Colwellia psychrerythraea strain 34H, a Gram-negative bacterium isolated from Arctic marine sediments, is considered a model to study the adaptation to cold environments. Recently, we demonstrated that C. psychrerythraea 34H produces two different extracellular polysaccharides, a capsular polysaccharide and a medium released polysaccharide, which confer cryoprotection to the bacterium. In this study, we report the structure of an additional capsular polysaccharide produced by Colwellia grown at a different temperature. The structure was determined using chemical methods, and one- and two-dimensional NMR spectroscopy. The results showed a trisaccharide repeating unit made up of only amino-sugar residues: N-acetyl-galactosamine, 2,4-diacetamido-2,4,6-trideoxy-glucose (bacillosamine), and 2-acetamido-2-deoxyglucuronic acid with the following structure: →4)-β-D-GlcpNAcA-(1 →3)-β-D-QuipNAc4NAc-(1 →3)-β-D-GalpNAc-(1 →. The 3D model, generated in accordance with 1H,1H-NOE NMR correlations and consisting of ten repeating units, shows a helical structure. In contrast with the other extracellular polysaccharides produced from Colwellia at 4 degrees C, this molecule displays only a low ice recrystallization inhibition activity.

NMR, extracellular polysaccharides, psychrophile, Anti-freeze activity, Cold adaptation

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
3,3,2	Ac	175.5	23.3
3,3	bDGlcpNA	103.3	56.0	72.3	81.6	77.6	174.3
3,2	Ac	174.6	23.3
3,4	Ac	175.9	23.3
3	bDQuipN4N	103.1	56.3	78.9	56.5	71.5	17.9
2	Ac	175.1	23.6
	bDGalpN	102.5	51.8	79.7	68.8	75.6	61.9


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
3,3,2	Ac	-	1.98
3,3	bDGlcpNA	4.43	3.58	3.68	3.77	3.60	-
3,2	Ac	-	2.04
3,4	Ac	-	1.97
3	bDQuipN4N	4.47	3.78	3.73	3.56	3.55	1.21
2	Ac	-	2.08
	bDGalpN	4.55	3.93	3.79	4.11	3.70	3.76


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
3,3,2	Ac		23.3/1.98
3,3	bDGlcpNA	103.3/4.43	56.0/3.58	72.3/3.68	81.6/3.77	77.6/3.60	
3,2	Ac		23.3/2.04
3,4	Ac		23.3/1.97
3	bDQuipN4N	103.1/4.47	56.3/3.78	78.9/3.73	56.5/3.56	71.5/3.55	17.9/1.21
2	Ac		23.6/2.08
	bDGalpN	102.5/4.55	51.8/3.93	79.7/3.79	68.8/4.11	75.6/3.70	61.9/3.76

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

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

648.619 g/mol (C₂₆H₄₀R₂N₄O₁₅, R = next and previous repeats, not counted in mol weight)

Microrganisms from sea ice, glacial and subglacial environments are currently under investigation due to their relevant ecological functions in these habitats, and to their potential biotechnological applications. The cold-adapted Colwellia psychrerythraea 34H produces extracellular polysaccharides with cryoprotection activity. We here describe the purification and detailed molecular primary and secondary structure of the exopolysaccharide (EPS) secreted by C. psychrerythraea 34H cells grown at 4 degrees C. The structure was determined by chemical analysis and NMR. The trisaccharide repeating unit of the EPS is constituted by a N-acetyl quinovosamine unit and two residues of galacturonic acid both decorated with alanine. In addition, the EPS was tested in vitro showing a significant inhibitory effect on ice recrystallization. In-depth NMR and computational analysis suggest a pseudohelicoidal structure which seems to prevent the local tetrahedral order of the water molecules in the first hydration shell, and could be responsible of the inhibition of ice recrystallization. As cell cryopreservation is an essential tool in modern biotechnology and medicine, the observations reported in this paper could pave the way for a biotechnological application of Colwellia EPS.

