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ABSTRACT Microorganisms often inhabit environments that are suboptimal for growth and reproduction. To survive when challenged by such conditions, individuals engage in dormancy, where they enter a metabolically inactive state. For this persistence strategy to confer an evolutionary advantage, microorganisms must be able to resuscitate and reproduce when conditions improve. Among bacteria in the phylum Actinomycetota, dormancy can be terminated by resuscitation-promoting factor (Rpf), an exoenzyme that hydrolyzes glycosidic bonds in the peptidoglycan of cell walls. We characterized Rpf fromMicrococcusKBS0714, a bacterium isolated from agricultural soil. The protein exhibited high substrate affinityin vitro, even though resuscitation was maximized in live-cell assays at micromolar concentrations. Site-directed mutations at conserved catalytic sites significantly reduced or eliminated resuscitation, as did the deletion of repeating motifs in a lectin-encoding linker region. We then tested the effects of recombinant Rpf fromMicrococcusKBS0714 on a diverse set of dormant soil bacteria. Patterns of resuscitation mapped onto strain phylogeny, which reflected core features of the cell envelope. Additionally, the direction and magnitude of the Rpf effect were associated with functional traits, in particular, aspects of the moisture niche and biofilm production, which are critical for understanding dormancy and the persistence of microbial populations in soils. These findings expand our understanding of how Rpf may affect seed bank dynamics with implications for the diversity and functioning of microorganisms in terrestrial ecosystems. IMPORTANCEDormancy is a process whereby individuals enter a reversible state of reduced metabolic activity. In fluctuating environments, dormancy protects individuals from unfavorable conditions, enhancing fitness and buffering populations against extinction. However, waking up from dormancy is a critical yet risky decision. Some bacteria resuscitate stochastically, while others rely on environmental cues or signals from neighboring cells to transition back to active growth. Resuscitation-promoting factor (Rpf) is an exoenzyme that cleaves bonds in the peptidoglycan of bacterial cell walls, facilitating dormancy termination and enabling regrowth. Although this family of proteins has been well characterized in model organisms and clinically relevant strains, our study characterizes Rpf from a soil bacterium and examines its effects on resuscitation across a diverse collection of bacteria, linking it to functional traits that may influence dormancy dynamics in both natural and managed ecosystems. 

Life has existed on Earth for most of the planet’s history, yet major gaps and unresolved questions remain about how it first arose and persisted. Early Earth posed numerous challenges for life, including harsh and fluctuating environments. Today, many organisms cope with such conditions by entering a reversible state of reduced metabolic activity, a phenomenon known as dormancy. This process protects inactive individuals and minimizes the risk of extinction by preserving information that stabilizes life-system dynamics. Here, we develop a framework for understanding dormancy on early Earth, beginning with a primer on dormancy theory and its core criteria. We hypothesize that dormancy-like mechanisms acting on chemical precursors in a prebiotic world may have facilitated the origin of life. Drawing on evidence from phylogenetic reconstructions and the fossil record, we demonstrate that dormancy is prevalent across the tree of life and throughout deep time. These observations lead us to consider how dormancy might have shaped nascent living systems by buffering stochastic processes in small populations, protecting against large-scale planetary disturbances, aiding dispersal in patchy landscapes and facilitating adaptive radiations. Given that dormancy is a fundamental and easily evolved property on Earth, it is also likely to be a feature of life elsewhere in the universe. 

The factors contributing to the persistence and stability of life are fundamental for understanding complex living systems. Organisms are commonly challenged by harsh and fluctuating environments that are suboptimal for growth and reproduction, which can lead to extinction. Many species contend with unfavourable and noisy conditions by entering a reversible state of reduced metabolic activity, a phenomenon known as dormancy. Here, we develop Spore Life, a model to investigate the effects of dormancy on population dynamics. It is based on Conway’s Game of Life (GoL), a deterministic cellular automaton where simple rules govern the metabolic state of an individual based on the metabolic state of its neighbours. For individuals that would otherwise die, Spore Life provides a refuge in the form of an inactive state. These dormant individuals (spores) can resuscitate when local conditions improve. The model includes a parameter $\alpha \in \left[\mathrm{0,1}\right]$that controls the survival probability of spores, interpolating between GoL ( $\alpha =0$) and Spore Life ( $\alpha =1$), while capturing stochastic dynamics in the intermediate regime ( $0<\alpha <1$). In addition to identifying the emergence of unique periodic configurations, we find that spore survival increases the average number of active individuals and buffers populations from extinction. Contrary to expectations, stabilization of the population is not the result of a large and long-lived seed bank. Instead, the demographic patterns in Spore Life only require a small number of resuscitation events. Our approach yields novel insight into what is minimally required for the origins of complex behaviours associated with dormancy and the seed banks that they generate. 

