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1. Resuscitation-promoting factor (Rpf) terminates dormancy among diverse soil bacteria

   https://doi.org/10.1101/2024.11.10.622857  

   Lennon, Jay T; Lehmkuhl, Brent K; Chen, Lingling; Illingworth, Melissa; Kuo, Venus; Muscarella, Mario E (November 2024, bioRxiv)

   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. 

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2. Tersicoccus phoenicis (Actinobacteria), a spacecraft clean room isolate, exhibits dormancy

   https://doi.org/10.1128/spectrum.01692-25  

   Tirumalai, Madhan; Ali, Sahar; Fox, George E; Widger, William (August 2025, Microbiology Spectrum)

   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. 

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3. Nascent flagellar basal bodies are immobilized by rod assembly in Bacillus subtilis

   https://doi.org/10.1128/mbio.00530-25  

   Dunn, Caroline M; Foust, Daniel J; Gao, Yongqiang; Biteen, Julie S; Shaw, Sidney L; Kearns, Daniel B (June 2025, mBio)

   Salama, Nina R (Ed.)

   ABSTRACT Flagella are complex, trans-envelope nanomachines that localize in species-specific patterns on the cell surface. Here, we study the localization dynamics of the earliest stage of basal body formation inBacillus subtilisusing a fluorescent fusion to the C-ring protein FliM. We find thatB. subtilisbasal bodies do not exhibit dynamic subunit exchange and are largely stationary at steady state, consistent with flagellar assembly through the peptidoglycan (PG). However, rare mobile basal bodies were observed, and the prevalence of mobile basal bodies is elevated both early in basal body assembly and when the rod is mutated. Thus, basal body mobility is a precursor to patterning, and we propose that rod polymerization probes the PG superstructure for pores of sufficient diameter to permit rod transit. Furthermore, mutation of the rod disrupts basal body patterning in a way that phenocopies mutation of the cytoplasmic flagellar patterning protein FlhF. We infer that rod synthesis and the cytoplasmic regulators coordinate flagellar assembly by interpreting a grid-like pattern of pores, pre-existent in the PG. IMPORTANCEBacteria insert flagella in a species-specific pattern on the cell body, but how patterns are achieved is poorly understood. In bacteria with a single polar flagellum, a marker protein localizes to the cell pole and nucleates the assembly of the flagellum at that site.Bacillus subtilisassembles ~25 basal bodies over the length of the cell in a grid-like pattern and lacks proteins required for their polar targeting. Here, we show thatB. subtilisbasal bodies are mobile soon after assembly and become immobilized when the flagellar rod transits the peptidoglycan (PG) wall. Moreover, defects in the flagellar rod lead to a more-random distribution of flagella and an increase in polar basal bodies. We conclude that the peritrichous patterning of flagella ofB. subtilisis different from the polar patterning of other bacteria, and we infer that theB. subtilisrod probes the PG for holes that can accommodate the machine. 

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4. Transcriptional and metabolic stasis define desiccation-induced dormancy in the soil bacterium Arthrobacter sp. AZCC_0090 until water vapor initiates resuscitation

   https://doi.org/10.1128/msystems.00493-25  

   Carini, Paul; Gomez-Buckley, Adriana; Guerrero, Christina R; Kridler, Melanie R; Viney, Isabella A; AminiTabrizi, Roya; Tfaily, Malak M; Moma, Peter; Meredith, Laura K; Louie, Katherine B; et al (February 2026, mSystems)

   Shade, Ashley (Ed.)

