Type IV-A3 CRISPR-Cas systems drive inter-plasmid conflicts by acquiring spacers in trans.

Plasmid-encoded type IV-A CRISPR-Cas systems lack an acquisition module, feature a DinG helicase instead of a nuclease, and form ribonucleoprotein complexes of unknown biological functions. Type IV-A3 systems are carried by conjugative plasmids that often harbor antibiotic-resistance genes and their CRISPR array contents suggest a role in mediating inter-plasmid conflicts, but this function remains unexplored. Here, we demonstrate that a plasmid-encoded type IV-A3 system co-opts the type I-E adaptation machinery from its host, Klebsiella pneumoniae (K. pneumoniae), to update its CRISPR array. Furthermore, we reveal that robust interference of conjugative plasmids and phages is elicited through CRISPR RNA-dependent transcriptional repression. By silencing plasmid core functions, type IV-A3 impacts the horizontal transfer and stability of targeted plasmids, supporting its role in plasmid competition. Our findings shed light on the mechanisms and ecological function of type IV-A3 systems and demonstrate their practical efficacy for countering antibiotic resistance in clinically relevant strains.

Long-term conservation tillage with reduced nitrogen fertilization intensity can improve winter wheat health via positive plant-microorganism feedback in the rhizosphere.

Microbiome-based solutions are regarded key for sustainable agroecosystems. However, it is unclear how agricultural practices affect the rhizosphere microbiome, plant-microorganism interactions and crop performance under field conditions. Therefore, we installed root observation windows in a winter wheat field cultivated either under long-term mouldboard plough (MP) or cultivator tillage (CT). Each tillage practice was also compared at two nitrogen (N) fertilization intensities, intensive (recommended N-supply with pesticides/growth regulators) or extensive (reduced N-supply, no fungicides/growth regulators). Shoot biomass, root exudates and rhizosphere metabolites, physiological stress indicators, and gene expression were analyzed together with the rhizosphere microbiome (bacterial/archaeal 16S rRNA gene, fungal ITS amplicon, and shotgun metagenome sequencing) shortly before flowering. Compared to MP, the rhizosphere of CT winter wheat contained more primary and secondary metabolites, especially benzoxazinoid derivatives. Potential copiotrophic and plant-beneficial taxa (e.g. Bacillus, Devosia, and Trichoderma) as well as functional genes (e.g. siderophore production, trehalose synthase, and ACC deaminase) were enriched in the CT rhizosphere, suggesting that tillage affected belowground plant-microorganism interactions. In addition, physiological stress markers were suppressed in CT winter wheat compared to MP. In summary, tillage practice was a major driver of crop performance, root deposits, and rhizosphere microbiome interactions, while the N-fertilization intensity was also relevant, but less important.

Draft genomes of seven isolates from Danish wastewater facilities belonging to Pseudomonas, Bacillus, Pseudochrobactrum, Brevundimonas, and Pandoraea.

We report here seven draft genomes of bacterial strains from two Danish wastewater facilities, two of which might be characterized as a new group within the Pseudomonas and Pseudochrobactrum genera, respectively. These genomes will provide useful references for understanding bacterial interactions and horizontal gene transfer within bacterial communities.

Warmer winters result in reshaping of the European beech forest soil microbiome (bacteria, archaea and fungi)-With potential implications for ecosystem functioning.

In temperate regions, climate warming alters temperature and precipitation regimes. During winter, a decline in insulating snow cover changes the soil environment, where especially frost exposure can have severe implications for soil microorganisms and subsequently for soil nutrient dynamics. Here, we investigated winter climate change responses in European beech forests soil microbiome. Nine study sites with each three treatments (snow exclusion, insolation, and ambient) were investigated. Long-term adaptation to average climate was explored by comparing across sites. Triplicated treatment plots were used to evaluate short-term (one single winter) responses. Community profiles of bacteria, archaea and fungi were created using amplicon sequencing. Correlations between the microbiome, vegetation and soil physicochemical properties were found. We identify core members of the forest-microbiome and link them to key processes, for example, mycorrhizal symbiont and specialized beech wood degraders (fungi) and nitrogen cycling (bacteria, archaea). For bacteria, the shift of the microbiome composition due to short-term soil temperature manipulations in winter was similar to the community differences observed between long-term relatively cold to warm conditions. The results suggest a strong link between the changes in the microbiomes and changes in environmental processes, for example, nitrogen dynamics, driven by variations in winter climate.

