Plasmids are so closely associated with pathogens and antibiotic resistance that their potential for conferring other traits is often overlooked. Few studies consider how the full suite of traits encoded by plasmids is related to a hosts environmental adaptation, particularly for Gram-positive bacteria. To investigate the role that plasmid traits might play in microbial communities from natural ecosystems, we identified plasmids carried by isolates of Curtobacterium (phylum Actinomycetota) from a variety of soil environments. We found that plasmids were common, but not ubiquitous, in the genus and varied greatly in their size and genetic diversity. There was little evidence of phylogenetic conservation among Curtobacterium plasmids even for closely related bacterial strains within the same ecotype, indicating that horizontal transmission of plasmids is common. The plasmids carried a wide diversity of traits that were not a random subset of the host chromosome. Furthermore, the composition of these plasmid traits was associated with the environmental context of the host bacterium. Together, the results indicate that plasmids contribute substantially to the microdiversity of a soil bacterium and that this diversity may play a role in niche differentiation and a bacteriums adaptation to its local environment.

Microbial community responses to environmental change are largely associated with ecological processes; however, the potential for microbes to rapidly evolve and adapt remains relatively unexplored in natural environments. To assess how ecological and evolutionary processes simultaneously alter the genetic diversity of a microbiome, we conducted two concurrent experiments in the leaf litter layer of soil over 18 mo across a climate gradient in Southern California. In the first experiment, we reciprocally transplanted microbial communities from five sites to test whether ecological shifts in ecotypes of the abundant bacterium, Curtobacterium, corresponded to past adaptive differentiation. In the transplanted communities, ecotypes converged toward that of the native communities growing on a common litter substrate. Moreover, these shifts were correlated with community-weighted mean trait values of the Curtobacterium ecotypes, indicating that some of the trait variation among ecotypes could be explained by local adaptation to climate conditions. In the second experiment, we transplanted an isogenic Curtobacterium strain and tracked genomic mutations associated with the sites across the same climate gradient. Using a combination of genomic and metagenomic approaches, we identified a variety of nonrandom, parallel mutations associated with transplantation, including mutations in genes related to nutrient acquisition, stress response, and exopolysaccharide production. Together, the field experiments demonstrate how both demographic shifts of previously adapted ecotypes and contemporary evolution can alter the diversity of a soil microbiome on the same timescale.

Viruses of marine cyanobacteria frequently contain auxiliary metabolic genes (AMGs) that augment host metabolism during infection, but little is known about their adaptive significance. We analyzed the distribution and genomic context of 33 AMGs across 60 cyanomyovirus genomes. Similarity in AMG content among cyanomyoviruses was only weakly correlated with phylogenetic relatedness; however, AMG content was generally conserved within the same operational taxonomic unit (OTU). A virus' AMG repertoire was also correlated with its isolation host and environment (coastal versus open ocean). A new analytical method based on shared co-linear blocks revealed that variation in the genomic location of an AMG was negatively correlated with its frequency across the genomes. We propose that rare AMGs are more frequently gained or lost as a result of fluctuating selection pressures, whereas common AMGs are associated with stable selection pressures. Finally, we describe a unique cyanomyovirus (S-CAM7) that lacks many AMGs including the photosynthesis gene psbA.

Bacteriophages are highly abundant in human microbiota where they coevolve with resident bacteria. Phage predation can drive the evolution of bacterial resistance, which can then drive reciprocal evolution in the phage to overcome that resistance. Such coevolutionary dynamics have not been extensively studied in human gut bacteria, and are of particular interest for both understanding and eventually manipulating the human gut microbiome. We performed experimental evolution of an Enterococcus faecium isolate from healthy human stool in the absence and presence of a single infecting Myoviridae bacteriophage, EfV12-phi1. Four replicates of E. faecium and phage were grown with twice daily serial transfers for 8 days. Genome sequencing revealed that E. faecium evolved resistance to phage through mutations in the yqwD2 gene involved in exopolysaccharide biogenesis and export, and the rpoC gene which encodes the RNA polymerase β' subunit. In response to bacterial resistance, phage EfV12-phi1 evolved varying numbers of 1.8 kb tandem duplications within a putative tail fiber gene. Host range assays indicated that coevolution of this phage-host pair resulted in arms race dynamics in which bacterial resistance and phage infectivity increased over time. Tracking mutations from population sequencing of experimental coevolution can quickly illuminate phage entry points along with resistance strategies in both phage and host - critical information for using phage to manipulate microbial communities.

Bacterial abundance is a fundamental metric for understanding the population dynamics of soil bacteria and their role in biogeochemical cycles. Despite its importance, methodological constraints hamper our ability to assess bacterial abundance in terrestrial environments. Here, we aimed to optimize the use of flow cytometry (FCM) to assay bacterial abundances in soil while providing a rigorous quantification of its limitations. Soil samples were spiked with Escherichia coli to evaluate the levels of recovery efficiency among three extraction approaches. The optimized method added a surfactant (a tetrasodium pyrophosphate [TSP] buffer) to 0.1 g of soil, applied an intermediate degree of agitation through shaking, and used a Nycodenz density gradient to separate the cells from background debris. This procedure resulted in a high (average, 89%) level of cell recovery. Recovery efficiencies did not differ significantly among sites across an elevation gradient but were positively correlated with percent carbon in the soil samples. Estimated abundances were also highly repeatable between technical replicates. The method was applied to samples from two field studies and, in both cases, was sensitive enough to detect treatment and site differences in bacterial abundances. We conclude that FCM offers a fast and sensitive method to assay soil bacterial abundance from relatively small amounts of soil. Further work is needed to assay differential biases of the method across a wider range of soil types.IMPORTANCE The ability to quantify bacterial abundance is important for understanding the contributions of microbial communities in soils, but such assays remain difficult and time-consuming. Flow cytometry offers a fast and direct way to count bacterial cells, but several concerns remain in applying the technique to soils. This study aimed to improve the efficiency of the method for soil while quantifying its limitations. We demonstrated that an optimized procedure was sensitive enough to capture differences in bacterial abundances among treatments and ecosystems in two field studies.

