Transcriptome-Stable Isotope Probing Provides Targeted Functional and Taxonomic Insights Into Microaerobic Pollutant-Degrading Aquifer Microbiota

While most studies using RNA-stable isotope probing (SIP) to date have focused on ribosomal RNA, the detection of 13C-labeled mRNA has rarely been demonstrated. This approach could alleviate some of the major caveats of current non-target environmental “omics.” Here, we demonstrate the feasibility of total RNA-SIP in an experiment where hydrocarbon-degrading microbes from a BTEX-contaminated aquifer were studied in microcosms with 13C-labeled toluene under microoxic conditions. From the total sequencing reads (∼30 mio. reads per density-resolved RNA fraction), an average of 1.2% of reads per sample were identified as non-rRNA, including mRNA. Members of the Rhodocyclaceae (including those related to Quatrionicoccus spp.) were most abundant and enriched in 13C-rRNA, while well-known aerobic degraders such as Pseudomonas spp. remained unlabeled. Transcripts related to cell motility, secondary metabolite formation and xenobiotics degradation were highly labeled with 13C. mRNA of phenol hydroxylase genes were highly labeled and abundant, while other transcripts of toluene-activation were not detected. Clear labeling of catechol 2,3-dioxygenase transcripts supported previous findings that some of these extradiol dioxygenases were adapted to low oxygen concentrations. We introduce a novel combination of total RNA-SIP with calculation of transcript-specific enrichment factors (EFs) in 13C-RNA, enabling a targeted approach to process-relevant gene expression in complex microbiomes

Functional Traits Co-Occurring with Mobile Genetic Elements in the Microbiome of the Atacama Desert

Mobile genetic elements (MGEs) play an essential role in bacterial adaptation and evolution. These elements are enriched within bacterial communities from extreme environments. However, very little is known if specific genes co-occur with MGEs in extreme environments and, if so, what their function is. We used shotgun-sequencing to analyse the metagenomes of 12 soil samples and characterized the composition of MGEs and the genes co-occurring with them. The samples ranged from less arid coastal sites to the inland hyperarid core of the Atacama Desert, as well as from sediments below boulders, protected from UV-irradiation. MGEs were enriched at the hyperarid sites compared with sediments from below boulders and less arid sites. MGEs were mostly co-occurring with genes belonging to the Cluster Orthologous Group (COG) categories “replication, recombination and repair,” “transcription” and “signal transduction mechanisms.” In general, genes coding for transcriptional regulators and histidine kinases were the most abundant genes proximal to MGEs. Genes involved in energy production were significantly enriched close to MGEs at the hyperarid sites. For example, dehydrogenases, reductases, hydrolases and chlorite dismutase and other enzymes linked to nitrogen metabolism such as nitrite- and nitro-reductase. Stress response genes, including genes involved in antimicrobial and heavy metal resistance genes, were rarely found near MGEs. The present study suggests that MGEs could play an essential role in the adaptation of the soil microbiome in hyperarid desert soils by the modulation of housekeeping genes such as those involved in energy production.EC/FP7/339231/EU/Habitability of Martian Environments: Exploring the Physiological and Environmental Limits of Life/HOM

CRISPR-Cas type I-A Cascade complex couples viral infection surveillance to host transcriptional regulation in the dependence of Csa3b

CRISPR-Cas (clustered regularly interspaced short palindromic repeats and the associated genes) constitute adaptive immune systems in bacteria and archaea and they provide sequence specific immunity against foreign nucleic acids. CRISPR-Cas systems are activated by viral infection. However, little is known about how CRISPR-Cas systems are activated in response to viral infection or how their expression is controlled in the absence of viral infection. Here, we demonstrate that both the transcriptional regulator Csa3b, and the type I-A interference complex Cascade, are required to transcriptionally repress the interference gene cassette in the archaeon Sulfolobus. Csa3b binds to two palindromic repeat sites in the promoter region of the cassette and facilitates binding of the Cascade to the promoter region. Upon viral infection, loading of Cascade complexes onto crRNA-matching protospacers leads to relief of the transcriptional repression. Our data demonstrate a mechanism coupling CRISPR-Cas surveillance of protospacers to transcriptional regulation of the interference gene cassette thereby allowing a fast response to viral infection

A long-term field experiment demonstrates the influence of tillage on the bacterial potential to produce soil structure-stabilizing agents such as exopolysaccharides and lipopolysaccharides

Background: Stable soil aggregates are essential for optimal crop growth and preventing soil erosion. However, tillage is often used in agriculture to loosen the soil, which disrupts the integrity of these aggregates. Soil aggregation can be enhanced by bacteria through their ability to produce exopolysaccharides and lipopolysaccharides. These compounds stabilize soil aggregates by “gluing” soil particles together. However, it has yet to be shown how tillage influences the bacterial potential to produce aggregate-stabilizing agents. Therefore, we sampled conventional and reduced tillage treatments at 0–10 cm, 10–20 cm and 20–50 cm from a long-term field trial in Frick, Switzerland. We compared the stable aggregate fraction of the soil and the bacterial potential to produce exopolysaccharides (EPS) and lipopolysaccharides (LPS) under different tillage regimes by employing a shotgun metagenomic approach. We established a method which combines hidden Markov model searches with blasts against sequences derived from the Kyoto Encyclopedia of Genes and Genomes database to analyze genes specific for the biosynthesis of these compounds. Results: Our data revealed that the stable aggregate fraction as well as the bacterial potential to produce EPS and LPS were comparable under both tillage regimes. The highest potential to produce these compounds was found in the upper soil layer, which was disturbed by tillage, but had higher content of organic carbon compared to the layer below the tillage horizon. Additionally, key players of EPS and LPS production differed at different sampling depths. Some families with high potential to produce EPS and LPS, such as Chitinophagaceae and Bradyrhizobiaceae, were more abundant in the upper soil layers, while others, e.g. Nitrospiraceae and Planctomycetaceae, preferred the lowest sampled soil depth. Each family had the potential to form a limited number of different aggregate-stabilizing agents. Conclusions: Our results indicate that conventional tillage and reduced tillage equally promote the bacterial potential to produce EPS and LPS in the tillage horizon. However, as major bacterial groups triggering EPS and LPS formation were not the same, it is likely that gene expression pattern differ in the different treatments due to various pathways of gene induction and transcription in different bacterial species

