Pectobacterium parmentieri is a pectinolytic plant pathogenic bacterium causing high economic losses of cultivated plants. The highly devastating potential of this phytopathogen results from the efficient production of plant cell wall-degrading enzymes, i.e., pectinases, cellulases and proteases, in addition to the impact of accessory virulence factors such as motility, siderophores, biofilm and lipopolysaccharide (LPS). LPS belongs to pathogen-associated molecular patterns (PAMPs) and plays an important role in plant colonization and interaction with the defense systems of the host. Therefore, we decided to investigate the heterogeneity of O-polysaccharides (OPS) of LPS of different strains of P. parmentieri, in search of an association between the selected genomic and phenotypic features of the strains that share an identical structure of the OPS molecule. In the current study, OPS were isolated from the LPS of two P. parmentieri strains obtained either in Finland in the 1980s (SCC3193) or in Poland in 2013 (IFB5432). The purified polysaccharides were analyzed by utilizing 1D and 2D NMR spectroscopy (1H, DQF-COSY, TOCSY, ROESY, HSQC, HSQC-TOCSY and HMBC) in addition to chemical methods. Sugar and methylation analyses of native polysaccharides, absolute configuration assignment of constituent monosaccharides and NMR spectroscopy data revealed that these two P. parmentieri strains isolated in different countries possess the same structure of OPS with a very rare residue of 5,7-diamino-3,5,7,9-tetradeoxy-l-glycero-l-manno-non-2-ulosonic acid (pseudaminic acid) substituted in the position C-8: →3)-β-D-Galf-(1→3)-α-D-Galp-(1→8)-β-Pse4Ac5Ac7Ac-(2→6)-α-D-Glcp-(1→6)-β-D-Glcp-(1→. The previous study indicated that three other P. parmentieri strains, namely IFB5427, IFB5408 and IFB5443, exhibit a different OPS molecule than SCC3193 and IFB5432. The conducted biodiversity-oriented assays revealed that the P. parmentieri IFB5427 and IFB5408 strains possessing the same OPS structure yielded the highest genome-wide similarity, according to average nucleotide identity analyses, in addition to the greatest ability to macerate chicory tissue among the studied P. parmentieri strains. The current research demonstrated a novel OPS structure, characteristic of at least two P. parmentieri strains (SCC3193 and IFB5432), and discussed the observed heterogenicity in the OPS of P. parmentieri in a broad genomic and phenotype-related context.

Lipopolysaccharide, virulence, pseudaminic acid, biodiversity, O-antigen chemical structure, Pectobacterium parmentieri SCC3193, soft rot Pectobacteriaceae

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6	C7	C8	C9
6,6,8,3	bDGalf	110.71	81.25	85.77	83.39	71.87	64.14
6,6,8	aDGalp	98.06	68.56	78.64	70.52	71.96	62.36
6,6,4	Ac	174.50	23.48
6,6,5	Ac	174.13-175.57	21.76-23.21
6,6,7	Ac	174.13-175.57	21.76-23.21
6,6	bXPsep	174.38	102.33	34.09	70.59	47.12	72.59	54.15	74.13	14.19
6	aDGlcp	99.27	72.84	74.45	71.39	71.87	64.78
	bDGlcp	103.29	74.29	77.19	70.49	75.74	66.79


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6	H7	H8	H9
6,6,8,3	bDGalf	5.241	4.373	4.303	4.199	3.932	3.686-3.722
6,6,8	aDGalp	5.088	3.892	3.854	4.138	4.209	3.756-3.756
6,6,4	Ac	-	1.956
6,6,5	Ac	-	2.008-2.14
6,6,7	Ac	-	2.008-2.14
6,6	bXPsep	-	-	1.784-2.489	4.911	4.325	4.157	4.325	4.267	1.293
6	aDGlcp	4.959	3.592	3.737	3.410	3.809	3.709-3.928
	bDGlcp	4.689	3.328	3.528	3.581	3.676	3.762-4.051


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6	C7/H7	C8/H8	C9/H9
6,6,8,3	bDGalf	110.71/5.241	81.25/4.373	85.77/4.303	83.39/4.199	71.87/3.932	64.14/3.686-3.722
6,6,8	aDGalp	98.06/5.088	68.56/3.892	78.64/3.854	70.52/4.138	71.96/4.209	62.36/3.756-3.756
6,6,4	Ac		23.48/1.956
6,6,5	Ac		21.76-23.21/2.008-2.14
6,6,7	Ac		21.76-23.21/2.008-2.14
6,6	bXPsep			34.09/1.784-2.489	70.59/4.911	47.12/4.325	72.59/4.157	54.15/4.325	74.13/4.267	14.19/1.293
6	aDGlcp	99.27/4.959	72.84/3.592	74.45/3.737	71.39/3.410	71.87/3.809	64.78/3.709-3.928
	bDGlcp	103.29/4.689	74.29/3.328	77.19/3.528	70.49/3.581	75.74/3.676	66.79/3.762-4.051

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

[*]O[C@H]1[C@H]([C@H](O)CO)O[C@@H](O[C@H]2[C@@H](O)[C@@H](CO)O[C@H](O[C@@H](C)[C@H](NC(C)=O)[C@@H]3O[C@@](OC[C@H]4O[C@H](OC[C@H]5O[C@@H]([*])[C@H](O)[C@@H](O)[C@@H]5O)[C@H](O)[C@@H](O)[C@@H]4O)(C(=O)O)C[C@H](OC(C)=O)[C@@H]3NC(C)=O)[C@@H]2O)[C@@H]1O

1006.911 g/mol (C₃₉H₆₂R₂N₂O₂₈, 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

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)