Rapid remodeling of the soil lipidome in response to a drying-rewetting event.

BACKGROUND: Microbiomes contribute to multiple ecosystem services by transforming organic matter in the soil. Extreme shifts in the environment, such as drying-rewetting cycles during drought, can impact the microbial metabolism of organic matter by altering microbial physiology and function. These physiological responses are mediated in part by lipids that are responsible for regulating interactions between cells and the environment. Despite this critical role in regulating the microbial response to stress, little is known about microbial lipids and metabolites in the soil or how they influence phenotypes that are expressed under drying-rewetting cycles. To address this knowledge gap, we conducted a soil incubation experiment to simulate soil drying during a summer drought of an arid grassland, then measured the response of the soil lipidome and metabolome during the first 3 h after wet-up. RESULTS: Reduced nutrient access during soil drying incurred a replacement of membrane phospholipids, resulting in a diminished abundance of multiple phosphorus-rich membrane lipids. The hot and dry conditions increased the prevalence of sphingolipids and lipids containing long-chain polyunsaturated fatty acids, both of which are associated with heat and osmotic stress-mitigating properties in fungi. This novel finding suggests that lipids commonly present in eukaryotes such as fungi may play a significant role in supporting community resilience displayed by arid land soil microbiomes during drought. As early as 10 min after rewetting dry soil, distinct changes were observed in several lipids that had bacterial signatures including a rapid increase in the abundance of glycerophospholipids with saturated and short fatty acid chains, prototypical of bacterial membrane lipids. Polar metabolites including disaccharides, nucleic acids, organic acids, inositols, and amino acids also increased in abundance upon rewetting. This rapid metabolic reactivation and growth after rewetting coincided with an increase in the relative abundance of firmicutes, suggesting that members of this phylum were positively impacted by rewetting. CONCLUSIONS: Our study revealed specific changes in lipids and metabolites that are indicative of stress adaptation, substrate use, and cellular recovery during soil drying and subsequent rewetting. The drought-induced nutrient limitation was reflected in the lipidome and polar metabolome, both of which rapidly shifted (within hours) upon rewet. Reduced nutrient access in dry soil caused the replacement of glycerophospholipids with phosphorus-free lipids and impeded resource-expensive osmolyte accumulation. Elevated levels of ceramides and lipids with long-chain polyunsaturated fatty acids in dry soil suggest that lipids likely play an important role in the drought tolerance of microbial taxa capable of synthesizing these lipids. An increasing abundance of bacterial glycerophospholipids and triacylglycerols with fatty acids typical of bacteria and polar metabolites suggest a metabolic recovery in representative bacteria once the environmental conditions are conducive for growth. These results underscore the importance of the soil lipidome as a robust indicator of microbial community responses, especially at the short time scales of cell-environment reactions. Video Abstract.

Trends in Microbial Community Composition and Function by Soil Depth.

Microbial communities play important roles in soil health, contributing to processes such as the turnover of organic matter and nutrient cycling. As soil edaphic properties such as chemical composition and physical structure change from surface layers to deeper ones, the soil microbiome similarly exhibits substantial variability with depth, with respect to both community composition and functional profiles. However, soil microbiome studies often neglect deeper soils, instead focusing on the top layer of soil. Here, we provide a synthesis on how the soil and its resident microbiome change with depth. We touch upon soil physicochemical properties, microbial diversity, composition, and functional profiles, with a special emphasis on carbon cycling. In doing so, we seek to highlight the importance of incorporating analyses of deeper soils in soil studies.

Development and Analysis of a Stable, Reduced Complexity Model Soil Microbiome.