Site-Specific Conditions Change the Response of Bacterial Producers of Soil Structure-Stabilizing Agents Such as Exopolysaccarides and Lipopolysaccarides to Tillage Intensity

Agro-ecosystems experience huge losses of land every year due to soil erosion induced by poor agricultural practices such as intensive tillage. Erosion can be minimized by the presence of stable soil aggregates, the formation of which can be promoted by bacteria. Some of these microorganisms have the ability to produce exopolysaccharides and lipopolysaccharides that "glue" soil particles together. However, little is known about the influence of tillage intensity on the bacterial potential to produce these polysaccharides, even though more stable soil aggregates are usually observed under less intense tillage. As the effects of tillage intensity on soil aggregate stability may vary between sites, we hypothesized that the response of polysaccharide-producing bacteria to tillage intensity is also determined by site-specific conditions. To investigate this, we performed a high-throughput shotgun sequencing of DNA extracted from conventionally and reduced tilled soils from three tillage system field trials characterized by different soil parameters. While we confirmed that the impact of tillage intensity on soil aggregates is site-specific, we could connect improved aggregate stability with increased absolute abundance of genes involved in the production of exopolysaccharides and lipopolysaccharides. The potential to produce polysaccharides was generally promoted under reduced tillage due to the increased microbial biomass. We also found that the response of most potential producers of polysaccharides to tillage was site-specific, e.g., Oxalobacteraceae had higher potential to produce polysaccharides under reduced tillage at one site, and showed the opposite response at another site. However, the response of some potential producers of polysaccharides to tillage did not depend on site characteristics, but rather on their taxonomic affiliation, i.e., all members of Actinobacteria that responded to tillage intensity had higher potential for exopolysaccharide and lipopolysaccharide production specifically under reduced tillage. This could be especially crucial for aggregate stability, as polysaccharides produced by different taxa have different "gluing" efficiency. Overall, our data indicate that tillage intensity could affect aggregate stability by both influencing the absolute abundance of genes involved in the production of exopolysaccharides and lipopolysaccharides, as well as by inducing shifts in the community of potential polysaccharide producers. The effects of tillage intensity depend mostly on site-specific conditions

Definition of Core Bacterial Taxa in Different Root Compartments of Dactylis glomerata, Grown in Soil under Different Levels of Land Use Intensity

Plant-associated bacterial assemblages are critical for plant fitness. Thus, identifying a consistent plant-associated core microbiome is important for predicting community responses to environmental changes. Our target was to identify the core bacterial microbiome of orchard grass Dactylis glomerata L. and to assess the part that is most sensitive to land management. Dactylis glomerata L. samples were collected from grassland sites with contrasting land use intensities but comparable soil properties at three different timepoints. To assess the plant-associated bacterial community structure in the compartments rhizosphere, bulk soil and endosphere, a molecular barcoding approach based on high throughput 16S rRNA amplicon sequencing was used. A distinct composition of plant-associated core bacterial communities independent of land use intensity was identified. Pseudomonas, Rhizobium and Bradyrhizobium were ubiquitously found in the root bacterial core microbiome. In the rhizosphere, the majority of assigned genera were Rhodoplanes, Methylibium, Kaistobacter and Bradyrhizobium. Due to the frequent occurrence of plant-promoting abilities in the genera found in the plant-associated core bacterial communities, our study helps to identify “healthy” plant-associated bacterial core communities. The variable part of the plant-associated microbiome, represented by the fluctuation of taxa at the different sampling timepoints, was increased under low land use intensity. This higher compositional variation in samples from plots with low land use intensity indicates a more selective recruitment of bacteria with traits required at different timepoints of plant development compared to samples from plots with high land use intensity

The Influence of Land Use Intensity on the Plant-Associated Microbiome of Dactylis glomerata L.

In this study, we investigated the impact of different land use intensities (LUI) on the root-associated microbiome of Dactylis glomerata (orchardgrass). For this purpose, eight sampling sites with different land use intensity levels but comparable soil properties were selected in the southwest of Germany. Experimental plots covered land use levels from natural grassland up to intensively managed meadows. We used 16S rRNA gene based barcoding to assess the plant-associated community structure in the endosphere, rhizosphere and bulk soil of D. glomerata. Samples were taken at the reproductive stage of the plant in early summer. Our data indicated that roots harbor a distinct bacterial community, which clearly differed from the microbiome of the rhizosphere and bulk soil. Our results revealed Pseudomonadaceae, Enterobacteriaceae and Comamonadaceae as the most abundant endophytes independently of land use intensity. Rhizosphere and bulk soil were dominated also by Proteobacteria, but the most abundant families differed from those obtained from root samples. In the soil, the effect of land use intensity was more pronounced compared to root endophytes leading to a clearly distinct pattern of bacterial communities under different LUI from rhizosphere and bulk soil vs. endophytes. Overall, a change of community structure on the plant–soil interface was observed, as the number of shared OTUs between all three compartments investigated increased with decreasing land use intensity. Thus, our findings suggest a stronger interaction of the plant with its surrounding soil under low land use intensity. Furthermore, the amount and quality of available nitrogen was identified as a major driver for shifts in the microbiome structure in all compartments