   ABSTRACT Microbes inhabiting soils experience periodic water deprivation. The effects of desiccation on DNA, protein, and membrane integrity are well-described. However, the effects of drying and rehydration on the composition of cellular RNA and metabolites are still poorly understood. Here, we describe how slow drying and rehydration with water vapor influence the composition of RNAs and metabolites in a soilArthrobacter. While drying reduced cultivability relative to hydrated controls, water vapor rehydration fully restored it. Ribosomal RNA proportions remained constant throughout all treatments, and mRNA profiles showed stable composition during desiccation—changing only during transitions into and out of desiccation-induced dormancy. Six transcriptional modules displayed distinct expression patterns in desiccated-rehydrated samples relative to hydrated controls, including desiccation-rehydration responsive and rehydration-specific profiles. Targeted intracellular metabolomics revealed similarly static profiles during desiccation, with a cluster of ribonucleosides and nucleobases increasing in response to desiccation and returning to baseline levels upon rehydration with water vapor. These findings demonstrate that both mRNA and metabolite profiles remain essentially frozen in desiccatedArthrobacter, with dynamic changes occurring only during state transitions. These results have important implications for environments with frequent drying cycles where stable mRNA in dormant cells combined with intracellular RNA recycling may obscure interpretations of RNA-based environmental analyses that use RNA as a marker of microbial activity. Our results suggest that RNA-based activity assessments in periodically dry environments require careful consideration of dormancy-associated molecular preservation.IMPORTANCEMetabolic activity quickly ceases in drying bacteria as they enter desiccation-induced dormancy. We show that mRNA and metabolite profiles were variable during drying and rewetting but did not change while desiccated. Additionally, water vapor stimulated the shift from the static to active state when exiting desiccation-induced dormancy. These shifts coincided with increased cultivability, indicating water vapor resuscitated dry cells. Because RNAs are transient, labile molecules that are turned over rapidly in growing bacteria, the presence of RNA in the environment is used as a marker for microbial activity. Our research shows this assumption may not hold for desiccated cells, indicating reliance on RNA as a marker of activity in environments that experience drying may obscure estimates ofin situmicrobial activity. 

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5. Mutant gltS alleles enable a Vibrio fischeri D-glutamate auxotroph to grow with lower requirements for exogenous D-glutamate

   https://doi.org/10.1128/spectrum.01025-25  

   Coppinger, Macey; Helm, Richard F; Yang, Liu; Ruby, Edward G; Popham, David L; Stabb, Eric V (October 2025, Microbiology Spectrum)

   Barquera, Blanca (Ed.)

   ABSTRACT D-glu is a key component of peptidoglycan (PG) and is essential for growth in most bacteria. To assess constraints on PG evolution and bacterial requirements for D-glu, we sought to artificially evolve PG biosynthesis, leading to either replacement of D-glu in the PG peptide or alternative pathways to D-glu incorporation. We previously found that suppression of D-glu auxotrophy in amurI racDmutant ofVibrio fischerigrown on lysogeny broth salts (LBS) medium was rare but could be accomplished by mutation ofbsrF, with restoration of wild-type PG structure. Here, we selected nine additional prototrophic suppressors of the samemurI racDmutant from 10¹⁰colony-forming units (CFU) plated on LBS supplemented with ~2.7 mM D-gln. Each suppressor had a mutation ingltS, which encodes a putative sodium:glutamate symporter. Increased copy numbers of mutantgltSalleles enabled growth on unsupplemented LBS and resulted in PG containing D-glu. Examination of media components suggests that D-gln supplementation had inadvertently added ~14 μM D-glu, and that LBS itself contains ~1.4 μM D-glu. The mutations ingltSenabled growth with similarly low D-glu concentrations, but also increased sensitivity to homocysteic acid, suggesting more promiscuous transport. Surprisingly, we discovered that expression of mutantgltSin the auxotroph leads to incorporation of lysine into PG, in addition to canonical D-glu. When seawater is supplemented with D-glu, thisV. fischerimutant still colonizedEuprymna scolopesand triggered PG-induced morphogenesis. Our results shed light on glutamate transport, highlight trade-offs in GltS structure and function, and reveal an unusual PG modification.IMPORTANCED-glu is an important building block in the peptidoglycan (PG) component of the bacterial cell wall, and its endogenous production is considered essential in most bacteria, even when grown in complex media. InVibrio fischeri,in transexpression of mutant GltS symporters allows D-glu auxotrophic strains to grow on lysogeny broth salts (LBS) medium without exogenous D-glu, although there is a fitness trade-off of increased sensitivity to homocysteic acid. Our finding that LBS contains sufficient D-glu to support robust growth highlights the undervalued importance of D-amino acid transport and the ubiquity of D-amino acids. Moreover, the discovery of D-lysine in the PG peptide is an unusual PG modification that warrants further study. 

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