The soil microbiome is central to the cycling of carbon and other nutrients and to the promotion of plant growth. Despite its importance, analysis of the soil microbiome is difficult due to its sheer complexity, with thousands of interacting species. Here, we reduced this complexity by developing model soil microbial consortia that are simpler and more amenable to experimental analysis but still represent important microbial functions of the native soil ecosystem. Samples were collected from an arid grassland soil and microbial communities (consisting mainly of bacterial species) were enriched on agar plates containing chitin as the main carbon source. Chitin was chosen because it is an abundant carbon and nitrogen polymer in soil that often requires the coordinated action of several microorganisms for complete metabolic degradation. Several soil consortia were derived that had tractable richness (30-50 OTUs) with diverse phyla representative of the native soil, including Actinobacteria, Bacteroidetes, Firmicutes, Proteobacteria, and Verrucomicrobia. The resulting consortia could be stored as glycerol or lyophilized stocks at -80°C and revived while retaining community composition, greatly increasing their use as tools for the research community at large. One of the consortia that was particularly stable was chosen as a model soil consortium (MSC-1) for further analysis. MSC-1 species interactions were studied using both pairwise co-cultivation in liquid media and during growth in soil under several perturbations. Co-abundance analyses highlighted interspecies interactions and helped to define keystone species, including Mycobacterium, Rhodococcus, and Rhizobiales taxa. These experiments demonstrate the success of an approach based on naturally enriching a community of interacting species that can be stored, revived, and shared. The knowledge gained from querying these communities and their interactions will enable better understanding of the soil microbiome and the roles these interactions play in this environment.

Deconstructing the Soil Microbiome into Reduced-Complexity Functional Modules.

The soil microbiome represents one of the most complex microbial communities on the planet, encompassing thousands of taxa and metabolic pathways, rendering holistic analyses computationally intensive and difficult. Here, we developed an alternative approach in which the complex soil microbiome was broken into components ("functional modules"), based on metabolic capacities, for individual characterization. We hypothesized that reproducible, low-complexity communities that represent functional modules could be obtained through targeted enrichments and that, in combination, they would encompass a large extent of the soil microbiome diversity. Enrichments were performed on a starting soil inoculum with defined media based on specific carbon substrates, antibiotics, alternative electron acceptors under anaerobic conditions, or alternative growing conditions reflective of common field stresses. The resultant communities were evaluated through 16S rRNA amplicon sequencing. Less permissive modules (anaerobic conditions, complex polysaccharides, and certain stresses) resulted in more distinct community profiles with higher richness and more variability between replicates, whereas modules with simple substrates were dominated by fewer species and were more reproducible. Collectively, approximately 27% of unique taxa present in the liquid soil extract control were found across functional modules. Taxa that were underrepresented or undetected in the source soil were also enriched across the modules. Metatranscriptomic analyses were carried out on a subset of the modules to investigate differences in functional gene expression. These results demonstrate that by dissecting the soil microbiome into discrete components it is possible to obtain a more comprehensive view of the soil microbiome and its biochemical potential than would be possible using more holistic analyses.IMPORTANCE The taxonomic and functional diversity inherent to the soil microbiome complicate assessments of the metabolic potential carried out by the community members. An alternative approach is to break down the soil microbiome into reduced-complexity subsets based on metabolic capacities (functional modules) prior to sequencing and analysis. Here, we demonstrate that this approach successfully identified specific phylogenetic and biochemical traits of the soil microbiome that otherwise remained hidden from a more top-down analysis.

Selection, Succession, and Stabilization of Soil Microbial Consortia.

Soil microorganisms play fundamental roles in cycling of soil carbon, nitrogen, and other nutrients, yet we have a poor understanding of how soil microbiomes are shaped by their nutritional and physical environment. In this study, we investigated the successional dynamics of a soil microbiome during 21 weeks of enrichment on chitin and its monomer, N-acetylglucosamine. We examined succession of the soil communities in a physically heterogeneous soil matrix as well as a homogeneous liquid medium. The guiding hypothesis was that the initial species richness would influence the tendency for the selected consortia to stabilize and maintain a relatively constant community structure over time. We also hypothesized that long-term, substrate-driven growth would result in consortia with reduced species richness compared to the parent microbiome and that this process would be deterministic with relatively little variation between replicates. We found that the initial species richness does influence the long-term community stability in both liquid media and soil and that lower initial richness results in a more rapid convergence to stability. Despite use of the same soil inoculum and access to the same major substrate, the resulting community composition differed greatly in soil from that in liquid medium. Hence, distinct selective pressures in soils relative to homogenous liquid media exist and can control community succession dynamics. This difference is likely related to the fact that soil microbiomes are more likely to thrive, with fewer compositional changes, in a soil matrix than in liquid environments. IMPORTANCE The soil microbiome carries out important ecosystem functions, but interactions between soil microbial communities have been difficult to study due to the high microbial diversity and complexity of the soil habitat. In this study, we successfully obtained stable consortia with reduced complexity that contained species found in the original source soil. These consortia and the methods used to obtain them can be a valuable resource for exploration of specific mechanisms underlying soil microbial community ecology. The results of this study also provide new experimental context to better inform how soil microbial communities are shaped by new environments and how a combination of initial taxonomic structure and physical environment influences stability.

