Diversity of soil microbial communities: In the perspective of targeting functional genes

Abstract Intensive land use in agriculture can lead to higher loss of biodiversity in soils and subsequently carbon due to tillage and application of pesticides and fertilizers. The microbial communities are pivotal to ecosystem processes in soil such as nutrient cycling, soil formation and plant productivity and thus are affected by land use. The main aim of this thesis is to understand the effects of land-use management on the diversity of both functions and taxonomy of soil microbial communities. Diversity of microbial enzymes involved is key to understand processes such as carbon cycling in soil. Due to current inefficient methods in obtaining the vast diversity in functional enzymes from environmental samples, we developed a molecular method based on sequence capture to address this issue. As this method is based on oligonucleotide probes, a bioinformatics pipeline to generate probes for targeting mainly diverse functional genes in environmental communities was designed. A web-based implementation of this pipeline was established to make it possible for other researchers to design custom oligos for their own study of interest in understanding ecosystems. The laboratory method ‘captured metagenomics’ was developed and optimized using two soils samples from probes designed to target genes coding for enzymes involved in organic matter degradation. Captured metagenomics was validated and it was superior to current genetic methods such as whole metagenome sequencing. The land-use management of soils affected the functional composition of microbes in degrading organic matter observed using captured metagenomics. The amount of nitrogen played an important role in defining the functional composition of SOM degrading enzymes while the amount of carbon played a role in defining the taxonomic composition of microbes in the soils. There was no correlation between the functional and taxonomic diversity of microbial communities in the soils that were part of this study. Land-use management also affected the taxonomic composition of AMF and agricultural practices decrease their diversity tremendously. Among the different farming systems, organic farming maintained a higher phylogenetic diversity of AMF with similar cereal production as other strategies. However, the land-use management of soils in this study did not affect bacterial taxonomic composition. These approaches have to be extended to understand microbial responses using enzyme expression to infer their behaviour and adaptation to environmental changes. Similar approaches on functional composition and diversity of microbes in more diverse soils would help us to understand different ecosystems and their functions more clearly

Captured metagenomics: large-scale targeting of genes based on 'sequence capture' reveals functional diversity in soils.

Microbial enzyme diversity is a key to understand many ecosystem processes. Whole metagenome sequencing (WMG) obtains information on functional genes, but it is costly and inefficient due to large amount of sequencing that is required. In this study, we have applied a captured metagenomics technique for functional genes in soil microorganisms, as an alternative to WMG. Large-scale targeting of functional genes, coding for enzymes related to organic matter degradation, was applied to two agricultural soil communities through captured metagenomics. Captured metagenomics uses custom-designed, hybridization-based oligonucleotide probes that enrich functional genes of interest in metagenomic libraries where only probe-bound DNA fragments are sequenced. The captured metagenomes were highly enriched with targeted genes while maintaining their target diversity and their taxonomic distribution correlated well with the traditional ribosomal sequencing. The captured metagenomes were highly enriched with genes related to organic matter degradation; at least five times more than similar, publicly available soil WMG projects. This target enrichment technique also preserves the functional representation of the soils, thereby facilitating comparative metagenomics projects. Here, we present the first study that applies the captured metagenomics approach in large scale, and this novel method allows deep investigations of central ecosystem processes by studying functional gene abundances

Divergent airway microbiomes in lung transplant recipients with or without pulmonary infection

BACKGROUND: Lung transplant (LTx) recipients are at increased risk for airway infections, but the cause of infection is often difficult to establish with traditional culture-based techniques. The objectives of the study was to compare the airway microbiome in LTx patients with and without ongoing airway infection and identify differences in their microbiome composition.METHODS: LTx recipients were prospectively followed with bronchoalveolar lavage (BAL) during the first year after transplantation. The likelihood of airway infection at the time of sampling was graded based on clinical criteria and BAL cultures, and BAL fluid levels of the inflammatory markers heparin-binding protein (HBP), IL-1β and IL-8 were determined with ELISA. The bacterial microbiome of the samples were analysed with 16S rDNA sequencing and characterized based on richness and evenness. The distance in microbiome composition between samples were determined using Bray-Curtis and weighted and unweighted UniFrac.RESULTS: A total of 46 samples from 22 patients were included in the study. Samples collected during infection and samples with high levels of inflammation were characterized by loss of bacterial diversity and a significantly different species composition. Burkholderia, Corynebacterium and Staphylococcus were enriched during infection and inflammation, whereas anaerobes and normal oropharyngeal flora were less abundant. The most common findings in BAL cultures, including Pseudomonas aeruginosa, were not enriched during infection.CONCLUSION: This study gives important insights into the dynamics of the airway microbiome of LTx recipients, and suggests that lung infections are associated with a disruption in the homeostasis of the microbiome

