Lloyd and Tahon recently criticized proposed bacterial phylum nomenclature changes (K.G. Lloyd, G. Tahon, Nat Rev Microbiol 20:123-124, 2022, https://doi.org/10.1038/s41579-022-00684-2 ) precipitated by the International Committee on Systematics of Prokaryotes (ICSP)’s official recognition of phylum nomenclature rules. Here, we extend the critique.
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.
Metabolic labeling paired with click chemistry is a powerful approach for selectively imaging the surfaces of diverse bacteria. Herein, we explored the feasibility of labeling the lipopolysaccharide (LPS) of Myxococcus xanthus —a Gram-negative predatory social bacterium known to display complex outer membrane (OM) dynamics—via growth in the presence of distinct azido (-N 3 ) analogues of 3-deoxy- d - manno -oct-2-ulosonic acid (Kdo). Determination of the LPS carbohydrate structure from strain DZ2 revealed the presence of one Kdo sugar in the core oligosaccharide, modified with phosphoethanolamine. The production of 8-azido-8-deoxy-Kdo (8-N 3 -Kdo) was then greatly improved over previous reports via optimization of the synthesis of its 5-azido-5-deoxy- d -arabinose precursor to yield gram amounts. The novel analogue 7-azido-7-deoxy-Kdo (7-N 3 -Kdo) was also synthesized, with both analogues capable of undergoing in vitro strain-promoted azide–alkyne cycloaddition (SPAAC) “click” chemistry reactions. Slower and faster growth of M. xanthus was displayed in the presence of 8-N 3 -Kdo and 7-N 3 -Kdo (respectively) compared to untreated cells, with differences also seen for single-cell gliding motility and type IV pilus-dependent swarm community expansion. While the surfaces of 8-N 3 -Kdo-grown cells were fluorescently labeled following treatment with dibenzocyclooctyne-linked fluorophores, the surfaces of 7-N 3 -Kdo-grown cells could not undergo fluorescent tagging. Activity analysis of the KdsB enzyme required to activate Kdo prior to its integration into nascent LPS molecules revealed that while 8-N 3 -Kdo is indeed a substrate of the enzyme, 7-N 3 -Kdo is not. Though a lack of M. xanthus cell aggregation was shown to expedite growth in liquid culture, 7-N 3 -Kdo-grown cells did not manifest differences in intrinsic clumping relative to untreated cells, suggesting that 7-N 3 -Kdo may instead be catabolized by the cells. Ultimately, these data provide important insights into the synthesis and cellular processing of valuable metabolic labels and establish a basis for the elucidation of fundamental principles of OM dynamism in live bacterial cells.
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.
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