Listeria monocytogenes exopolysaccharide: origin, structure, biosynthetic machinery and c‐di‐GMP‐dependent regulation

Summary Elevated levels of the second messenger c‐di‐GMP activate biosynthesis of an unknown exopolysaccharide (EPS) in the food‐borne pathogen Listeria monocytogenes. This EPS strongly protects cells against disinfectants and desiccation, indicating its potential significance for listerial persistence in the environment and for food safety. We analyzed the potential phylogenetic origin of this EPS, determined its complete structure, characterized genes involved in its biosynthesis and hydrolysis and identified diguanylate cyclases activating its synthesis. Phylogenetic analysis of EPS biosynthesis proteins suggests that they have evolved within monoderms. Scanning electron microscopy revealed that L. monocytogenes EPS is cell surface‐bound. Secreted carbohydrates represent exclusively cell‐wall debris. Based on carbohydrate composition, linkage and NMR analysis, the structure of the purified EPS is identified as a β‐1,4‐linked N‐acetylmannosamine chain decorated with terminal α‐1,6‐linked galactose. All genes of the pssA‐E operon are required for EPS production and so is a separately located pssZ gene. We show that PssZ has an EPS‐specific glycosylhydrolase activity. Exogenously added PssZ prevents EPS‐mediated cell aggregation and disperses preformed aggregates, whereas an E72Q mutant in the presumed catalytic residue is much less active. The diguanylate cyclases DgcA and DgcB, whose genes are located next to pssZ, are primarily responsible for c‐di‐GMP‐dependent EPS production. The c‐di‐GMP‐activated exopolysaccharide from the food‐borne pathogen Listeria monocytogenes strongly protects cells against disinfectants. We present the structure of this novel exopolysaccharide as a β‐1,4‐linked N‐acetylmannosamine chain decorated with terminal α‐1,6‐linked galactose. We also characterize the exopolysaccharide hydrolase that may be used for breaking down exopolysaccharide‐based cell aggregates thus increasing bacterial susceptibility to disinfectants.