Anne Schöler

Analysis of soil microbial communities based on amplicon sequencing of marker genes

The use of cultivation independent methods has revolutionized soil biology in the last decades. Most popular approaches are based on directly extracted DNA from soil and subsequent analysis of PCR-amplified marker genes by next-generation sequencing. While these high-throughput methods offer novel possibilities over cultivation-based approaches, several key points need to be considered to minimize potential biases during library preparation and downstream bioinformatic analysis. This opinion paper highlights crucial steps that should be considered for accurate analysis and data interpretation.

Making big data smart—how to use metagenomics to understand soil quality

Next-generation sequencing (NGS) has revolutionized the field of biology over the last decade. The Genomes OnLine Database (GOLD) that monitors sequencing projects worldwide has grown from just 1575 sequencing projects in 2005 to over 70,000 in 2015 (Reddy et al. 2015). This is partly caused by a rapid drop in the price of high-throughput sequencing (Hayden 2014), but also an increase of free user-friendly bioinformatical tools such as MG-RAST (Meyer et al. 2008), MEGAN (Huson et al. 2016) and user fora such as seqanswers.com, biostars.org etc. This Bbrave new world^ was introduced into soil sciences more than 10 years ago (Daniel 2005) and is becoming increasingly popular, as it is the only approach known, which allows a direct assessment of microbial community composition and function on various trophic levels. Today, according to the web of science, more than 900 papers have been published on soil metagenomes. In early times, sequencing depth was in the range of less than 1 Gbase and often resulted in the identification of only major functional traits and house keeping genes; today in recent publications up to 100 Gbases have been sequenced (Hultman et al. 2015), which allowed even a partly reconstruction of genomes of single microbes from the obtained reads. However, the interpretation of soil metagenomics data is still a challenge, given the often complex composition of the microbiomes, as well as their huge dynamics in time and space (Ebrahimi and Or 2016). Previous papers have focused on specific aspects of metagenomic data generation or analysis, such as the impact of the DNA extraction methods and read annotation stringency on the apparent composition of a metagenome (Delmont et al. 2013), the importance of coverage estimation (Rodriguez-R and Konstantinidis 2014a) or the change from the current use of gene-centric snapshots towards genomecentric temporal studies (Prosser 2015). Major steps and recommendations for further reading are summarized in Table 1. In this paper, we discuss some basic guidelines for the experimental design of metagenomic surveys to characterize community composition and function of soil microbiomes, without losing the environmental context.

Phosphorus depletion in forest soils shapes bacterial communities towards phosphorus recycling systems

Phosphorus (P) is an important macronutrient for all biota on earth but similarly a finite resource. Microorganisms play on both sides of the fence as they effectively mineralize organic and solubilize precipitated forms of soil phosphorus but conversely also take up and immobilize P. Therefore, we analysed the role of microbes in two beech forest soils with high and low P content by direct sequencing of metagenomic deoxyribonucleic acid. For inorganic P solubilization, a significantly higher microbial potential was detected in the P-rich soil. This trait especially referred to Candidatus Solibacter usiatus, likewise one of the dominating species in the data sets. A higher microbial potential for efficient phosphate uptake systems (pstSCAB) was detected in the P-depleted soil. Genes involved in P starvation response regulation (phoB, phoR) were prevalent in both soils. This underlines the importance of effective phosphate (Pho) regulon control for microorganisms to use alternative P sources during phosphate limitation. Predicted genes were primarily harboured by Rhizobiales, Actinomycetales and Acidobacteriales.

Microevolution of Anthrax from a Young Ancestor (M.A.Y.A.) Suggests a Soil-Borne Life Cycle of Bacillus anthracis