An easily modifiable conjugative plasmid for studying horizontal gene transfer.

Horizontal gene transfer is an important mechanism in bacterial evolution and can occur at striking frequencies when mediated by mobile genetic elements. Conjugative plasmids are mobile genetic elements that are main drivers of horizontal transfer and a major facilitator in the spread of antibiotic resistance genes. However, conjugative plasmid models that readily can be genetically modified with the aim to study horizontal transfer are not currently available. The aim of this study was to develop a conjugative plasmid model where the insertion of gene cassettes such as reporter genes (e.g., fluorescent proteins) or antibiotic resistance genes would be efficient and convenient. Here, we introduced a single attTn7 site into the conjugative broad-host-range IncP-1 plasmid pKJK5 in a non-disruptive manner. Furthermore, a version with lower transfer rate and a non-conjugative version of pKJK5-attTn7 were also constructed. The advantage of having the attTn7 sites is that genes of interest can be introduced in a single step with very high success rate using the Tn7 transposition system. In addition, larger genetic fragments can be inserted. To illustrate the efficacy of the constructed pKJK5 plasmids, they were complemented with sfGFP (a gene encoding superfolder green fluorescent protein) in addition to seven different ß-lactamase genes representing the four known classes of ß-lactamases.

Horizontal Gene Transfer of an IncP1 Plasmid to Soil Bacterial Community Introduced by Escherichia coli through Manure Amendment in Soil Microcosms.

The quantification and identification of new plasmid-acquiring bacteria in representative mating conditions is critical to characterize the risk of horizontal gene transfer in the environment. This study aimed to quantify conjugation events resulting from manure application to soils and identify the transconjugants resulting from these events. Conjugation was quantified at multiple time points by plating and flow cytometry, and the transconjugants were recovered by fluorescence-activated cell sorting and identified by 16S rRNA sequencing. Overall, transconjugants were only observed within the first 4 days after manure application and at values close to the detection limits of this experimental system (1.00-2.49 log CFU/g of manured soil, ranging between 10-5 and 10-4 transconjugants-to-donor ratios). In the pool of recovered transconjugants, we found amplicon sequence variants (ASVs) of genera whose origin was traced to soils (Bacillus and Nocardioides) and manure (Comamonas and Rahnella). This work showed that gene transfer from fecal to soil bacteria occurred despite the less-than-optimal conditions faced by manure bacteria when transferred to soils, but these events were rare, mainly happened shortly after manure application, and the plasmid did not colonize the soil community. This study provides important information to determine the risks of AMR spread via manure application.

Barley Rhizosphere Microbiome Transplantation - A Strategy to Decrease Susceptibility of Barley Grown in Soils With Low Microbial Diversity to Powdery Mildew.

Beneficial bacteria in the rhizosphere are known to trigger faster and stronger plant immune responses to biotic and abiotic stressors. In the present study, we aimed to test the hypothesis that a rhizosphere microbiome transplant (RMT) may improve the immune response and reduce the disease rates of barley (Hordeum vulgare). This hypothesis was tested in a greenhouse system with the powdery mildew-causing fungus Blumeria graminis f. sp. hordei (Bgh). Detached rhizosphere microbiome from barley grown in a field soil was transplanted to barley seedlings grown in potting soil with reduced microbial diversity. Saline-treated plants served as control. At the three-leaf stage, barley was infected with Bgh. Decreased susceptibility to Bgh was observed for barley treated with the RMT as displayed by lower Bgh pustule counts in a detached leaf assay. A trend toward enhanced relative transcript abundances of the defense-related genes PR1b and PR17b was observed in leaves, 24 h after the Bgh challenge, when compared to the control. Moreover, 10 days after the Bgh challenge, the barley rhizosphere microbiome was harvested and analyzed by sequencing of 16S rRNA gene amplicons. The microbial community composition was significantly influenced by the RMT and displayed higher microbial diversity compared to the control. Furthermore, microbial beta-diversity and predicted functional profiles revealed a treatment-dependent clustering. Bacterial isolates from the RMT showed in vitro plant beneficial traits related to induced resistance. Our results showed that transplantation of a rhizosphere microbiome could be a sustainable strategy to improve the health of plants grown in potting soil with low microbial diversity under greenhouse conditions.