1 Abstract Mutualisms evolve over time when individuals belonging to different species derive fitness benefits through the exchange of resources and services. Although prevalent in natural and managed ecosystems, mutualisms can be destabilized by environmental fluctuations that alter the costs and benefits of maintaining the symbiosis. In the rhizobia-legume mutualism, bacteria provide reduced nitrogen to the host plant in exchange for photosynthates that support bacterial metabolism. However, this relationship can be disrupted by the addition of external nitrogen sources to the soil, such as fertilizers. While the molecular mechanisms underpinning the rhizobia-legume symbiosis are well-characterized, the genome-wide fitness effects of nitrogen enrichment on symbiotic rhizobia are less clear. Here, we inoculated a randomly barcoded transposon-site sequencing (RB-TnSeq) library of the bacteriumEnsifer(Sinorhizobium)melilotiinto soils containing a host plant, alfalfa (Medicago sativa), under conditions of low and high nitrogen availability. Although plant performance remained robust to fertilization, nitrogen enrichment altered gene fitness for specific traits and functions in the rhizobial partner. Genes involved in carbohydrate metabolism showed increased fitness irrespective of soil nutrient content, whereas fitness gains in quorum-sensing genes were only observed in high-nitrogen environments. We also documented reductions in the fitness of nucleotide metabolism and cell-growth genes, while genes from oxidative phosphorylation and various amino-acid biosynthesis pathways were detrimental to fitness under elevated soil nitrogen, underscoring the complex trade-offs in rhizobial responses to nutrient enrichment. Our experimental functional genomics approach identified gene functions and pathways across allE. melilotireplicons that may be associated with the disruption of an agronomically important mutualism. 2ImportanceUnderstanding the evolutionary dynamics of the rhizobia-legume mutualism is important for elucidating how plant-soil-microbe interactions operate in natural and managed ecosystems. Legumes constitute a significant portion of global food production and generate 25% of all terrestrially fixed nitrogen. The application of chemical fertilizers can disrupt the mutualism by altering the selective pressures experienced by symbiotic rhizobia, potentially affecting gene fitness throughout the microbial genome and leading to the evolution of less productive or cooperative mutualists. To investigate how exogenous nitrogen inputs influence gene fitness during the complex rhizobial lifecycle, we used a barcoded genome-wide mutagenesis screen to quantify gene-level fitness across the rhizobial genome during symbiosis and identify metabolic functions affected by nitrogen enrichment. Our findings provide genomic insight into potential eco-evolutionary mechanisms by which symbioses are maintained or degraded over time in response to changing environmental conditions. 

Abstract Along the river–sea continuum, microorganisms are directionally dispersed by water flow while being exposed to strong environmental gradients. To compare the two assembly mechanisms that may strongly and differently influence metacommunity dynamics, namely homogenizing dispersal and heterogeneous selection, we characterized the total (16S rRNA gene) and putatively active (16S rRNA transcript) bacterial communities in the Pearl River–South China Sea Continuum, during the wet (summer) and dry (winter) seasons using high-throughput sequencing. Moreover, well-defined sampling was conducted by including freshwater, oligohaline, mesohaline, polyhaline, and marine habitats. We found that heterogeneous selection exceeded homogenizing dispersal in both the total and active fractions of bacterial communities in two seasons. However, homogeneous selection was prevalent (the dominant except in active bacterial communities during summer), which was primarily due to the bacterial communities’ tremendous diversity (associated with high rarity) and our specific sampling design. In either summer or winter seasons, homogeneous and heterogeneous selection showed higher relative importance in total and active communities, respectively, implying that the active bacteria were more responsive to environmental gradients than were the total bacteria. In summary, our findings provide insight into the assembly of bacterial communities in natural ecosystems with high spatial connectivity and environmental heterogeneity. 