During an anthrax outbreak at the Pollino National Park (Basilicata, Italy) in 2004, diseased cattle were buried and from these anthrax-foci Bacillus anthracis endospores still diffuse to the surface resulting in local accumulations. Recent data suggest that B. anthracis multiplies in soil outside the animal-host body. This notion is supported by the frequent isolation of B. anthracis from soil lacking one or both virulence plasmids. Such strains represent an evolutionary dead end, as they are likely no longer able to successfully infect new hosts. This loss of virulence plasmids is explained most simply by postulating a soil-borne life cycle of the pathogen. To test this hypothesis we investigated possible microevolution at two natural anthrax foci from the 2004 outbreak. If valid, then genotypes of strains isolated from near the surface at these foci should be on a different evolutionary trajectory from those below residing in deeper-laying horizons close to the carcass. Thus, the genetic diversity of B. anthracis isolates was compared conducting Progressive Hierarchical Resolving Assays using Nucleic Acids (PHRANA) and next generation Whole Genome Sequencing (WGS). PHRANA was not discriminatory enough to resolve the fine genetic relationships between the isolates. Conversely, WGS of nine isolates from near-surface and nine from near-carcass revealed five isolate specific SNPs, four of which were found only in different near-surface isolates. In support of our hypothesis, one surface-isolate lacked plasmid pXO1 and also harbored one of the unique SNPs. Taken together, our results suggest a limited soil-borne life cycle of B. anthracis.

Metagenomic analyses reveal no differences in genes involved in cellulose degradation under different tillage treatments

Incorporation of plant litter is a frequent agricultural practice to increase nutrient availability in soil, and relies heavily on the activity of cellulose-degrading microorganisms. Here we address the question of how different tillage treatments affect soil microbial communities and their cellulose-degrading potential in a long-term agricultural experiment. To identify potential differences in microbial taxonomy and functionality, we generated six soil metagenomes of conventional (CT) and reduced (RT) tillage-treated topsoil samples, which differed in their potential extracellular cellulolytic activity as well as their microbial biomass. Taxonomic analysis of metagenomic data revealed few differences between RT and CT, and a dominance of Proteobacteria and Actinobacteria, whereas eukaryotic phyla were not prevalent. Prediction of cellulolytic enzymes revealed glycoside hydrolase families 1, 3 and 94, auxiliary activity family 8 and carbohydrate-binding module 2 as the most abundant in soil. These were annotated mainly to the phyla of Proteobacteria, Actinobacteria and Bacteroidetes. These results suggest that the observed higher cellulolytic activity in RT soils can be explained by a higher microbial biomass or changed expression levels but not by shifts in the soil microbiome. Overall, this study reveals the stability of soil microbial communities and cellulolytic gene composition under the investigated tillage treatments.

The Microbiome of Endophytic, Wood Colonizing Bacteria from Pine Trees as Affected by Pine Wilt Disease.

Pine wilt disease (PWD) is a devastating forest disease present worldwide. In this study we analyzed the effects of the invasion of the pinewood nematode Bursaphelenchus xylophilus, the major pathogen causing PWD, on the endophytic microbiome of adult P. pinaster trees. Wood samples from trees with different degrees of PWD disease were collected at two sites (A and M) in Portugal. Endophytic bacteria were characterized based on directly extracted DNA by fingerprinting and barcoding using the 16S rRNA gene as marker. Furthermore, cultivation-based approaches were used to obtain isolates of the major taxa to study their ecophysiology. The endophytic microbiome from P. pinaster trees differed significantly between the two sampling sites. Main bacterial OTUs belonged to the Proteobacteria (39% (site M) - 97% (site A)), and Firmicutes (0.70% (site A) - 44% (site M)). However, consequences of the invasion with the pathogen were comparable. Interestingly diversity of wood endophytic bacteria increased with the severity of the diseases, with highest diversity levels observed in in the most affected trees. Our results suggest that in the first stages of the disease, the defence mechanisms of plants are repressed by the pathogen, resulting in a colonization of the wood interior by soil microorganisms.

Dominant Groups of Potentially Active Bacteria Shared by Barley Seeds become Less Abundant in Root Associated Microbiome

Endophytes are microorganisms colonizing plant internal tissues. They are ubiquitously associated with plants and play an important role in plant growth and health. In this work, we grew five modern cultivars of barley in axenic systems using sterile sand mixture as well as in greenhouse with natural soil. We characterized the potentially active microbial communities associated with seeds and roots using rRNA based amplicon sequencing. The seeds of the different cultivars share a great part of their microbiome, as we observed a predominance of a few bacterial OTUs assigned to Phyllobacterium, Paenibacillus, and Trabusiella. Seed endophytes, particularly members of the Enterobacteriacea and Paenibacillaceae, were important members of root endophytes in axenic systems, where there were no external microbes. However, when plants were grown in soil, seed endophytes became less abundant in root associated microbiome. We observed a clear enrichment of Actinobacteriacea and Rhizobiaceae, indicating a strong influence of the soil bacterial communities on the composition of the root microbiome. Two OTUs assigned to Phyllobacteriaceae were found in all seeds and root samples growing in soil, indicating a relationship between seed-borne and root associated microbiome in barley. Even though the role of endophytic bacteria remains to be clarified, it is known that many members of the genera detected in our study produce phytohormones, shape seedling exudate profile and may play an important role in germination and establishment of the seedlings.

