Lennon Jay T, Lehmkuhl Brent K, Chen Lingling, Illingworth Melissa, Kuo Venus, Muscarella Mario E
Department of Biology, Indiana University, Bloomington, Indiana, USA.
Department of Molecular and Cellular Biochemistry, Indiana University, Bloomington, Indiana, USA.
mSystems. 2025 May 20;10(5):e0151724. doi: 10.1128/msystems.01517-24. Epub 2025 Apr 16.
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 KBS0714, a bacterium isolated from agricultural soil. The protein exhibited high substrate affinity , 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 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.
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.
微生物常常栖息于不利于生长和繁殖的环境中。为了在受到此类条件挑战时生存下来，个体进入休眠状态，即进入代谢不活跃状态。为了使这种持久性策略赋予进化优势，微生物必须能够在条件改善时复苏并繁殖。在放线菌门细菌中，休眠可以通过复苏促进因子（Rpf）来终止，Rpf是一种外切酶，可水解细胞壁肽聚糖中的糖苷键。我们对从农业土壤中分离出的细菌KBS0714的Rpf进行了表征。该蛋白表现出高底物亲和力，尽管在活细胞测定中微摩尔浓度时复苏效果最佳。保守催化位点的定点突变显著降低或消除了复苏能力，凝集素编码连接区中重复基序的缺失也有同样效果。然后，我们测试了来自KBS0714的重组Rpf对多种休眠土壤细菌的影响。复苏模式映射到菌株系统发育上，这反映了细胞包膜的核心特征。此外，Rpf效应的方向和大小与功能性状相关，特别是水分生态位和生物膜产生方面，这对于理解土壤中微生物种群的休眠和持久性至关重要。这些发现扩展了我们对Rpf如何影响种子库动态的理解，对陆地生态系统中微生物的多样性和功能具有重要意义。
休眠是个体进入代谢活动降低的可逆状态的过程。在波动的环境中，休眠保护个体免受不利条件影响，增强适应性并缓冲种群免于灭绝。然而，从休眠中苏醒是一个关键但有风险的决定。一些细菌随机复苏，而其他细菌则依赖环境线索或来自邻近细胞的信号来转变回活跃生长状态。复苏促进因子（Rpf）是一种外切酶，可切割细菌细胞壁肽聚糖中的键，促进休眠终止并实现再生长。尽管该蛋白家族在模式生物和临床相关菌株中已得到充分表征，但我们的研究对来自土壤细菌的Rpf进行了表征，并研究了其对多种细菌复苏的影响，将其与可能影响自然和管理生态系统中休眠动态的功能性状联系起来。
