| Abstract | 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 (-N₃) 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-N₃-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-N₃-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-N₃-Kdo and 7-N₃-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-N₃-Kdo-grown cells were fluorescently labeled following treatment with dibenzocyclooctyne-linked fluorophores, the surfaces of 7-N₃-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-N₃-Kdo is indeed a substrate of the enzyme, 7-N₃-Kdo is not. Though a lack of M. xanthus cell aggregation was shown to expedite growth in liquid culture, 7-N₃-Kdo-grown cells did not manifest differences in intrinsic clumping relative to untreated cells, suggesting that 7-N₃-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. |
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