Monitoring the “health” of an ecological community is a critical component of conservation planning. We propose that aggregating intraspecific genetic variation across all species of an ecological community (Community Genetic Distribution; CGD) provides a new way to measure biodiversity that is unifying across taxa, economically scalable, and geographically transferable. Such community-scale data provides information about past dynamics that can unveil processes structuring contemporary biodiversity, and can identify communities that are resilient to perturbation. Using the CGD, high-throughput biodiversity genetic inventories (e.g. metabarcoding/eDNA) can be leveraged to identify the genetic signatures of pristine and disturbed systems. We show examples of the CGD from empirical systems, how it responds through space and time to human disturbance, and how it successfully recovers restoration and succession gradients from metabarcoding datasets with the goal of obtaining insight on community genetic health and developing indicator metrics which can identify communities that are resilient to perturbation. We outline ways in which the CGD complements and extends information in the suite of currently described essential biodiversity variables, and how it can contribute to the targets of the Kunming-Montreal Global Biodiversity Framework. 

ABSTRACT Much of life on Earth is at the mercy of currents and flow. Residence time (τ) estimates how long organisms and resources stay within a system based on the ratio of volume (V) to flow rate (Q). Short residence times promote immigration but may prevent the establishment of species that cannot quickly reproduce, or resist being washed out. In contrast, long residence times reduce resource input, selecting for species that can survive on a low supply of energy and nutrients. Theory suggests that these opposing forces shape the abundance, diversity, and function of flowing systems. In this study, we subjected chemostats inoculated with a complex lake microbial community to a residence time gradient spanning seven orders of magnitude. Microbial abundance, richness, and evenness increased with residence time, while functions like productivity and resource consumption decreased along the gradient. Microbial taxa were non- randomly distributed, forming distinct clusters of short-τ and long-τ specialists, reflecting a pattern of niche partitioning. Consistent with theoretical predictions, we demonstrate that residence time shapes assembly processes with direct implications for biodiversity and community function. These insights are crucial for understanding and managing flowing environments, such as animal gut microbiomes, soil litter invertebrate communities, and plankton in freshwater and marine ecosystems. 

Abstract Microorganisms often inhabit environments that are suboptimal for growth and reproduction. To survive when challenged by such conditions, individuals may engage in dormancy where they enter a metabolically inactive state. For this persistence strategy to confer an evolutionary advantage, microorganisms must be able to resuscitate and reproduce when conditions improve. Among bacteria in the phylum Actinomycetota, dormancy can be terminated by resuscitation-promoting factor (Rpf), an exoenzyme that hydrolyzes glycosidic bonds in the peptidoglycan of the cell wall. We characterized Rpf fromMicrococcusKBS0714, a bacterium isolated from agricultural soil. Compared to previous studies, the Rpf elicited activity at relatively high concentrations, yet demonstrated high substrate affinity. Site-directed mutations at conserved catalytic sites significantly reduced or abolished resuscitation, as did the deletion of repeating motifs in a lectin-encoding linker region. We then tested the effects of recombinant Rpf fromMicrococcusKBS0714 on a diverse set of dormant soil bacteria. Patterns of resuscitation mapped onto strain phylogeny, which reflected core features of the cell envelope. Additionally, the direction and magnitude of the Rpf effect were associated with functional traits, in particular, aspects of the moisture niche and biofilm production, which are critical for understanding persistence and resuscitation during dormancy. These findings expand our understanding of how Rpf may affect seed-bank dynamics and have implications for the diversity and functioning of soil ecosystems. 

I. ABSTRACT Bacteriophage (phage) infect, lyse, and propagate within bacterial populations. However, physiological changes in bacterial cell state can protect against infection even within genetically susceptible populations. One such example is the generation of endospores byBacillusand its relatives, characterized by a reversible state of reduced metabolic activity that protects cells against stressors including desiccation, energy limitation, antibiotics, and infection by phage. Here we tested how sporulation at the cellular scale impacts phage dynamics at population scales when propagating amongstB. subtilisin spatially structured environments. Initially, we found that plaques resulting from infection and lysis were approximately 3-fold smaller on lawns of sporulating wild-type bacteria vs. non-sporulating bacteria. Notably, plaque size was reduced due to an early termination of expanding phage plaques rather than the reduction of plaque growth speed. Microscopic imaging of the plaques revealed ‘sporulation rings’, i.e., spores enriched around plaque edges relative to phage-free regions. We developed a series of mathematical models of phage, bacteria, spore, and small molecules that recapitulate plaque dynamics and identify a putative mechanism: sporulation rings arise in response to lytic activity. In aggregate, sporulation rings inhibit phage from accessing susceptible cells even when sufficient resources are available for further infection and lysis. Together, our findings identify how dormancy can self-limit phage infections at population scales, opening new avenues to explore the entangled fates of phages and their bacterial hosts in environmental and therapeutic contexts.