Evaluation of Azido 3-Deoxy-d- manno -oct-2-ulosonic Acid (Kdo) Analogues for Click Chemistry-Mediated Metabolic Labeling of Myxococcus xanthus DZ2 Lipopolysaccharide.

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 -a Gram-negative predatory social bacterium known to display complex outer membrane (OM) dynamics-via growth in the presence of distinct azido (-N) analogues of 3-deoxy-d--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-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-Kdo) was also synthesized, with both analogues capable of undergoing strain-promoted azide-alkyne cycloaddition (SPAAC) "click" chemistry reactions. Slower and faster growth of was displayed in the presence of 8-N-Kdo and 7-N-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-Kdo-grown cells were fluorescently labeled following treatment with dibenzocyclooctyne-linked fluorophores, the surfaces of 7-N-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-Kdo is indeed a substrate of the enzyme, 7-N-Kdo is not. Though a lack of cell aggregation was shown to expedite growth in liquid culture, 7-N-Kdo-grown cells did not manifest differences in intrinsic clumping relative to untreated cells, suggesting that 7-N-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.