Exploring the intersection between necromass reutilization and genome protection reveals novel proteins that...

Recovery of microbial populations following severe stress and extensive loss of viability-termed RUS (repopulation upon shattering)-represents a fundamental biological challenge. Successful RUS depends on the ability of surviving cells to acquire and recycle material released from dead cells. However, depending on the nature and intensity of stress, the liberated substrates may contain energy-rich yet potentially hazardous or genotoxic compounds. Previous studies in the basidiomycete fungus Ustilago maydis indicated that effective RUS requires not only metabolic recycling but genome-protection mechanisms. The principal aim of this study was to further investigate the link between RUS and genome stability.

Supernatants derived from peroxide-treated cells induced significant DNA damage in untreated wild-type cells, demonstrating that substrates released from dead cells possess intrinsic genotoxic potential. Analysis of mutants deficient in genome-protection pathways revealed that deletion of several homologous recombination and excision repair factors caused minor impairment of substrate reutilization. In contrast, loss of key regulatory components-including the small acidic protein Dss1, the recombination- and cell cycle-associated factor Rec1, and especially the apical checkpoint kinase Atr1-resulted in strong defects in growth under supernatant-induced stress. Transcriptome-guided analysis identified additional genes linking RUS to genome protection. Deletion of a Nudix hydrolase (designated Ndx1) and a Pirin-like protein (Pir1) significantly impaired growth on substrates, while disruption of UMAG_05976 caused a severe RUS phenotype and sensitivity to DNA-damaging agents. UMAG_05976 belongs to a set of supernatant-induced genes lacking detectable conserved domains but with homologs restricted to closely related smut fungi within Ustilaginales. UMAG_05976 was designated Rdf1 (RUS-deployed factor 1). A complementary genetic screen identified mutations in genes encoding a class V myosin (Myo2), a PX-domain-containing protein (Lec1), a ubiquitin-specific protease (Usp1), and a leucine biosynthetic enzyme (Leu4), which all impaired substrate re-utilization and increased genotoxic sensitivity.

Together, these findings demonstrate that growth on nutrients derived from damaged cells inherently challenges genome integrity and requires coordinated activity of checkpoint signaling, stress-response regulators, ubiquitin-mediated regulation, and membrane-associated processes. This study strengthens the conceptual link between RUS and genome protection and provides a framework for future investigations of microbial survival after extreme stress.