6 research outputs found
Evaluation of Azido 3-Deoxy- d - Manno-oct-2-ulosonic Acid (Kdo) Analogues for Click Chemistry-Mediated Metabolic Labeling of Myxococcus xanthus DZ2 Lipopolysaccharide
[Image: see text] 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 xanthusâa Gram-negative predatory social bacterium known to display complex outer membrane (OM) dynamicsâvia 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 azideâalkyne cycloaddition (SPAAC) âclickâ 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
Modulation of bacterial multicellularity via spatio-specific polysaccharide secretion
International audienceThe development of multicellularity is a key evolutionary transition allowing for differentiation of physiological functions across a cell population that confers survival benefits; among uni-cellular bacteria, this can lead to complex developmental behaviors and the formation of higher-order community structures. Herein, we demonstrate that in the social ÎŽ-proteobac-terium Myxococcus xanthus, the secretion of a novel biosurfactant polysaccharide (BPS) is spatially modulated within communities, mediating swarm migration as well as the formation of multicellular swarm biofilms and fruiting bodies. BPS is a type IV pilus (T4P)-inhibited acidic polymer built of randomly acetylated ÎČ-linked tetrasaccharide repeats. Both BPS and exopolysaccharide (EPS) are produced by dedicated Wzx/Wzy-dependent polysaccharide-assembly pathways distinct from that responsible for spore-coat assembly. While EPS is preferentially produced at the lower-density swarm periphery, BPS production is favored in the higher-density swarm interior; this is consistent with the former being known to stimulate T4P retraction needed for community expansion and a function for the latter in promoting initial cell dispersal. Together, these data reveal the central role of secreted polysaccharides in the intricate behaviors coordinating bacterial multicellularity
Evaluation of Azido 3âDeoxyâd-<i>manno</i>-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 Myxococcus xanthusa Gram-negative
predatory social bacterium known to display complex outer membrane
(OM) dynamicsvia 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
azideâalkyne cycloaddition (SPAAC) âclickâ 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
Evaluation of Azido 3âDeoxyâd-<i>manno</i>-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 Myxococcus xanthusa Gram-negative
predatory social bacterium known to display complex outer membrane
(OM) dynamicsvia 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
azideâalkyne cycloaddition (SPAAC) âclickâ 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