The treasure inside barley seeds: microbial diversity and plant beneficial bacteria.

BACKGROUND: Bacteria associated with plants can enhance the plants' growth and resistance against phytopathogens. Today, growers aim to reduce the use of mineral fertilizers and pesticides. Since phytopathogens cause severe yield losses in crop production systems, biological alternatives gain more attention. Plant and also seed endophytes have the potential to influence the plant, especially seed-borne bacteria may express their beneficiary impact at initial plant developmental stages. In the current study, we assessed the endophytic seed microbiome of seven genetically diverse barley accessions by 16S rRNA gene amplicon sequencing and verified the in vitro plant beneficial potential of isolated seed endophytes. Furthermore, we investigated the impact of the barley genotype and its seed microbiome on the rhizosphere microbiome at an early growth stage by 16S rRNA gene amplicon sequencing. RESULTS: The plant genotype displayed a significant impact on the microbiota in both barley seed and rhizosphere. Consequently, the microbial alpha- and beta-diversity of the endophytic seed microbiome was highly influenced by the genotype. Interestingly, no correlation was observed between the endophytic seed microbiome and the single nucleotide polymorphisms of the seven genotypes. Unclassified members of Enterobacteriaceae were by far most dominant. Other abundant genera in the seed microbiome belonged to Curtobacterium, Paenibacillus, Pantoea, Sanguibacter and Saccharibacillus. Endophytes isolated from barley seeds were affiliated to dominant genera of the core seed microbiome, based on their 16S rRNA gene sequence. Most of these endophytic isolates produced in vitro plant beneficial secondary metabolites known to induce plant resistance. CONCLUSION: Although barley accessions representing high genetic diversity displayed a genotype-dependent endophytic seed microbiome, a core seed microbiome with high relative abundances was identified. Endophytic isolates were affiliated to members of the core seed microbiome and many of them showed plant beneficial properties. We propose therefore that new breeding strategies should consider genotypes with high abundance of beneficial microbes.

Impact of Tellurite on the Metabolism of Paenibacillus pabuli AL109b With Flagellin Production Explaining High Reduction Capacity.

Tellurium (Te) is a metalloid with scarce and scattered abundance but with an increased interest in human activity for its uses in emerging technologies. As is seen for other metals and metalloids, the result of mining activity and improper disposal of high-tech devices will lead to niches with increased abundance of Te. This metalloid will be more available to bacteria and represent an increasing selective pressure. This environmental problem may constitute an opportunity to search for microorganisms with genetic and molecular mechanisms of microbial resistance to Te toxic anions. Organisms from Te-contaminated niches could provide tools for Te remediation and fabrication of Te-containing structures with added value. The objective of this study was to determine the ability of a high metal-resistant Paenibacillus pabuli strain ALJ109b, isolated from high metal content mining residues, to reduce tellurite ion, and to evaluate the formation of metallic tellurium by cellular reduction, isolate the protein responsible, and determine the metabolic response to tellurite during growth. P. pabuli ALJ109b demonstrated to be resistant to Te (IV) at concentrations higher than reported for its genus. It can efficiently remove soluble Te (IV) from solution, over 20% in 8 h of growth, and reduce it to elemental Te, forming monodisperse nanostructures, verified by scattering electron microscopy. Cultivation of P. pabuli ALJ109b in the presence of Te (IV) affected the general protein expression pattern, and hence the metabolism, as demonstrated by high-throughput proteomic analysis. The Te (IV)-induced metabolic shift is characterized by an activation of ROS response. Flagellin from P. pabuli ALJ109b demonstrates high Te (0) forming activity in neutral to basic conditions in a range of temperatures from 20°C to 37°C. In conclusion, the first metabolic characterization of a strain of P. pabuli response to Te (IV) reveals a highly resistant strain with a unique Te (IV) proteomic response. This strain, and its flagellin, display, all the features of potential tools for Te nanoparticle production.

Tillage shapes the soil and rhizosphere microbiome of barley-but not its susceptibility towards Blumeria graminis f. sp. hordei.