Development of a Stable Lung Microbiome in Healthy Neonatal Mice

The lower respiratory tract has been previously considered sterile in a healthy state, but advances in culture-independent techniques for microbial identification and characterization have revealed that the lung harbors a diverse microbiome. Although research on the lung microbiome is increasing and important questions were already addressed, longitudinal studies aiming to describe developmental stages of the microbial communities from the early neonatal period to adulthood are lacking. Thus, little is known about the early-life development of the lung microbiome and the impact of external factors during these stages. In this study, we applied a barcoding approach based on high-throughput sequencing of 16S ribosomal RNA gene amplicon libraries to determine age-dependent differences in the bacterial fraction of the murine lung microbiome and to assess potential influences of differing “environmental microbiomes” (simulated by the application of used litter material to the cages). We could clearly show that the diversity of the bacterial community harbored in the murine lung increases with age. Interestingly, bacteria belonging to the genera Delftia and Rhodococcus formed an age-independent core microbiome. The addition of the used litter material influenced the lung microbiota of young mice but did not significantly alter the community composition of adult animals. Our findings elucidate the dynamic nature of the early-life lung microbiota and its stabilization with age. Further, this study indicates that even slight environmental changes modulate the bacterial community composition of the lung microbiome in early life, whereas the lung microbes of adults demonstrate higher resilience towards environmental variations.

Reconstruction of Transformation Processes Catalyzed by the Soil Microbiome Using Metagenomic Approaches

Microorganisms are central players in the turnover of nutrients in soil and drive the decomposition of complex organic materials into simpler forms that can be utilized by other biota. Therefore microbes strongly drive soil quality and ecosystem services provided by soils, including plant yield and quality. Thus it is one of the major goals of soil sciences to describe the most relevant enzymes that are involved in nutrient mobilization and to understand the regulation of gene expression of the corresponding genes. This task is however impeded by the enormous microbial diversity in soils. Indeed, we are far to appreciate the number of species present in 1 g of soil, as well as the major functional traits they carry. Here, also most next-generation sequencing (NGS) approaches fail as immense sequencing efforts are needed to fully uncover the functional diversity of soils. Thus even if a gene of interest can be identified by BLAST similarity analysis, the obtained number of reads by NGS is too low for a quantitative assessment of the gene or for a description of its taxonomic diversity. Here we present an integrated approach, which we termed the second-generation full cycle approach, to quantify the abundance and diversity of key enzymes involved in nutrient mobilization. This approach involves the functional annotation of metagenomic data with a relative low coverage (5 Gbases or less) and the design of highly targeted primer systems to assess the abundance or diversity of enzyme-coding genes that are drivers for a particular transformation step in nutrient turnover.

The Impact of the Diurnal Cycle on the Microbial Transcriptome in the Rhizosphere of Barley

While root exudation follows diurnal rhythms, little is known about the consequences for the microbiome of the rhizosphere. In this study, we used a metatranscriptomic approach to analyze the active microbial communities, before and after sunrise, in the rhizosphere of barley. We detected increased activities of many prokaryotic microbial taxa and functions at the pre-dawn stage, compared to post-dawn. Actinomycetales, Planctomycetales, Rhizobiales, and Burkholderiales were the most abundant and therefore the most active orders in the barley rhizosphere. The latter two, as well as Xanthomonadales, Sphingomonadales, and Caulobacterales showed a significantly higher abundance in pre-dawn samples compared to post-dawn samples. These changes in taxonomy coincide with functional changes as genes involved in both carbohydrate and amino acid metabolism were more abundant in pre-dawn samples compared to post-dawn samples. This study significantly enhances our present knowledge on how rhizospheric microbiota perceives and responds to changes in the soil during dark and light periods.