Secretion and regulation of synthesis of exopolysaccharides in Myxococcus xanthus
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Philipps-Universität Marburg
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Abstract
The properties of Gram-negative bacteria are significantly shaped by their surface polysaccharides. These polysaccharides have fundamental functions in biofilm formation, protection against biotic and abiotic stresses, virulence, adhesion, motility and in symbiotic and pathogenic host-microbe interactions. Their synthesis is costly and, therefore, tightly regulated. Synthesis of secreted polysaccharides by the ubiquitous Wzx/Wzy-dependent pathway is initiated by a phosphoglycosyltransferase (PGT), catalyzing the transfer of an activated monosaccharide to undecaprenylphosphate (Und-P). Once assembled, the polysaccharides are secreted across the outer membrane (OM) by an OM polysaccharide export translocon, known as OPX. While the canonical OPX protein is an octameric exporter featuring a periplasmic channel and an integral OM α-helical pore, the distinct synthase-dependent pathway for synthesis of secreted polysaccharides utilizes 16- or 18- stranded β barrel proteins for translocating polysaccharides across the OM.
In the Gram-negative bacterium Myxococcus xanthus, the secreted exopolysaccharide (EPS) regulates crucial biological traits, including type IV pilus (T4P)-dependent motility, adhesion and biofilm formation. EPS is synthesized and secreted by the Wzx/Wzy-dependent EPS pathway. In this study, a novel component of the EPS pathway, EpsX, was characterized as an integral OM 18-stranded β-barrel protein important for EPS biosynthesis. AlgE, the OM polysaccharide translocon of a synthase dependent pathway, was identified as its closest structural homolog. Based on computational structural biology, EpsY, the OPX protein of the EPS pathway, comprises only periplasmic domains and lacks the domain required to form the integral OM α-helical pore. Experimental evidence supports that EpsX and EpsY mutually stabilize each other and directly interact. These observations and structural modeling support that EpsX and EpsY form a bipartite translocon for EPS export in which EpsX forms the OM pore and EpsY the periplasmic channel. Computational genomics indicates that similar bipartite translocons are found in many Gram-negative bacterial phyla.
EPS biosynthesis is initiated by the PGT EpsZ, transferring galactose-1-P to Und-P. The chemosensory-like Dif system stimulates EPS biosynthesis via the phosphorylated response regulator EpsW upon T4P extension by an unknown mechanism. Here, using global transcriptomic and proteomic analyses, it was demonstrated that EpsW regulates EPS biosynthesis at the post-translational level. MiniTurbo-based proximity labeling experiments strongly support that phosphorylated EpsW interacts with EpsZ. Previous structural characterization of the EpsZ homolog WbaP in Salmonella enterica supports that it forms a functional homodimer and that dimerization involves a distinct cytoplasmic β-hairpin. Computational structural biology indicates that EpsZ lacks this characteristic β-hairpin and likely cannot dimerize on its own. Strikingly, these computational analyses also support that EpsW, by directly interacting with EpsZ, promotes the formation of the active EpsZ dimer. Altogether, these findings support that EpsW, upon T4P extension and phosphorylation by the DifE kinase, activates EPS synthesis at its initial step by facilitating the formation of the active conformation of the PGT EpsZ.
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Created: 2024Issued: 2025-06-04Updated: 2025-06-04
Faculty
Fachbereich Biologie
Publisher
Philipps-Universität Marburg
Language
eng
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DoctoralThesis
DFG-subjects
PolysaccharideMikrobiologie
DDC-Numbers
500
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Schwabe, Johannes: Secretion and regulation of synthesis of exopolysaccharides in Myxococcus xanthus. : Philipps-Universität Marburg 2025-06-04. DOI: https://doi.org/10.17192/z2024.0215.