Altered gut microbiota community structure and correlated immune system changes in dibutyl phthalate exposed mice

Di-n-butyl phthalate (DBP) is a ubiquitous environmental contaminant linked with various adverse health effects, including immune system dysfunction. Gut microbial dysbiosis can contribute to a wide range of pathogenesis, particularly immune disease. Here, we investigated the impact of DBP on the gut microbiome and examined correlations with immune system changes after five weeks oral exposure (10 or 100 mg/kg/day) in adult male mice. The fecal microbiome composition was characterized using 16S rRNA sequencing. DBP-treated mice displayed a significantly distinct microbial community composition, indicated by Bray-Curtis distance. Numerous amplicon sequence variants (ASVs) at the genus level were altered. Compared to the vehicle control group, the 10 mg/kg/day DBP group had 63 more abundant and 65 less abundant ASVs, while 60 ASVs were increased and 76 ASVs were decreased in the 100 mg/kg/day DBP group. Both DBP treatment groups showed higher abundances of ASVs assigned to Desulfovibrio (Proteobacteria phylum) and Enterorhabdus genera, while ASVs belonging to Parabacteroides, Lachnospiraceae UCG-006 and Lachnoclostridium were less common compared to the control group. Interestingly, an ASV belonging to Rumniniclostridium 6, which was less abundant in DBP-treated mice, demonstrated a negative correlation with the increased number of non-classical monocytes observed in the blood of DBP-treated animals. In addition, an ASV from Lachnospiraceae UCG-001, which was more abundant in the DBP-treated animals, showed a positive correlation with the non-classical monocyte increase. This study shows that DBP exposure greatly modifies the gut bacterial microbiome and indicates a potential contribution of microbial dysbiosis to DBP-induced immune system impairment, illustrating the importance of investigating how interactions between exposome components can affect health

An Okinawan-Based Nordic Diet Leads to Profound Effects on Gut Microbiota and Plasma Metabolites Linked to Glucose and Lipid Metabolism

Dietary interventions modify gut microbiota and clinical outcomes. Weight reduction and improved glucose and lipid homeostasis were observed after adopting an Okinawan-based Nordic diet (O-BN) in individuals with type 2 diabetes. The aim of the present study was to explore changes in metabolomics and gut microbiota during O-BN and correlate changes with clinical outcomes. A total of 30 patients (17 women), aged 57.5 ± 8.2 years, diabetes duration 10.4 ± 7.6 years, 90% over-weight, were included. Participants were provided an O-BN for 12 weeks. Before and after intervention, and 16 weeks afterwards, anthropometry and clinical data were estimated and questionnaires were collected, as well as samples of blood and stool. Plasma metabolomics were determined by gas- (GC-MS) or liquid- (LC-MS) chromatography-based mass spectrometry and fecal microbiota determination was based on 16S rRNA amplicons from regions V1-V2. During the intervention, weight (6.8%), waist circumference (6.1%), and levels of glucose, HbA1c, insulin, triglycerides, and cholesterol were decreased. Of 602 metabolites, 323 were changed for any or both periods; 199 (101 lipids) metabolites were decreased while 58 (43 lipids) metabolites were increased during the intervention. Changes in glucose homeostasis were linked to changes in, e.g., 1,5-anhydroglucitol, thyroxine, and chiro-inositol. Changes of microbe beta diversity correlated positively with food components and negatively with IL-18 (p = 0.045). Abundance differences at phylum and genus levels were found. Abundances of Actinobacteria, Bacteroidetes, Firmicutes, and Verrucomicrobia correlated with anthropometry, HbA1c, lipids, inflammation, and food. Changes in metabolites and microbiota were reversed after the intervention. The O-BN-induced changes in metabolomics and gut microbiota correspond to clinical outcomes of reduced weight and inflammation and improved glucose and lipid metabolism

