Bacterial secretion of extracellular polysaccharides is essential for surface colonization, biofilm formation, and pathogenesis. In diderm bacteria, such polymers traverse the periplasm and outer membrane (OM) through outer-membrane polysaccharide export (OPX) proteins that form secretion pores. Among them, Class-3 OPX proteins are the most widespread but lack an OM-spanning pore domain, leaving their mechanisms poorly understood. Here, we characterize WzaB from Myxococcus xanthus as a model for Class-3 OPX-mediated secretion. Structural and molecular dynamics analyses reveal that WzaB exists as a rigid monomer in solution, in contrast to the constitutive octamerization observed in Class-1 OPX proteins. Biochemical, biophysical, and in vivo analyses show that WzaB oligomerizes in a lipidation-dependent manner and directly interacts with the OM porin WzpB and the inner-membrane co-polymerase WzcB, with binding determinants mapped for both partners. Together, these proteins assemble into a trans-envelope polysaccharide secretion complex, redefining OPX function and revealing a distinct translocon architecture for Class-3 OPX systems.

Center for Basic and Applied Research Faculty of Informatics and Management University of Hradec Králové Hradec Králové Czech Republic

Institut National de la Recherche Scientifique Centre Armand Frappier Santé Biotechnologie Institut Pasteur International Network Laval QC Canada

Laboratory of Molecular Modeling Applied to the Chemical and Biological Defense Military Institute of Engineering Rio de Janeiro RJ Brazil

PROTEO the Quebec Network for Research on Protein Function Structure and Engineering Montréal QC Canada

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