Background: Pseudomonas aeruginosa is a Gram-negative bacterium and an opportunistic pathogen, which causespersisting life-threatening infections in cystic fibrosis (CF) patients. Biofilm mode of growth facilitates its survival in a variety of environments. Most P. aeruginosa isolates, including the non-mucoid laboratory strain PA14, are able to form a thick pellicle, which results in a surface-associated biofilm at the air-liquid (A-L) interface in standing liquid cultures.Exopolysaccharides (EPS) are considered as key components in the formation of this biofilm pellicle. In the non-mucoid P.aeruginosa strain PA14, the "scaffolding" polysaccharides of the biofilm matrix, and the molecules responsible for the structural integrity of rigid A-L biofilm have not been identified. Moreover, the role of LPS in this process is unclear, and the chemical structure of the LPS O-antigen of PA14 has not yet been elucidated. Principal Findings: In the present work we carried out a systematic analysis of cellular and extracellular (EC) carbohydrates of P. aeruginosa PA14. We also elucidated the chemical structure of the LPS O-antigen by chemical methods and 2-D NMR spectroscopy. Our results showed that it is composed of linear trisaccharide repeating units, identical to those described for P. aeruginosa Lanyi type O:2a,c (Lanyi-Bergman O-serogroup 10a, 10c; IATS serotype 19) and having the following structure:-4)-a-L-GalNAcA-(1-3)-a-D-QuiNAc-(1-3)-a-L-Rha-(1-. Furthermore, an EC O-antigen polysaccharide (EC O-PS) and the glycerol-phosphorylated cyclic b-(1,3)-glucans were identified in the culture supernatant of PA14, grown statically inminimal medium. Finally, the extracellular matrix of the thick biofilm formed at the A-L interface contained, in addition to eDNA, important quantities (at least, 20% of dry weight) of LPS-like material. Conclusions: We characterized the chemical structure of the LPS O-antigen and showed that the O-antigen polysaccharide is an abundant extracellular carbohydrate of PA14. We present evidence that LPS-like material is found as a component of a biofilm matrix of P. aeruginosa.

Pseudomonas aeruginosa, NMR spectroscopy, exopolysaccharides, cystic fibrosis, Biofilm

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
3,4	aLRhap	102.3	68.8	77.1	71.4	70.5	17.8
3,2	Ac
3	aLGalpNA	98.2	50.7	67.6	80.0	71.6	?
2	Ac
	aDQuipN	95.9	54.9	76.4	75.2	69.2	17.8


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
3,4	aLRhap	4.84	3.95	3.78	3.50	4.05	1.26
3,2	Ac
3	aLGalpNA	5.16	4.17	4.10	4.40	4.97	-
2	Ac
	aDQuipN	4.87	4.12	3.90	3.28	4.10	1.26


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
3,4	aLRhap	102.3/4.84	68.8/3.95	77.1/3.78	71.4/3.50	70.5/4.05	17.8/1.26
3,2	Ac
3	aLGalpNA	98.2/5.16	50.7/4.17	67.6/4.10	80.0/4.40	71.6/4.97	
2	Ac
	aDQuipN	95.9/4.87	54.9/4.12	76.4/3.90	75.2/3.28	69.2/4.10	17.8/1.26

The spectrum also has 1 signal at unknown position (not plotted).

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

550.514 g/mol (C₂₂H₃₄R₂N₂O₁₄, R = next and previous repeats, not counted in mol weight)