The conserved Pss exopolysaccharide is a common component of Listeria biofilms

Listeria monocytogenes (Lm) is a major foodborne pathogen capable of persisting on fresh produce and in food-processing environments. Although exopolysaccharides (EPSs) are recognized as central components of resilient bacterial biofilms, Lm biofilms have long been considered to be EPS-less or EPS-poor. We previously identified a listerial EPS, Pss, whose biosynthesis is encoded by the highly conserved pss operon and is stimulated by elevated c-di-GMP levels. However, the prevalence of Pss in wild-type strains remained unclear, in part, due to the lack of a specific detection probe. Here, we engineered a genetically encoded fluorescent probe, PssZ*-mS, based on a catalytically inactive variant (E72Q) of the Pss hydrolase PssZ. Structural modeling predicted that the E72Q substitution abolishes catalysis without disrupting Pss binding. PssZ*-mS bound purified Pss with high affinity (K_d, 50 nM) and showed no detectable interaction with other tested EPSs, including cellulose, pectin, maltodextrin, or alginate. Using PssZ*-mS, we demonstrate that the reference wild-type strain EGDe produces substantial Pss under standard conditions in liquid minimal glucose-containing medium and on diverse abiotic surfaces relevant to food environments. A survey of Lm isolates revealed that Pss production is common, though not universal, among Lm strains and is also present in non-monocytogenes Listeria species. Notably, three genetically distinct strains from the infamous 2011 whole-cantaloupe listeriosis outbreak synthesize Pss. These findings challenge the prevailing view of listerial biofilms and identify Pss as a widespread component of Lm biofilms, underscoring the need for targeted anti-Pss strategies to enhance food safety.IMPORTANCEListeria monocytogenes persists on fresh produce and in food-processing environments despite rigorous sanitation, yet the molecular basis of this resilience remains poorly understood. We developed a specific fluorescent probe for the conserved Pss exopolysaccharide (EPS) and show that Pss is a common component of listerial biofilms. These findings challenge the long-standing view that Listeria forms primarily EPS-less or EPS-poor biofilms and identify Pss as an important biofilm component. By enabling direct visualization of Pss, this work provides a new tool for studying listerial biofilms and opens avenues for targeted antibiofilm strategies to improve food safety and public health.