environment, specific biochemical reactions involved in decomposition can be linked to individual fungal taxa. Here we describe the use of these methods to explore the cellulolytic fungal community in forest litter and soil.

• Klíčová slova
• Cellulose, Metagenomics, Metatranscriptomics, Microbial communities, Organic matter decomposition, Soil ecology, Stable isotope probing,
• MeSH
• celulosa metabolismus   MeSH
• DNA fungální genetika   MeSH
• fylogeneze MeSH
• houby MeSH
• izotopové značení metody   MeSH
• metagenomika * MeSH
• půdní mikrobiologie * MeSH
• regulace genové exprese u hub MeSH
• substrátová specifita MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• celulosa MeSH
• DNA fungální MeSH