Javier A. Ceja-Navarro

Ecosystem retrogression drives nutrient depletion, reduced productivity, and profound reorganization of soil microbial communities. Using amplicon sequencing and genome‐resolved metagenomics, we examined how cross‐domain microbial networks and functional gene potential respond to long‐term phosphorus and nitrogen limitation along the well‐characterized Ecological Staircase chronosequence in Mendocino, California, USA. Microbial diversity and abundance declined sharply with terrace age for prokaryotes, predatory protists, and bacteriophages, whereas fungi and phototrophic protists increased in nutrient‐depleted, acidic soils. These compositional shifts were accompanied by major changes in reconstructed microbial networks: relative modularity increased alongside robustness, indicating adaptive reorganization that may sustain ecosystem function under resource scarcity. Fungi emerged as central stabilizers in these restructured networks, carrying enriched genetic potential to degrade plant polymers and mobilize phosphorus and nitrogen. Despite a decline in overall phage diversity, the relative abundance of phages encoding phosphorus‐mobilizing auxiliary metabolic genes increased, suggesting that viral contributions to host phosphorus metabolism may be enhanced under nutrient limitation. Together, these results demonstrate that ecosystem retrogression drives cross‐domain microbial reorganization toward fewer but more interconnected lineages, characterized by greater integration of functional genetic potential. This reorganization enhances the potential for functional resilience under extreme nutrient limitation, revealing how microbial networks adapt to maintain the capacity for nutrient cycling and stability as soils age and fertility declines.

Protists are ubiquitous soil predators that regulate bacterial communities and biogeochemical cycling, yet how their predation alters expression of nutrient-cycling genes and traits linked to antibiotic resistance genes (ARGs), virulence factors (VFs), and mobile genetic elements (MGEs) in natural soils remains poorly understood. Here, we used a short-term soil microcosm experiment to distinguish the effects of moisture-stimulated resident protists from enhanced predation by an introduced exogenous predatory protist community. Using quantitative stable isotope probing (qSIP) and metagenomic and metatranscriptomic analyses, we tracked protist activity and microbial responses over 3 days. Enhanced predation rapidly reshaped transcriptionally active microbial communities, increasing expression of nitrogen and phosphorus cycling genes while concurrently elevating diversity and transcription of ARGs, VFs, and MGEs, including multidrug-efflux systems, motility-, biofilm-related traits, and phage-associated elements. Metagenome-assembled genome─resolved analyses showed that some resident soil populations were activated by wet-up and remained transcriptionally active under predation pressure, encoding nutrient-cycling, resistance, virulence, and mobility traits that contributed to the functional background of wetted soils. These results suggest that, even over short time periods, protist predation links soil nutrient processing with environmentally relevant resistance and genetic mobility pathways, acting as a crucial ecological driver of gene expression related to nutrient processing and microbial interaction traits during environmental change.

Around the world, and for the past ~2 billion years, surfaces of many soils have a “living skin” made by tiny plants and microbes called a biological soil crust (biocrust). Biocrusts are crucially important for helping to support the ecosystems they inhabit, for example making soil fertile, storing or redirecting water and stopping erosion. Biocrusts also boost the variety of living things present in an area (biodiversity). The CrustNet project will determine what controls the biodiversity of biocrusts globally for the first time, and its outcomes. CrustNet is a networked, distributed study of biocrust ecology, with participants around the world. Participants conduct the same set of studies and collect the same types of data to be pooled together to create an unprecedented global database about biocrusts. CrustNet addresses: (1) The determinants of the global scale functional biodiversity of biocrusts (2) determinants of the variability and shape of the relationship between biodiversity and ecosystem function across ecosystems, (3) effects of biocrust functional biodiversity on ecosystem resistance and resilience to physical disturbance and climate change, and (4) determinants of plant-biocrust co-occurrence patterns. CrustNet uses a tiered research protocol, including low-cost observational studies and manipulative experiments. Tier 1 includes mandatory detailed surveys of the composition of biocrusts, measurement of ecosystem functions along a biocrust development gradient, and contribution of samples to a trait database. Tier 2 includes low-cost experimentally-applied physical disturbance of the soil and subsequent tracking of the response of biocrusts. Tier 3 includes experimental climate manipulations using reciprocal transplantations and rainfall reduction using passive shelters. These studies will be foundational to our understanding of determinants of biocrust diversity, function and response to disturbance. To date, 14 sites in 5 nations have already been established and sampled, and pledged sites are ever increasing (23 additional sites have been proposed by international sampler-partners). In the process of conducting this research, a world-wide collaboration is being established, leading to greater participation of researchers from diverse backgrounds, and unparalleled training opportunities.

Codon and nucleotide frequencies are known to relate to the rate of gene transcription, yet how these traits shape transcriptional profiles of soil microbial communities remains unclear. Here we test the prediction that functional genes with high codon optimization and energetically lower cost nucleotides ( i.e ., nucleotides requiring less adenosine triphosphate (ATP) for synthesis) have higher transcriptional expression in a soil microbial community. In laboratory incubations, we subjected an agricultural soil to two separate short-term environmental changes: labile carbon (glucose) addition or a sudden 30-min increase in temperature from 20 °C to 60 °C. Using the total genomic codon frequencies to predict preferred codon usage for each taxon, we then estimated codon optimization for each transcript. On the community level, we found a higher average level of codon optimization after the addition of glucose. Synonymous nucleotide composition in the transcript pool also shifted towards energetically cheaper nucleotides, favoring uracil (U) over adenine (A) and cytosine (C) over guanine (G). Similarly, we found that encoded amino acid usage shifted towards energetically cheaper amino acids in response to labile carbon. In contrast, in communities responding to heat shock, there were no significant differences in the averaged gene traits of expressed transcripts. We used metagenome-assembled-genomes to further examine the ability of gene traits to predict transcriptional responses within and between taxa. We found that traits of individual genes could not reliably predict the level of transcription of a gene within or between taxa—highlighting the limits of this approach. However, we did find that when traits were averaged across several related genes, codon optimization was able to predict levels of transcription in metabolic pathways associated with growth and nutrient uptake in response to glucose. Similar relationships were not observed in response to heat, or for functions associated with stress—such as genes associated with sporulation or heat shock. These results demonstrate that gene traits, such as codon usage, nucleotide selection, and amino acid selection, relate to the transcriptional expression of genes in soil microbial communities and suggests that these relationships may be dependent on both gene function and the specific type of environmental stimuli.

