Diverse bacteria assemble and secrete polysaccharides that alter their physiologies through modulation of motility, biofilm formation, and host immune system evasion. Most such pathways require outer membrane (OM) polysaccharide export (OPX) proteins for sugar-polymer transport to the cell surface.

The presence of an exopolysaccharide (EPS) layer surrounding bacterial cells, termed a “glycocalyx”, confers protection against toxic molecules. However, the effect of glycocalyx integrity on the tolerance to such agents is poorly understood. Using a modified disc-diffusion assay, we tested the susceptibility to a panel of antibiotics and oxidative stress-inducing compounds of various mutant strains of the social predatory Gram-negative soil bacterium Myxococcus xanthus; the selected mutants were those that manifest different physical states of their respective EPS glycocalyces. While the overall presence of an EPS layer was indeed beneficial for tolerance, the integrity of this layer was also found to affect the susceptibility of the bacterium to killing; however, this finding was not universal, and instead was dependent on the specific compound tested. Thus, the integrity of the cell-surface EPS glycocalyx plays an important role in the tolerance of M. xanthus to harmful compounds.

A bstract: Integrins are crucial for eukaryotic cell attachment and motility within the extracellular matrix (ECM) via focal-adhesion formation, with their evolutionary emergence important for the development of multicellularity. Intriguingly, single gliding cells of the predatory deltaproteobacterium Myxococcus xanthus form bacterial focal-adhesion (bFA) sites; therein, helically-trafficked motors become immobilized at anchored locations through Glt apparatus association with cell-surface integrin αI-domain-like adhesin CglB. Using traction-force, bead-force, and total internal reflection-fluorescence microscopies combined with biochemical approaches, we herein identify the von Willebrand A domain-containing cell-surface lipoprotein CglD to be a β-integrin-like outer-membrane lipoprotein that functionally associates with and anchors the trans-envelope Glt–CglB gliding apparatus, stabilizing and efficiently anchoring this assembly at bFAs. Calcium dependence governs CglD importance, consistent with its integrated ECM eukaryotic cartilage oligomeric matrix protein domains. CglD thus confers mechanosensory and mechanotransductory capabilities to the gliding apparatus, helping explain bFA-mediated trans-envelope force transduction, from inner-membrane-embedded motors to the cell surface.

A bstract: The predatory deltaproteobacterium Myxococcus xanthus uses a helically-trafficked motor at bacterial focal adhesion (bFA) sites to power gliding motility. Using TIRF and force microscopy, we herein identify the integrin αI-domain-like outer-membrane (OM) lipoprotein CglB as an essential substratum-coupling protein of the gliding motility complex. Similar to most known OM lipoproteins, CglB is anchored on the periplasmic side of the OM and thus a mechanism must exist to secrete it to the cell surface in order for it to interact with the underlying substratum. We reveal this process to be mediated by a predicted OM β-barrel structure of the gliding complex. This OM platform was found to regulate the conformational activation and secretion of CglB across the OM. These data suggest that the gliding complex promotes surface exposure of CglB at bFAs, thus explaining the manner by which forces exerted by inner-membrane motors are transduced across the cell envelope to the substratum; they also uncover a novel protein secretion mechanism, highlighting the ubiquitous connection between secretion and bacterial motility.

Secretion of high-molecular-weight polysaccharides across the bacterial envelope is ubiquitous as it enhances prokaryotic survival in (a)biotic settings. Such polymers are often assembled by Wzx/Wzy- or ABC transporter-dependent schemes that implicate outer-membrane (OM) polysaccharide export (OPX) proteins in polymer translocation to the cell surface. In the social predatory bacterium Myxococcus xanthus , exopolysaccharide (EPS)-pathway WzaX, major spore coat (MASC)-pathway WzaS, and biosurfactant polysaccharide-pathway WzaB were herein found to be truncated OPX homologues of Escherichia coli Wza lacking OM-spanning α-helices. Comparative genomics across all bacteria, complemented with cryo-electron tomography cell- envelope analyses, revealed WzaX/S/B architecture to be the most common amongst three defined OPX-protein structural classes independent of periplasmic thickness. Fold-recognition and deep- learning analyses revealed the conserved M. xanthus proteins MXAN_7418/3226/1916 (encoded adjacent to WzaX/S/B) to be integral OM β-barrels, with structural homology to the poly- N -acetyl-D- glucosamine synthase-dependent pathway porin PgaA. Such porins were identified in bacteria near numerous genes for all three OPX-protein classes. Interior MXAN_7418/3226/1916 β-barrel electrostatics were found to match known properties of their associated polymers. With MXAN_3226 essential for MASC export, and MXAN_7418 absence shown herein to compromise EPS translocation, these data support a novel secretion paradigm for Wzx/Wzy-dependent pathways in which those containing an OPX component that cannot span the OM instead utilize a β-barrel porin to mediate polysaccharide transport across the OM.

Bacterial surface exopolysaccharide (EPS) layers are key determinants of biofilm establishment and maintenance, leading to the formation of higher-order 3D structures conferring numerous survival benefits to a cell community. In addition to a specific EPS glycocalyx, we recently revealed that the social δ-proteobacterium Myxococcus xanthus secretes a novel biosurfactant polysaccharide (BPS), with both EPS and BPS polymers required for type IV pilus (T4P)-dependent swarm expansion via spatio-specific biofilm expression profiles. Thus the synergy between EPS and BPS secretion somehow modulates the multicellular lifecycle of M. xanthus . Herein, we demonstrate that BPS secretion functionally-activates the EPS glycocalyx via its destabilization, fundamentally altering the characteristics of the cell surface. This impacts motility behaviours at the single-cell level as well as the aggregative capacity of cells in groups via EPS fibril formation and T4P assembly. These changes modulate structuration of swarm biofilms via cell layering, likely contributing to the formation of internal swarm polysaccharide architecture. Together, these data reveal the manner by which the interplay between two secreted polymers induces single-cell changes that modulate swarm biofilm communities.