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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. 

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.