Long-term agricultural practices are assumed to shape the rhizosphere microbiome of crops with implications for plant health. In a long-term field experiment, we investigated the effect of different tillage and fertilization practices on soil and barley rhizosphere microbial communities by means of amplicon sequencing of 16S rRNA gene fragments from total community DNA. Differences in the microbial community composition depending on the tillage practice, but not the fertilization intensity were revealed. To examine whether these soil and rhizosphere microbiome differences influence the plant defense response, barley (cultivar Golden Promise) was grown in field or standard potting soil under greenhouse conditions and challenged with Blumeria graminis f. sp. hordei (Bgh). Amplicon sequence analysis showed that preceding tillage practice, but also aboveground Bgh challenge significantly influenced the microbial community composition. Expression of plant defense-related genes PR1b and PR17b was higher in challenged compared to unchallenged plants. The Bgh infection rates were strikingly lower for barley grown in field soil compared to potting soil. Although previous agricultural management shaped the rhizosphere microbiome, no differences in plant health were observed. We propose therefore that the management-independent higher microbial diversity of field soils compared to potting soils contributed to the low infection rates of barley.

Microbial community analysis of soils under different soybean cropping regimes in the Argentinean south-eastern Humid Pampas.

Soil microbial communities are key players of ecosystem processes and important for crop and soil health. The Humid Pampas region in Argentina concentrates 75% of the national soybean production, which is based on intensive use of agrochemicals, monocropping and no-till. A long-term field experiment under no-till management in the southeast of the Argentinean Pampas provides a unique opportunity to compare soybean under monocropping with cultivation including alternating cover crops or in a three-phase rotation. We hypothesized that cropping regimes and season affect soil microbial community composition and diversity. Amplicon sequencing of 16S rRNA genes and internal transcribed spacer fragments showed a stronger microbial seasonal dynamic in conservation regimes compared to monocropping. In addition, several bacterial (e.g. Catenulispora, Streptomyces and Bacillus) and fungal genera (e.g. Exophiala) with cropping regime-dependent differential relative abundances were identified. Despite a temporal shift in microbial and chemical parameters, this study shows that long-term cropping regimes shaped the soil microbiota. This might have important implications for soil quality and soybean performance and should therefore be considered in the development of sustainable agricultural managements.

You don't have the guts: a diverse set of fungi survive passage through Macrotermes bellicosus termite guts.

BACKGROUND: Monoculture farming poses significant disease challenges, but fungus-farming termites are able to successfully keep their monoculture crop free from contamination by other fungi. It has been hypothesised that obligate gut passage of all plant substrate used to manure the fungal symbiont is key to accomplish this. Here we refute this hypothesis in the fungus-farming termite species Macrotermes bellicosus. RESULTS: We first used ITS amplicon sequencing to show that plant substrate foraged on by termite workers harbour diverse fungal communities, which potentially could challenge the farming symbiosis. Subsequently, we cultivated fungi from dissected sections of termite guts to show that fungal diversity does not decrease during gut passage. Therefore, we investigated if healthy combs harboured these undesirable fungal genera, and whether the presence of workers affected fungal diversity within combs. Removal of workers led to a surge in fungal diversity in combs, implying that termite defences must be responsible for the near-complete absence of other fungi in functioning termite gardens. CONCLUSIONS: The rapid proliferation of some of these fungi when colonies are compromised indicates that some antagonists successfully employ a sit-and-wait strategy that allows them to remain dormant until conditions are favourable. Although this strategy requires potentially many years of waiting, it prevents these fungi from engaging in an evolutionary arms race with the termite host, which employs a series of complementary behavioural and chemical defences that may prove insurmountable.

Snow microbiome functional analyses reveal novel aspects of microbial metabolism of complex organic compounds.