Bacterial communities in the organic leaf litter layer and bulk (mineral and organic) soil are sensitive to environmental change. However, despite close interactions between these communities, the leaf litter layer has historically been studied in isolation from the bulk soil. Whether bacterial response to environmental change is uniform throughout the surface soil remains unclear. Here, we simultaneously characterized how bacterial community composition in three surface soil layers (the leaf litter layer, 0–2 cm of bulk soil, and 0–10 cm of bulk soil) responded to a wildfire burning through a 13-year drought simulation in two adjacent ecosystems, a grassland and coastal sage scrubland. We found that bacterial communities in all three surface soil layers were distinct in composition and varied with drought, ecosystem type, and temporal variation. Moreover, the impact of these environmental changes on bacterial community composition decreased with depth in the surface soil. Bacterial response to drought was three-fold higher in the leaf litter layer than in the top 10 cm of bulk soil, with the drought treatment explaining 4.8% and 1.6% of the compositional variation, respectively. Wildfire altered bacterial composition in the leaf litter layer but not within the top 10 cm of bulk soil. Further, previous exposure of the bacterial communities in the leaf litter layer to drought did not influence its response to the wildfire. Thus, considering soil depth when assessing the impact of environmental conditions on the surface soil microbiome may improve predictions about the degree to which microbial communities, and therefore soil carbon, will respond to future environmental change.

Leaf litter microbes collectively degrade plant polysaccharides, influencing land-atmosphere carbon exchange. An open question is how substrate complexity-defined as the structure of the saccharide and the amount of external processing by extracellular enzymes-influences species interactions. We tested the hypothesis that monosaccharides (i.e. xylose) promote negative interactions through resource competition, and polysaccharides (i.e. xylan) promote neutral or positive interactions through resource partitioning or synergism among extracellular enzymes. We assembled a three-species community of leaf litter-degrading bacteria isolated from a grassland site in Southern California. In the polysaccharide xylan, pairs of species stably coexisted and grew equally in coculture and in monoculture. Conversely, in the monosaccharide xylose, competitive exclusion and negative interactions prevailed. These pairwise dynamics remained consistent in a three-species community: all three species coexisted in xylan, while only two species coexisted in xylose, with one species capable of using peptone. A mathematical model showed that in xylose these dynamics could be explained by resource competition. Instead, the model could not predict the coexistence patterns in xylan, suggesting other interactions exist during biopolymer degradation. Overall, our study shows that substrate complexity influences species interactions and patterns of coexistence in a synthetic microbial community of leaf litter degraders.

In many ecosystems, global changes are likely to profoundly affect microorganisms. In Southern California, changes in precipitation and nitrogen deposition may influence the composition and functional potential of microbial communities and their resulting ability to degrade plant material. To test whether such environmental changes impact the distribution of functional groups involved in leaf litter degradation, we determined how the genomic diversity of microbial communities in a semi-arid grassland ecosystem changed under reduced precipitation or increased N deposition. We monitored communities seasonally over a period of 2 years to place environmental change responses into the context of natural variation. Fungal and bacterial communities displayed strong seasonal patterns, Fungi being mostly detected during the dry season whereas Bacteria were common during wet periods. Most putative cellulose degraders were associated with 33 bacterial genera and predicted to constitute 18% of the microbial community. Precipitation reduction reduced bacterial abundance and cellulolytic potential whereas nitrogen addition did not affect the cellulolytic potential of the microbial community. Finally, we detected a strong correlation between the frequencies of genera of putative cellulose degraders and cellulase genes. Thus, microbial taxonomic composition was predictive of cellulolytic potential. This work provides a framework for how environmental changes affect microorganisms responsible for plant litter deconstruction.

Global changes such as increased drought and atmospheric nitrogen deposition perturb both the microbial and plant communities that mediate terrestrial ecosystem functioning. However, few studies consider how microbial responses to global changes may be influenced by interactions with plant communities. To begin to address the role of microbial–plant interactions, we tested the hypothesis that the response of microbial communities to global change depends on the plant community. We characterized bacterial and fungal communities from 395 plant litter samples taken from the Loma Ridge Global Change Experiment, a decade-long global change experiment in Southern California that manipulates rainfall and nitrogen levels across two adjacent ecosystems, a grassland and a coastal sage scrubland. The differences in bacterial and fungal composition between ecosystems paralleled distinctions in plant community composition. In addition to the direct main effects, the global change treatments altered microbial composition in an ecosystem-dependent manner, in support of our hypothesis. The interaction between the drought treatment and ecosystem explained nearly 5% of the variation in bacterial community composition, similar to the variation explained by the ecosystem-independent effects of drought. Unexpectedly, we found that the main effect of drought was approximately four times as strong on bacterial composition as that of nitrogen addition, which did not alter fungal or plant composition. Overall, the findings underscore the importance of considering plant–microbe interactions when considering the transferability of the results of global change experiments across ecosystems.