Agricultural management practices influence AMF diversity and community composition with cascading effects on plant productivity

Understanding the effects of different agricultural practices on the mycorrhizal symbiosis is important for agricultural production and the sustainable use of soil. We investigated the composition and diversity of arbuscular mycorrhizal fungi (AMF) in soils from fields under different agricultural practices (conventional and organic cereal fields, leys and permanent pastures) in southern Sweden. The diversity of AMF was found to be greatest in permanent pastures, corroborating evidence that agricultural practices such as tillage impair AMF diversity. Neither geographical location nor soil type nor any of the major soil characteristics we measured impacted AMF diversity or community composition. AMF community composition was significantly affected by the different agricultural practices, particularly conventional management, which reduced AMF diversity. Of the cereal fields sampled, those under organic management held the greatest AMF diversity, and in a glasshouse experiment this greater diversity was positively related to barley phosphorus uptake and grain biomass production. Our results demonstrate the impact of different agricultural practices on AMF communities. In particular, we demonstrate the ability of organic farming to sustain greater AMF diversity relative to conventional farming, and the potential importance of this increased diversity for sustainable cereal production

Agricultural land use determines functional genetic diversity of soil microbial communities

Microbial communities play a major role in the degradation of soil organic matter (SOM) in soils. Despite its significance, the functional diversity of the highly diverse microbial communities is poorly understood. To address this, we applied a recently developed technique, captured metagenomics, to determine the effects of land-use on the functional genetic diversity of genes involved in the carbon degradation of SOM in five pairs of agricultural soils with either winter wheat or grass as management. In addition, 16S rRNA based amplicon sequencing was used to study the taxonomic composition in the same soils. The functional genes resulting from the captured metagenomes had a higher abundance and diversity of sequences coding for enzymes degrading SOM in the grasslands compared to the wheat soils. Though the taxonomic diversity did not correlate with the land use. Amounts of C and N (organic matter content) in the soils affected both functional and taxonomic diversity of the microbial communities, where N was highly correlated to their functions and C was highly correlated to their taxonomy. Captured metagenomic analyses of the functional genes may provide a measure of the potential SOM degradation capacity by soil microbial communities at a high resolution. This can be used for assessments of how agricultural management affects the functioning of soil communities

MetCap: a bioinformatics probe design pipeline for large-scale targeted metagenomics

Background: Massive sequencing of genes from different environments has evolved metagenomics as central to enhancing the understanding of the wide diversity of micro-organisms and their roles in driving ecological processes. Reduced cost and high throughput sequencing has made large-scale projects achievable to a wider group of researchers, though complete metagenome sequencing is still a daunting task in terms of sequencing as well as the downstream bioinformatics analyses. Alternative approaches such as targeted amplicon sequencing requires custom PCR primer generation, and is not scalable to thousands of genes or gene families. Results: In this study, we are presenting a web-based tool called MetCap that circumvents the limitations of amplicon sequencing of multiple genes by designing probes that are suitable for large-scale targeted metagenomics sequencing studies. MetCap provides a novel approach to target thousands of genes and genomic regions that could be used in targeted metagenomics studies. Automatic analysis of user-defined sequences is performed, and probes specifically designed for metagenome studies are generated. To illustrate the advantage of a targeted metagenome approach, we have generated more than 300,000 probes that match more than 400,000 publicly available sequences related to carbon degradation, and used these probes for target sequencing in a soil metagenome study. The results show high enrichment of target genes and a successful capturing of the majority of gene families. MetCap is freely available to users from: http://soilecology.biol.lu.se/metcap/. Conclusion: MetCap is facilitating probe-based target enrichment as an easy and efficient alternative tool compared to complex primer-based enrichment for large-scale investigations of metagenomes. Our results have shown efficient large-scale target enrichment through MetCap-designed probes for a soil metagenome. The web service is suitable for any targeted metagenomics project that aims to study several genes simultaneously. The novel bioinformatics approach taken by the web service will enable researchers in microbial ecology to tap into the vast diversity of microbial communities using targeted metagenomics as a cost-effective alternative to whole metagenome sequencing