Microbes active in extreme cold are not as well explored as those of other extreme environments. Studies have revealed a substantial microbial diversity and identified cold-specific microbiome molecular functions. We analyzed the metagenomes and metatranscriptomes of 20 snow samples collected in early and late spring in Svalbard, Norway using mi-faser, our read-based computational microbiome function annotation tool. Our results reveal a more diverse microbiome functional capacity and activity in the early- vs. late-spring samples. We also find that functional dissimilarity between the same-sample metagenomes and metatranscriptomes is significantly higher in early than late spring samples. These findings suggest that early spring samples may contain a larger fraction of DNA of dormant (or dead) organisms, while late spring samples reflect a new, metabolically active community. We further show that the abundance of sequencing reads mapping to the fatty acid synthesis-related microbial pathways in late spring metagenomes and metatranscriptomes is significantly correlated with the organic acid levels measured in these samples. Similarly, the organic acid levels correlate with the pathway read abundances of geraniol degradation and inversely correlate with those of styrene degradation, suggesting a possible nutrient change. Our study thus highlights the activity of microbial degradation pathways of complex organic compounds previously unreported at low temperatures.

Compatibility of X-ray computed tomography with plant gene expression, rhizosphere bacterial communities and enzyme activities.

Non-invasive X-ray computed tomography (XRCT) is increasingly used in rhizosphere research to visualize development of soil-root interfaces in situ. However, exposing living systems to X-rays can potentially impact their processes and metabolites. In order to evaluate these effects, we assessed the responses of rhizosphere processes 1 and 24 h after a low X-ray exposure (0.81 Gy). Changes in root gene expression patterns occurred 1 h after exposure with down-regulation of cell wall-, lipid metabolism-, and cell stress-related genes, but no differences remained after 24 h. At either time point, XRCT did not affect either root antioxidative enzyme activities or the composition of the rhizosphere bacterial microbiome and microbial growth parameters. The potential activities of leucine aminopeptidase and phosphomonoesterase were lower at 1 h, but did not differ from the control 24 h after exposure. A time delay of 24 h after a low X-ray exposure (0.81 Gy) was sufficient to reverse any effects on the observed rhizosphere systems. Our data suggest that before implementing novel experimental designs involving XRCT, a study on its impact on the investigated processes should be conducted.

Do Organic Substrates Drive Microbial Community Interactions in Arctic Snow?

The effect of nutrients on microbial interactions, including competition and collaboration, has mainly been studied in laboratories, but their potential application to complex ecosystems is unknown. Here, we examined the effect of changes in organic acids among other parameters on snow microbial communities in situ over 2 months. We compared snow bacterial communities from a low organic acid content period to that from a higher organic acid period. We hypothesized that an increase in organic acids would shift the dominant microbial interaction from collaboration to competition. To evaluate microbial interactions, we built taxonomic co-variance networks from OTUs obtained from 16S rRNA gene sequencing. In addition, we tracked marker genes of microbial cooperation (plasmid backbone genes) and competition (antibiotic resistance genes) across both sampling periods in metagenomes and metatranscriptomes. Our results showed a decrease in the average connectivity of the network during late spring compared to the early spring that we interpreted as a decrease of cooperation. This observation was strengthened by the significantly more abundant plasmid backbone genes in the metagenomes from the early spring. The modularity of the network from the late spring was also found to be higher than the one from the early spring, which is another possible indicator of increased competition. Antibiotic resistance genes were significantly more abundant in the late spring metagenomes. In addition, antibiotic resistance genes were also positively correlated to the organic acid concentration of the snow across both seasons. Snow organic acid content might be responsible for this change in bacterial interactions in the Arctic snow community.

Sources and selection of snow-specific microbial communities in a Greenlandic sea ice snow cover.

Sea ice and its snow cover are critical for global processes including climate regulation and biogeochemical cycles. Despite an increase in studies focused on snow microorganisms, the ecology of snow inhabitants remains unclear. In this study, we investigated sources and selection of a snowpack-specific microbial community by comparing metagenomes from samples collected in a Greenlandic fjord within a vertical profile including atmosphere, snowpack with four distinct layers of snow, sea ice brine and seawater. Microbial communities in all snow layers derived from mixed sources, both marine and terrestrial, and were more similar to atmospheric communities than to sea ice or seawater communities. The surface snow metagenomes were characterized by the occurrence of genes involved in photochemical stress resistance, primary production and metabolism of diverse carbon sources. The basal saline snow layer that was in direct contact with the sea ice surface harbored a higher abundance of cells than the overlying snow layers, with a predominance of Alteromonadales and a higher relative abundance of marine representatives. However, the overall taxonomic structure of the saline layer was more similar to that of other snow layers and the atmosphere than to underlying sea ice and seawater. The expulsion of relatively nutrient-rich sea ice brine into basal snow might have stimulated the growth of copiotrophic psychro- and halotolerant snow members. Our study indicates that the size, composition and function of snowpack microbial communities over sea ice were influenced primarily by atmospheric deposition and inflow of sea ice brine and that they form a snow-specific assemblage reflecting the particular environmental conditions of the snowpack habitat.

