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.

Lipopolysaccharide, synthesis, oligosaccharide, structure, Research, bioinformatics, vaccine, application, optimization

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
4	aDGlcp	101.8	73.2	73.9	70.7	72.4	66.8
2	Ac
6	60%Me	59.7
	aDGalpN	98.5	51.1	68.4	79.6	70.5	72.0


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
4	aDGlcp	4.92	3.53	3.81	3.54	4.24	3.63-3.98
2	Ac
6	60%Me	3.41
	aDGalpN	4.99	4.25	4.03	4.04	4.17	3.75-3.82


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
4	aDGlcp	101.8/4.92	73.2/3.53	73.9/3.81	70.7/3.54	72.4/4.24	66.8/3.63-3.98
2	Ac
6	60%Me	59.7/3.41
	aDGalpN	98.5/4.99	51.1/4.25	68.4/4.03	79.6/4.04	70.5/4.17	72.0/3.75-3.82

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

[*]OC[C@H]1O[C@H](O[C@H]2[C@@H](COC)O[C@H]([*])[C@H](NC(C)=O)[C@H]2O)[C@H](O)[C@@H](O)[C@@H]1O

379.362 g/mol (C₁₅H₂₅R₂NO₁₀, R = next and previous repeats, not counted in mol weight)

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.

Lipopolysaccharide, synthesis, oligosaccharide, structure, Research, bioinformatics, vaccine, application, optimization

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

R1 = LIP
Warning = sub-repeat ending at aDGlcpN: unknown unit count was assumed to be 3
[1*]N[C@H]1[C@H](OC[C@H]2O[C@H](OP(=O)(O)O)[C@H](N[1*])[C@@H](O)[C@@H]2O)O[C@H](CO[C@]2(C(=O)O)C[C@@H](O)[C@@H](O[C@H]3O[C@H](COP(=O)(O)O[C@H]4O[C@H](CO)[C@@H](O)[C@H](O)[C@@H]4O[C@H]4O[C@H](CO)[C@@H](O)[C@H](O)[C@@H]4O)[C@H](O)[C@H](O[C@@H]4O[C@H](CO)[C@@H](O)[C@H](O)[C@H]4O[C@@H]4OC[C@@H](O[C@H]5O[C@H](COC)[C@@H](O[C@H]6O[C@H](CO[C@H]7O[C@H](COC)[C@@H](O[C@H]8O[C@H](CO[C@H]9O[C@H](COC)[C@@H](O[C@H]%10O[C@H](CO[C@H]%11O[C@H](COC)[C@@H](O[C@H]%12O[C@H](CO)[C@@H](O)[C@H](O)[C@H]%12O)[C@H](O)[C@H]%11NC(C)=O)[C@@H](O)[C@H](O)[C@H]%10O)[C@H](O)[C@H]9NC(C)=O)[C@@H](O)[C@H](O)[C@H]8O)[C@H](O)[C@H]7NC(C)=O)[C@@H](O)[C@H](O)[C@H]6O)[C@H](O)[C@H]5NC(C)=O)[C@H](O)[C@H]4O)[C@H]3NC(C)=O)[C@@H]([C@H](O)COP(=O)(O)OCCN)O2)[C@@H](O)[C@@H]1O

3180.676 g/mol (C₁₁₃H₁₉₃R₂N₈O₈₉P₃, R = residue superclass(es), not counted in mol weight)