The rice seedling blight fungus Rhizopus microsporus has an unusual symbiosis with a bacterium, Burkholderia rhizoxinica, which lives within the fungal cytosol and produces a potent phytotoxin that causes severe losses in agriculture. To gain insight into symbiosis factors we investigated the endosymbiont's exopolysaccharide (EPS), a secreted matrix that plays pivotal roles in mediating cell-environment interactions. By a combination of homo- and heteronuclear 2D NMR experiments, we elucidated a previously unknown EPS structure: a repeating tetrasaccharide unit bearing a nonstoichiometric acetyl group on a mannose residue. We also analyzed the EPS biosynthesis gene cluster and generated a targeted mutant to compare the phenotypes. Scanning electron microscope images revealed a reduced ability of the mutant to form extracellular polymers around cell aggregates. Phylogenetic analyses suggest that the symbiont's EPS genes are retained through evolutionary processes.

Burkholderia, Oligosaccharides, NMR spectroscopy, exopolysaccharides, bacteria-fungi interactions, Rhizopus

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
4,3,2	70%Ac	173.8	20.9
4,3	aDManp	97.99	72.0	76.17	66.07	72.7	60.12
4	bDGlcp	102.3	72.3	82.3	70.27	76.37	61.00
3	aDGalp	101.6	69.12	70.3	69.2	71.8	61.2
	aDManp	101.8	70.8	78.8	71.6	70.14	61.2


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
4,3,2	70%Ac	-	2.11
4,3	aDManp	5.17	5.22	4.10	3.84	3.90	3.77
4	bDGlcp	4.45	3.30	3.57	3.37	3.45	3.65-3.91
3	aDGalp	5.08	3.66	3.85	3.94	4.00	3.68
	aDManp	5.04	4.22	3.77	3.99	3.83	3.68


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
4,3,2	70%Ac		20.9/2.11
4,3	aDManp	97.99/5.17	72.0/5.22	76.17/4.10	66.07/3.84	72.7/3.90	60.12/3.77
4	bDGlcp	102.3/4.45	72.3/3.30	82.3/3.57	70.27/3.37	76.37/3.45	61.00/3.65-3.91
3	aDGalp	101.6/5.08	69.12/3.66	70.3/3.85	69.2/3.94	71.8/4.00	61.2/3.68
	aDManp	101.8/5.04	70.8/4.22	78.8/3.77	71.6/3.99	70.14/3.83	61.2/3.68

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

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

690.601 g/mol (C₂₆H₄₂R₂O₂₁, R = next and previous repeats, not counted in mol weight)

The rice seedling blight fungus Rhizopus microsporus has an unusual symbiosis with a bacterium, Burkholderia rhizoxinica, which lives within the fungal cytosol and produces a potent phytotoxin that causes severe losses in agriculture. To gain insight into symbiosis factors we investigated the endosymbiont's exopolysaccharide (EPS), a secreted matrix that plays pivotal roles in mediating cell-environment interactions. By a combination of homo- and heteronuclear 2D NMR experiments, we elucidated a previously unknown EPS structure: a repeating tetrasaccharide unit bearing a nonstoichiometric acetyl group on a mannose residue. We also analyzed the EPS biosynthesis gene cluster and generated a targeted mutant to compare the phenotypes. Scanning electron microscope images revealed a reduced ability of the mutant to form extracellular polymers around cell aggregates. Phylogenetic analyses suggest that the symbiont's EPS genes are retained through evolutionary processes.

Burkholderia, Oligosaccharides, NMR spectroscopy, exopolysaccharides, bacteria-fungi interactions, Rhizopus

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
4,3	aDManp	101.2	69.7	78.5	65.5	72.7	60.4
4	bDGlcp	102.4	72.2	82.0	70.4	76.3	60.7
3	aDGalp	101.2	69.07	69.9	69.2	72.2	61.0
	aDManp	101.98	70.2	78.8	72.0	69.4	60.4


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
4,3	aDManp	5.08	4.08	3.84	3.72	3.89	3.72
4	bDGlcp	4.24	3.24	3.54	3.31	3.43	3.63-3.87
3	aDGalp	5.09	3.62	3.80	3.87	3.98	3.64
	aDManp	4.99	4.22	3.99	3.97	3.86	3.72


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
4,3	aDManp	101.2/5.08	69.7/4.08	78.5/3.84	65.5/3.72	72.7/3.89	60.4/3.72
4	bDGlcp	102.4/4.24	72.2/3.24	82.0/3.54	70.4/3.31	76.3/3.43	60.7/3.63-3.87
3	aDGalp	101.2/5.09	69.07/3.62	69.9/3.80	69.2/3.87	72.2/3.98	61.0/3.64
	aDManp	101.98/4.99	70.2/4.22	78.8/3.99	72.0/3.97	69.4/3.86	60.4/3.72

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

[*]O[C@H]1[C@H](O)[C@@H](CO)O[C@H](O[C@@H]2[C@@H](O)[C@H](O[C@@H]3[C@@H](CO)O[C@H]([*])[C@@H](O)[C@H]3O[C@H]3O[C@H](CO)[C@H](O)[C@H](O)[C@H]3O)O[C@H](CO)[C@H]2O)[C@H]1O

648.564 g/mol (C₂₄H₄₀R₂O₂₀, R = next and previous repeats, not counted in mol weight)

The rice seedling blight fungus Rhizopus microsporus has an unusual symbiosis with a bacterium, Burkholderia rhizoxinica, which lives within the fungal cytosol and produces a potent phytotoxin that causes severe losses in agriculture. To gain insight into symbiosis factors we investigated the endosymbiont's exopolysaccharide (EPS), a secreted matrix that plays pivotal roles in mediating cell-environment interactions. By a combination of homo- and heteronuclear 2D NMR experiments, we elucidated a previously unknown EPS structure: a repeating tetrasaccharide unit bearing a nonstoichiometric acetyl group on a mannose residue. We also analyzed the EPS biosynthesis gene cluster and generated a targeted mutant to compare the phenotypes. Scanning electron microscope images revealed a reduced ability of the mutant to form extracellular polymers around cell aggregates. Phylogenetic analyses suggest that the symbiont's EPS genes are retained through evolutionary processes.

Burkholderia, Oligosaccharides, NMR spectroscopy, exopolysaccharides, bacteria-fungi interactions, Rhizopus

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

[*]O[C@H]1[C@H](O)[C@@H](CO)O[C@H](O[C@@H]2[C@@H](O)[C@H](O[C@@H]3[C@@H](CO)O[C@H]([*])[C@@H](O)[C@H]3O)O[C@H](CO)[C@H]2O)[C@H]1O

486.423 g/mol (C₁₈H₃₀R₂O₁₅, R = next and previous repeats, not counted in mol weight)