Snow and ice ecosystems: not so extreme.

Snow and ice environments cover up to 21% of the Earth's surface. They have been regarded as extreme environments because of their low temperatures, high UV irradiation, low nutrients and low water availability, and thus, their microbial activity has not been considered relevant from a global microbial ecology viewpoint. In this review, we focus on why snow and ice habitats might not be extreme from a microbiological perspective. Microorganisms interact closely with the abiotic conditions imposed by snow and ice habitats by having diverse adaptations, that include genetic resistance mechanisms, to different types of stresses in addition to inhabiting various niches where these potential stresses might be reduced. The microbial communities inhabiting snow and ice are not only abundant and taxonomically diverse, but complex in terms of their interactions. Altogether, snow and ice seem to be true ecosystems with a role in global biogeochemical cycles that has likely been underestimated. Future work should expand past resistance studies to understanding the function of these ecosystems.

Reconstructing rare soil microbial genomes using in situ enrichments and metagenomics.

Despite extensive direct sequencing efforts and advanced analytical tools, reconstructing microbial genomes from soil using metagenomics have been challenging due to the tremendous diversity and relatively uniform distribution of genomes found in this system. Here we used enrichment techniques in an attempt to decrease the complexity of a soil microbiome prior to sequencing by submitting it to a range of physical and chemical stresses in 23 separate microcosms for 4 months. The metagenomic analysis of these microcosms at the end of the treatment yielded 540 Mb of assembly using standard de novo assembly techniques (a total of 559,555 genes and 29,176 functions), from which we could recover novel bacterial genomes, plasmids and phages. The recovered genomes belonged to Leifsonia (n = 2), Rhodanobacter (n = 5), Acidobacteria (n = 2), Sporolactobacillus (n = 2, novel nitrogen fixing taxon), Ktedonobacter (n = 1, second representative of the family Ktedonobacteraceae), Streptomyces (n = 3, novel polyketide synthase modules), and Burkholderia (n = 2, includes mega-plasmids conferring mercury resistance). Assembled genomes averaged to 5.9 Mb, with relative abundances ranging from rare (<0.0001%) to relatively abundant (>0.01%) in the original soil microbiome. Furthermore, we detected them in samples collected from geographically distant locations, particularly more in temperate soils compared to samples originating from high-latitude soils and deserts. To the best of our knowledge, this study is the first successful attempt to assemble multiple bacterial genomes directly from a soil sample. Our findings demonstrate that developing pertinent enrichment conditions can stimulate environmental genomic discoveries that would have been impossible to achieve with canonical approaches that focus solely upon post-sequencing data treatment.

Potential drivers of microbial community structure and function in Arctic spring snow.

The Arctic seasonal snowpack can extend at times over a third of the Earth's land surface. This chemically dynamic environment interacts constantly with different environmental compartments such as atmosphere, soil and meltwater, and thus, strongly influences the entire biosphere. However, the microbial community associated with this habitat remains poorly understood. Our objective was to investigate the functional capacities, diversity and dynamics of the microorganisms in snow and to test the hypothesis that their functional signature reflects the snow environment. We applied a metagenomic approach to nine snow samples taken over 2 months during the spring season. Fungi, Bacteroidetes, and Proteobacteria were predominant in metagenomic datasets and changes in community structure were apparent throughout the field season. Functional data that strongly correlated with chemical parameters like mercury or nitrogen species supported that this variation could be explained by fluctuations in environmental conditions. Through inter-environmental comparisons we examined potential drivers of snowpack microbial community functioning. Known cold adaptations were detected in all compared environments without any apparent differences in their relative abundance, implying that adaptive mechanisms related to environmental factors other than temperature may play a role in defining the snow microbial community. Photochemical reactions and oxidative stress seem to be decisive parameters in structuring microbial communities inside Arctic snowpacks.