Background: Plant cell walls are interwoven structures recalcitrant to degradation. Native and adapted microbiomes can be particularly effective at plant cell wall deconstruction. Although most understanding of biological cell wall deconstruction has been obtained from isolates, cultivated microbiomes that break down cell walls have emerged as new sources for biotechnologically relevant microbes and enzymes. These microbiomes provide a unique resource to identify key interacting functional microbial groups and to guide the design of specialized synthetic microbial communities. Results: To establish a system assessing comparative microbiome performance, parallel microbiomes were cultivated on sorghum (Sorghum bicolor L. Moench) from compost inocula. Biomass loss and biochemical assays indicated that these microbiomes diverged in their ability to deconstruct biomass. Network reconstructions from gene expression dynamics identified key groups and potential interactions within the adapted sorghum-degrading communities, including Actinotalea, Filomicrobium, and Gemmatimonadetes populations. Functional analysis demonstrated that the microbiomes proceeded through successive stages that are linked to enzymes that deconstruct plant cell wall polymers. The combination of network and functional analysis highlighted the importance of cellulose-degrading Actinobacteria in differentiating the performance of these microbiomes. Conclusions: The two-tier cultivation of compost-derived microbiomes on sorghum led to the establishment of microbiomes for which community structure and performance could be assessed. The work reinforces the observation that subtle differences in community composition and the genomic content of strains may lead to significant differences in community performance.

Plant cell walls are interwoven structures recalcitrant to degradation. Both native and adapted microbiomes are particularly effective at plant cell wall deconstruction. Studying these deconstructive microbiomes provides an opportunity to assess microbiome performance and relate it to specific microbial populations and enzymes. To establish a system assessing comparative microbiome performance, parallel microbiomes were cultivated on sorghum (Sorghum bicolor L. Moench) from compost inocula. Biomass loss and biochemical assays indicated that these microbiomes diverged in their ability to deconstruct biomass. Network reconstructions from time-dependent gene expression identified key deconstructive groups within the adapted sorghum-degrading communities, including Actinotalea, Filomicrobium, and Gemmanimonadetes populations. Functional analysis of gene expression demonstrated that the microbiomes proceeded through successional stages that are linked to enzymes that deconstruct plant cell wall polymers. This combination of network and functional analysis highlighted the importance of celluloseactive Actinobacteria in differentiating the performance of these microbiomes.

Some insects form symbioses in which actinomycetes provide defense against pathogens by making antimicrobials. The range of chemical strategies employed across these associations, and how these strategies relate to insect lifestyle, remains underexplored. We assessed subsocial passalid beetles of the species Odontotaenius disjunctus, and their frass (fecal material), which is an important food resource within their galleries, as a model insect/actinomycete system. Through chemical and phylogenetic analyses, we found that O. disjunctus frass collected across eastern North America harbored multiple lineages of Streptomyces and diverse antimicrobials. Metabolites detected in frass displayed synergistic and antagonistic inhibition of a fungal entomopathogen, Metarhizium anisopliae, and multiple streptomycete isolates inhibited this pathogen when co-cultivated directly in frass. These findings support a model in which the lifestyle of O. disjunctus accommodates multiple Streptomyces lineages in their frass, resulting in a rich repertoire of antimicrobials that likely insulates their galleries against pathogenic invasion.

Background: Despite their widespread distribution and ecological importance, protists remain one of the least understood components of the soil and rhizosphere microbiome. Knowledge of the roles that protists play in stimulating organic matter decomposition and shaping microbiome dynamics continues to grow, but there remains a need to understand the extent to which biological and environmental factors mediate protist community assembly and dynamics. We hypothesize that protists communities are filtered by the influence of plants on their rhizosphere biological and physicochemical environment, resulting in patterns of protist diversity and composition that mirror previously observed diversity and successional dynamics in rhizosphere bacterial communities. Results: We analyzed protist communities associated with the rhizosphere and bulk soil of switchgrass (SG) plants (Panicum virgatum) at different phenological stages, grown in two marginal soils as part of a large-scale field experiment. Our results reveal that the diversity of protists is lower in rhizosphere than bulk soils, and that temporal variations depend on soil properties but are less pronounced in rhizosphere soil. Patterns of significantly prevalent protists groups in the rhizosphere suggest that most protists play varied ecological roles across plant growth stages and that some plant pathogenic protists and protists with omnivorous diets reoccur over time in the rhizosphere. We found that protist co-occurrence network dynamics are more complex in the rhizosphere compared to bulk soil. A phylogenetic bin-based null model analysis showed that protists’ community assembly in our study sites is mainly controlled by homogenous selection and dispersal limitation, with stronger selection in rhizosphere than bulk soil as SG grew and senesced. Conclusions: We demonstrate that environmental filtering is a dominant determinant of overall protist community properties and that at the rhizosphere level, plant control on the physical and biological environment is a critical driver of protist community composition and dynamics. Since protists are key contributors to plant nutrient availability and bacterial community composition and abundance, mapping and understanding their patterns in rhizosphere soil is foundational to understanding the ecology of the root-microbe-soil system.