Mario E. Muscarella

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 from Micrococcus KBS0714, a bacterium isolated from agricultural soil. The protein exhibited high substrate affinity in 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 from Micrococcus KBS0714 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. IMPORTANCE: Dormancy 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.

Permafrost microbial research has flourished in the past decades, due in part to improvements in sampling and molecular techniques, but also the increased focus on the permafrost greenhouse gas feedback to climate change and other ecological processes in high latitude and alpine permafrost soils. Permafrost microorganisms are adapted to these extreme environments and remain active at low temperatures and when resources are limited. They are also an important component of global elemental cycles as they regulate organic matter turnover and greenhouse gas production, particularly as permafrost thaws. Here we review the permafrost microbiology literature coupled with an exploration of its historical aspects, with a particular focus on a new understanding advanced by molecular biology techniques. We further identify knowledge gaps and ways forward to improve our understanding of microbial contributions to ecosystem biogeochemistry of permafrost‐affected systems.

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 from Micrococcus KBS0714, 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 from Micrococcus KBS0714 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.

Metabolism in stream ecosystems alters the fate of organic carbon (OC) received from surrounding landscapes, but our understanding of in‐stream metabolic processes in boreal ecosystems remains limited. Determining the factors that regulate OC metabolism will help predict how the C balance of boreal streams may respond to future environmental change. In this study, we addressed the question: what controls OC metabolism in boreal headwater streams draining catchments with discontinuous permafrost? We hypothesized that metabolism is collectively regulated by OC reactivity, phosphorus availability, and temperature, with discharge modulating each of these conditions. We tested these hypotheses using a combination of laboratory experiments and whole‐stream ecosystem metabolism measurements throughout the Caribou‐Poker Creeks Research Watershed (CPCRW) in Interior Alaska, USA. In the laboratory experiments, respiration and dissolved OC (DOC) removal were both co‐limited by the supply of reactive C and phosphorus, but temperature and residence time acted as stronger controls of DOC removal. Ecosystem respiration (ER) was largely predicted by discharge and site, with some variance explained by gross primary production (GPP) and temperature. Both ER and GPP varied inversely with watershed permafrost extent, with an inverse relationship between temperature and permafrost extent providing one plausible explanation. Our results provide some of the first evidence of a functional response to permafrost thaw in stream ecosystems and suggest the role of metabolism in landscape C cycling may increase as climate change progresses.

Carbon (C) emissions from headwater streams are derived from both terrestrial inputs and in-stream metabolism of organic C (OC), but the role of metabolism in boreal stream C fluxes remains uncertain. Determining the factors that regulate OC metabolism will help predict how the C balance of boreal streams will respond to future environmental change. In this study, we addressed the question: what controls OC metabolism in boreal headwater streams draining catchments with discontinuous permafrost? We hypothesized that metabolism is collectively regulated by OC reactivity, phosphorus availability, and temperature, with discharge modulating each of these conditions. We tested these hypotheses using a combination of laboratory experiments and whole-stream ecosystem metabolism measurements throughout the Caribou-Poker Creeks Research Watershed (CPCRW) in Interior Alaska, USA. In the laboratory experiments, respiration and dissolved organic carbon (DOC) removal were both co-limited by the supply of reactive C and phosphorus, but temperature and residence time acted as stronger controls of DOC removal. Ecosystem respiration (ER) was largely predicted by discharge and site, with some variance explained by gross primary production (GPP) and temperature. Both ER and GPP varied inversely with watershed permafrost extent, with an inverse relationship between temperature and permafrost extent providing the most plausible explanation. Our results provide some of the first evidence of a functional response to permafrost thaw in stream ecosystems and suggest that the contribution of metabolism to stream C emissions may increase as climate change progresses.

Climate change induced salinization events are predicted to intensify and lead to increased salt stress in freshwater aquatic ecosystems. As a consequence, formerly distinct abiotic conditions and associated biotic communities merge, and the emergence, loss, and persistence of microbial taxa modify the types and rates of ecosystem processes. This study examined how bacterial taxonomic and phylogenetic diversity and ecosystem function respond to acute salinization events where freshwater and estuarine communities and environments coalesce. We hypothesized that when the salinity change outpaces microbial adaptation or saline microbial populations are not yet established in formerly freshwater conditions, then these aquatic communities will exhibit diminished carbon cycling rates, decreased microbial diversity, and altered composition of microbial communities compared to historically freshwater communities. We used an experimental mesocosm approach to determine how salinity and the merging of distinct communities influenced resultant bacterial community structure and function. Each mesocosm represented different salinities (0, 5, 9, 13 psu). Two dispersal treatments, representing aquatic communities sourced from brackish 13 psu ponds and a mix of 13 psu and freshwater ponds, were added to all salinity levels and replicated four times. Results revealed that salinity, but not dispersal, decreased bacterial taxonomic and phylogenetic diversity. Carbon mineralization rates were highest in freshwater conditions and associated to bacterial taxa represented in low relative abundance. Acute salinity changes, such as localized flooding due to storm surge, will more negatively affect freshwater aquatic communities compared to chronic exposure to salinization where the communities have had time to adapt or turnover. IMPORTANCE STATEMENT: Climate change induced salinization results in the mixing of formerly distinct environmental conditions and aquatic communities. This study examined the consequence of short-term, acute salinity stress on aquatic bacterial taxonomic and phylogenetic diversity and ecosystem function using an experimental approach. Results revealed that salinity, but not the source of aquatic communities, decreased bacterial taxonomic and phylogenetic diversity. Carbon mineralization rates, which represented ecosystem function, were highest in freshwater conditions and also associated with indicator bacterial taxa in low abundance relative to the total microbial community. Taken together, acute salinity changes will more negatively affect freshwater aquatic communities compared to chronic exposure to salinization where the communities have had time to adapt or turnover resulting in recovered biogeochemical functions.

Every seed germinating in soils, wastewater treatment, and stream confluence exemplify microbial community coalescence—the blending of previously isolated communities. Here, we present theoretical and experimental knowledge on how separated microbial communities mix, with particular focus on managed ecosystems.

As the most abundant and diverse form of life on Earth, microorganisms commonly inhabit energy-limited environments where cellular maintenance and growth is highly constrained. To gain insight into how microorganisms persist under such conditions, we derived demographic parameters from a diverse collection of bacteria by censusing 100 populations in a closed system for 1,000 days. All but one taxon survived prolonged resource scarcity, yielding estimated times-to-extinction ranging over four orders of magnitude from 10 ⁰ – 10 ⁵ years. These findings corroborate reports of long-lived bacteria that have been recovered from ancient environmental samples, while providing insight into mechanisms of persistence. Critically, we found that as death rates declined over time, lifespan was extended through the scavenging of dead cells. Although growth and reproduction were dramatically suppressed in the absence of an exogenous resource supply, bacterial populations continued to evolve. Hundreds of mutations were acquired, contributing to genome-wide signatures of negative selection as well as molecular signals of adaptation. Remarkable consistency in the ecological and evolutionary dynamics indicate that distantly related bacteria respond to energy-limitation in a similar and predictable manner, which likely contributes to the stability and robustness of microbial life.

Hydrologic changes modify microbial community structure and ecosystem functions, especially in wetland systems. Here, we present 24 metagenomes from a coastal freshwater wetland experiment in which we manipulated hydrologic conditions and plant presence. These wetland soil metagenomes will deepen our understanding of how hydrology and vegetation influence microbial functional diversity.