Drought delays development of the sorghum root microbiome and enriches for monoderm bacteria.

Drought stress is a major obstacle to crop productivity, and the severity and frequency of drought are expected to increase in the coming century. Certain root-associated bacteria have been shown to mitigate the negative effects of drought stress on plant growth, and manipulation of the crop microbiome is an emerging strategy for overcoming drought stress in agricultural systems, yet the effect of drought on the development of the root microbiome is poorly understood. Through 16S rRNA amplicon and metatranscriptome sequencing, as well as root metabolomics, we demonstrate that drought delays the development of the early sorghum root microbiome and causes increased abundance and activity of monoderm bacteria, which lack an outer cell membrane and contain thick cell walls. Our data suggest that altered plant metabolism and increased activity of bacterial ATP-binding cassette (ABC) transporter genes are correlated with these shifts in community composition. Finally, inoculation experiments with monoderm isolates indicate that increased colonization of the root during drought can positively impact plant growth. Collectively, these results demonstrate the role that drought plays in restructuring the root microbiome and highlight the importance of temporal sampling when studying plant-associated microbiomes.

Drought and host selection influence bacterial community dynamics in the grass root microbiome.

Root endophytes have been shown to have important roles in determining host fitness under periods of drought stress, and yet the effect of drought on the broader root endosphere bacterial community remains largely uncharacterized. In this study, we present phylogenetic profiles of bacterial communities associated with drought-treated root and rhizosphere tissues of 18 species of plants with varying degrees of drought tolerance belonging to the Poaceae family, including important crop plants. Through 16S rRNA gene profiling across two distinct watering regimes and two developmental time points, we demonstrate that there is a strong correlation between host phylogenetic distance and the microbiome dissimilarity within root tissues, and that drought weakens this correlation by inducing conserved shifts in bacterial community composition. We identify a significant enrichment in a wide variety of Actinobacteria during drought within the roots of all hosts, and demonstrate that this enrichment is higher within the root than it is in the surrounding environments. Furthermore, we show that this observed enrichment is the result of an absolute increase in Actinobacterial abundance and that previously hypothesized mechanisms for observed enrichments in Actinobacteria in drought-treated soils are unlikely to fully account for the phenomena observed here within the plant root.

Drought Stress and Root-Associated Bacterial Communities.

Root-associated bacterial communities play a vital role in maintaining health of the plant host. These communities exist in complex relationships, where composition and abundance of community members is dependent on a number of factors such as local soil chemistry, plant genotype and phenotype, and perturbations in the surrounding abiotic environment. One common perturbation, drought, has been shown to have drastic effects on bacterial communities, yet little is understood about the underlying causes behind observed shifts in microbial abundance. As drought may affect root bacterial communities both directly by modulating moisture availability, as well as indirectly by altering soil chemistry and plant phenotypes, we provide a synthesis of observed trends in recent studies and discuss possible directions for future research that we hope will provide for more knowledgeable predictions about community responses to future drought events.

The role of the plant-specific ALTERED XYLOGLUCAN9 protein in Arabidopsis cell wall polysaccharide O-acetylation.

A mutation in the ALTERED XYLOGLUCAN9 (AXY9) gene was found to be causative for the decreased xyloglucan acetylation phenotype of the axy9.1 mutant, which was identified in a forward genetic screen for Arabidopsis (Arabidopsis thaliana) mutants. The axy9.1 mutant also exhibits decreased O-acetylation of xylan, implying that the AXY9 protein has a broad role in polysaccharide acetylation. An axy9 insertional mutant exhibits severe growth defects and collapsed xylem, demonstrating the importance of wall polysaccharide O-acetylation for normal plant growth and development. Localization and topological experiments indicate that the active site of the AXY9 protein resides within the Golgi lumen. The AXY9 protein appears to be a component of the plant cell wall polysaccharide acetylation pathway, which also includes the REDUCED WALL ACETYLATION and TRICHOME BIREFRINGENCE-LIKE proteins. The AXY9 protein is distinct from the TRICHOME BIREFRINGENCE-LIKE proteins, reported to be polysaccharide acetyltransferases, but does share homology with them and other acetyltransferases, suggesting that the AXY9 protein may act to produce an acetylated intermediate that is part of the O-acetylation pathway.