An official website of the United States government
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.
more »
« less
This content will become publicly available on May 20, 2026
Resuscitation-promoting factor (Rpf) terminates dormancy among diverse soil bacteria
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.
more »
« less
- Award ID(s):
- 2022049
- PAR ID:
- 10629064
- Editor(s):
- Albright, Michaeline_B N
- Publisher / Repository:
- American Society for Microbiology
- Date Published:
- Journal Name:
- mSystems
- Volume:
- 10
- Issue:
- 5
- ISSN:
- 2379-5077
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
Tischler, Dirk (Ed.)ABSTRACT Space missions or spacecraft equipment destined for sensitive environments, such as Mars, Europa, or Enceladus, are required to be designed to avoid forward contamination. Spacecraft are assembled in clean rooms (SACs) employing treatments to eliminate microbial contamination. However, some organisms can survive the cleaning procedures. Characterization of these populations, through both culture-based and sequencing methods, reveals that the majority consists of spore-forming bacteria. However, a smaller group of non-spore-forming organisms, primarily classified within the orderMicrococcalesof the phylumActinobacteria(Actinomycetota), exists in some SACs. Despite their repeated occurrence and isolation, actinobacterial strains associated with SACs have not been studied for their dormancy potential. Here, we show for the first time that a non-spore-forming SAC isolate,Tersicoccus phoenicis(Micrococcales), enters dormancy under nutrient starvation. Dormancy inMicrococcus luteusinvolves a universal stress protein and a resuscitation-promoting factor (Rpf). Genes for these proteins are widely found in actinobacteria, includingT. phoenicis. We show that dormantT. phoenicis(Micrococcales) can be revived through the addition of the Rpf to the media. Dormancy, as observed in the SAC actinobacterial isolateT. phoenicis, could well be a common trait adopted by other actinobacterial strains under the stressful conditions of spacecraft clean rooms or the ISS (International Space Station). This has implications for the persistence, identification, and recovery of such microbes from cleanroom facilities.IMPORTANCENASA’s long-range goal of a human mission to the Mars surface raises issues relating to planetary protection. The concerns of forward contamination require that spacecraft assembly clean rooms (SACs) be maintained to inhibit microbial survival. However, despite these efforts, distinct microbial communities persist. Here, we show that a SAC isolate,T. phoenicis, exhibits dormancy, a state in which the cells are viable but not cultivable. Dormancy may help non-spore-forming organisms survive in the clean room environments utilized for space missions. This has implications for improving cleaning procedures.more » « less
-
Abstract While microorganisms are recognized for driving belowground processes that influence the productivity and fitness of plant populations, the vast majority of bacteria and fungi in soil belong to a seed bank consisting of dormant individuals. However, plant performance may be affected by microbial dormancy through its effects on the activity, abundance, and diversity of soil microorganisms. To test how microbial seed banks influence plant‐soil interactions, we purified recombinant resuscitation promoting factor (Rpf), a bacterial protein that terminates dormancy. In a factorially designed experiment, we then applied the Rpf to soil containing field mustard (Brassicarapa), an agronomically important plant species. Plant biomass was ~33% lower in the Rpf treatment compared to plants grown with an unmanipulated microbial seed bank. In addition, Rpf reduced soil respiration, decreased bacterial abundance, and increased fungal abundance. These effects of Rpf on plant performance were accompanied by shifts in bacterial community composition, which may have diluted mutualists or resuscitated pathogens. Our findings suggest that changes in microbial seed banks may influence the magnitude and direction of plant‐soil feedbacks in ways that affect above‐ and belowground biodiversity and function.more » « less
-
Becker, Anke (Ed.)ABSTRACT Agrobacterium fabrum is a phytopathogen that causes crown gall disease. In the rhizosphere, it encounters plant exudates, some of which are toxic, such as 4-hydroxybenzaldehyde (4HBA). Others, including 4-hydroxybenzoate (4HB), participate in the induction of virulence genes.A. fabrum encodes the transcription factor PecS, which has been reported to enhance bacterial fitness in the rhizosphere. The gene encoding PecS is divergent from pecM, which encodes an efflux pump. PecS represses both pecS and pecM, as evidenced by increased expression in the presence of the PecS ligand urate and by elevated pecM expression in a pecS disruption strain. We report here that the expression ofpecM is induced selectively by 4HBA. Expression of genes encoding enzymes involved in the degradation of 4HB is induced by both 4HBA and 4HB, as expected; however, overexpression ofpecM attenuates the induction by 4HBA, suggesting that 4HBA is a substrate for PecM. Consistent with this inference, untargeted metabolomics shows that 4HBA accumulates intracellularly whenpecM is disrupted. Analysis of PecS by thermal stability assay and DNase I footprinting suggests that 4HBA is not a ligand for PecS. Taken together, our data suggest that 4HBA is a substrate for PecM.IMPORTANCEPlant roots secrete a number of compounds that may be toxic to bacteria residing in the surrounding soil. One such bacterium is Agrobacterium fabrum, which infects plants and induces tumor formation. We show here that an A. fabrum strain in which the efflux pump PecM has been disrupted accumulates 4-hydroxybenzaldehyde, and that this plant root exudate induces the expression of pecM. Our data suggest that PecM and PecS, a transcription factor that regulates pecM expression, both function to promote A. fabrum fitness in the rhizosphere. As a competitive advantage in the rhizosphere is a prerequisite for subsequent plant infection, our data contribute to a more complete understanding of the A. fabrum infection process.more » « less
-
Bose, Arpita (Ed.)ABSTRACT The marine cyanobacteriumProchlorococcusnumerically dominates the phytoplankton communities in all lower latitude, open ocean environments. Having lost the catalase gene,Prochlorococcusis highly susceptible to exogenous hydrogen peroxide (H2O2) produced at the ocean’s surface. Protection by H2O2-scavenging heterotrophic “helper” bacteria has been demonstrated in laboratory cultures and implicated as an important mechanism ofProchlorococcussurvival in the ocean. Importantly, some other phytoplankton can also scavenge H2O2, suggesting these competing microbes may inadvertently protectProchlorococcus. In this study, we assessed the ability of co-occurring phytoplankton, the cyanobacteriumSynechococcusand picoeukaryotesMicromonasandOstreococcus, to protectProchlorococcusfrom H2O2exposure when cocultured at ecologically relevant abundances. All three genera could significantly degrade H2O2and diminishProchlorococcusmortality during H2O2exposures simulating photochemical production and rainfall events. We suggest that these phytoplankton groups contribute significantly to the H2O2microbial sink of the open ocean, thus complicating their relationships with and perhaps contributing to the evolutionary history ofProchlorococcus.IMPORTANCEThe marine cyanobacteriumProchlorococcusis the most abundant photosynthetic organism on the planet and is crucially involved in microbial community dynamics and biogeochemical cycling in most tropical and subtropical ocean waters. This success is due, in part, to the detoxification of the reactive oxygen species hydrogen peroxide (H2O2) performed by “helper” organisms. Earlier work identified heterotrophic bacteria as helpers, and here, we demonstrate that rival cyanobacteria and picoeukaryotic phytoplankton can also contribute to the survival ofProchlorococcusduring exposure to H2O2. Whereas heterotrophic bacteria helper organisms can benefit directly from promoting the survival of carbon-fixingProchlorococcuscells, phytoplankton helpers may suffer a twofold injury: production of H2O2degrading enzymes constrains already limited resources in oligotrophic environments, and the activity of these enzymes bolsters the abundance of their numerically dominant competitor. These findings build toward a better understanding of the intricate dynamics and interactions that shape microbial community structure in the open ocean.more » « less