NMR spectroscopy, extracellular polysaccharide, structure-activity relationship, EPS, psychrophilic, Alanine decoration, Cold-adaptation, Colwellia psychrerythraea 34H, cryopreservation, Exopolymer

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
4,4,2	Ac	175.9	24.0
4,4	bDQuipN	103.5	56.0	81.2	77.7	72.5	18.4
4,6	xLAla?	181.2	52.2	19.2
4	aDGalpA	100.8	69.6	70.7	79.4	73.0	171.0
6	xLAla?	180.3	52.0	19.2
	aDGalpA	100.9	69.0	69.8	77.9	72.0	170.2


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
4,4,2	Ac	-	1.91
4,4	bDQuipN	4.56	3.74	3.72	3.39	3.37	1.27
4,6	xLAla?	-	4.22	1.42
4	aDGalpA	4.98	3.63	3.95	4.30	4.90	-
6	xLAla?	-	4.23	1.39
	aDGalpA	5.54	3.87	3.97	4.46	4.28	-


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
4,4,2	Ac		24.0/1.91
4,4	bDQuipN	103.5/4.56	56.0/3.74	81.2/3.72	77.7/3.39	72.5/3.37	18.4/1.27
4,6	xLAla?		52.2/4.22	19.2/1.42
4	aDGalpA	100.8/4.98	69.6/3.63	70.7/3.95	79.4/4.30	73.0/4.90	
6	xLAla?		52.0/4.23	19.2/1.39
	aDGalpA	100.9/5.54	69.0/3.87	69.8/3.97	77.9/4.46	72.0/4.28	

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

[*]O[C@H]1[C@H](O)[C@@H](C)O[C@@H](O[C@H]2[C@@H](C(=O)N[C@@H](C)C(=O)O)O[C@H](O[C@H]3[C@@H](C(=O)N[C@@H](C)C(=O)O)O[C@H]([*])[C@H](O)[C@H]3O)[C@H](O)[C@H]2O)[C@@H]1NC(C)=O

681.601 g/mol (C₂₆H₃₉R₂N₃O₁₈, R = next and previous repeats, not counted in mol weight)

The low temperatures of polar regions and high-altitude environments, especially icy habitats, present challenges for many microorganisms. Their ability to live under subfreezing conditions implies the production of compounds conferring cryotolerance. Colwellia psychrerythraea 34H, a gamma-proteobacterium isolated from subzero Arctic marine sediments, provides a model for the study of life in cold environments. We report here the identification and detailed molecular primary and secondary structures of capsular polysaccharide from C. psychrerythraea 34H cells. The polymer was isolated in the water layer when cells were extracted by phenol/water and characterized by one- and two-dimensional NMR spectroscopy together with chemical analysis. Molecular mechanics and dynamics calculations were also performed. The polysaccharide consists of a tetrasaccharidic repeating unit containing two amino sugars and two uronic acids bearing threonine as substituent. The structural features of this unique polysaccharide resemble those present in antifreeze proteins and glycoproteins. These results suggest a possible correlation between the capsule structure and the ability of C. psychrerythraea to colonize subfreezing marine environments.

capsular polysaccharide, Marine bacterium, Colwellia psychrerythraea

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
3,2,3	bDGlcpA	104.5	73.9	74.9	81.0	77.3	175.4
3,2,2	Ac	176.0-176.4	23.7-23.8
3,2	bDGlcpN	104.7	56.0	83.3	69.8	76.8	62.1
3,6	xLThr	177.2	60.8	69.8	20.5
3	aDGalpA	98.3	79.2	69.2	71.2	72.9	171.5
2	Ac	176.0-176.4	23.7-23.8
	bDGalpN	102.3	52.0	79.3	66.1	76.1	62.3


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
3,2,3	bDGlcpA	4.35	3.23	3.48	3.65	3.59	-
3,2,2	Ac	-	1.77-1.86
3,2	bDGlcpN	4.50	3.75	3.62	3.38	3.35	3.61-3.78
3,6	xLThr	-	4.18	4.15	1.05
3	aDGalpA	5.31	3.72	3.79	4.09	4.16	-
2	Ac	-	1.77-1.86
	bDGalpN	4.36	3.84	3.76	4.19	3.56	3.64-3.71


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
3,2,3	bDGlcpA	104.5/4.35	73.9/3.23	74.9/3.48	81.0/3.65	77.3/3.59	
3,2,2	Ac		23.7-23.8/1.77-1.86
3,2	bDGlcpN	104.7/4.50	56.0/3.75	83.3/3.62	69.8/3.38	76.8/3.35	62.1/3.61-3.78
3,6	xLThr		60.8/4.18	69.8/4.15	20.5/1.05
3	aDGalpA	98.3/5.31	79.2/3.72	69.2/3.79	71.2/4.09	72.9/4.16	
2	Ac		23.7-23.8/1.77-1.86
	bDGalpN	102.3/4.36	52.0/3.84	79.3/3.76	66.1/4.19	76.1/3.56	62.3/3.64-3.71

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

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

859.741 g/mol (C₃₂H₄₉R₂N₃O₂₄, R = next and previous repeats, not counted in mol weight)