PrtA-mediated flagellar turnover is essential for robust biofilm development in <i>Serratia marcescens</i>

Biofilm formation is crucial for bacterial persistence, requiring precise regulatory mechanisms to transition from motility to sessility. Here, we uncover the role of the metalloprotease PrtA in <i>Serratia marcescens</i> biofilm development and its interaction with flagellar components. Loss of PrtA leads to reduced biofilm biomass, thickness, and viable cell counts, as shown through high-resolution confocal microscopy. The biofilm-deficient phenotype is rescued by wild-type PrtA expression but not by a proteolytically inactive PrtA_E177A mutant, underscoring the essential role of PrtA's enzymatic activity. Exogenous addition of purified PrtA restores biofilm formation, confirming its enzymatic necessity. Proteomic profiling identified flagellar proteins as primary PrtA targets, with an overrepresentation of flagellar components in <i>prtA</i> mutant biofilms. In addition, PrtA selectively degrades depolymerized flagellar filaments, facilitating biofilm progression by removing excess flagellar material. Transcriptional analysis reveals an inverse expression pattern of flagellar master regulator (<i>flhDC</i>) and <i>prtA</i> during biofilm establishment, suggesting a coordinated regulatory axis that suppresses flagellar function while promoting biofilm development. Confocal microscopy at the liquid-air interface shows increased flagellar content in <i>prtA</i> mutant biofilms, supporting PrtA's role in matrix organization and biofilm integrity. Collectively, these findings establish PrtA as a crucial mediator of flagellar turnover and extracellular proteolysis, linking motility suppression to robust biofilm formation. This work not only advances our understanding of biofilm regulation in <i>S. marcescens</i> but also identifies PrtA as a potential target for novel biofilm control strategies.IMPORTANCEBiofilms are central to the persistence and pathogenicity of <i>Serratia marcescens</i>, particularly in clinical settings where they contribute to chronic infections and antimicrobial resistance. This study identifies the metalloprotease PrtA as a critical regulator of biofilm development, acting through the selective degradation of flagellar components to mediate the transition from motility to sessility. By demonstrating that PrtA's proteolytic activity is essential for proper biofilm architecture and viability, and that it directly targets excess flagellar material, we provide mechanistic insight into how biofilm maturation is coordinated with motility suppression. The discovery of an inverse regulatory relationship between <i>prtA</i> and the flagellar master regulator <i>flhDC</i> further supports the existence of a finely tuned system controlling biofilm establishment. Together, these findings enhance our understanding of biofilm regulation in <i>Serratia marcescens</i>, an opportunistic human pathogen increasingly associated with antibiotic resistance, and highlight PrtA as a promising target for novel anti-biofilm strategies.