Biofilm formation is a complex, ordered process. In the opportunistic pathogen Pseudomonas aeruginosa, Psl and Pel exopolysaccharides and extracellular DNA (eDNA) serve as structural components of the biofilm matrix. Despite intensive study, Pel's chemical structure and spatial localization within mature biofilms remain unknown. Using specialized carbohydrate chemical analyses, we unexpectedly found that Pel is a positively charged exopolysaccharide composed of partially acetylated 1→4 glycosidic linkages of N-acetylgalactosamine and N-acetylglucosamine. Guided by the knowledge of Pel's sugar composition, we developed a tool for the direct visualization of Pel in biofilms by combining Pel-specific Wisteria floribunda lectin staining with confocal microscopy. The results indicate that Pel cross-links eDNA in the biofilm stalk via ionic interactions. Our data demonstrate that the cationic charge of Pel is distinct from that of other known P. aeruginosa exopolysaccharides and is instrumental in its ability to interact with other key biofilm matrix components.biofilms | exopolysaccharide | extracellular DNA | Pel | Psl

Background Pseudomonas aeruginosa is a Gram-negative bacterium and an opportunistic pathogen, which causes persisting 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 FindingsIn 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 Lanýi type O:2a,c (Lanýi-Bergman O-serogroup 10a, 10c; IATS serotype 19) and having the following structure: -4)-α-L-GalNAcA-(1–3)-α-D-QuiNAc-(1–3)- α-L-Rha-(1-. Furthermore, an EC O-antigen polysaccharide (EC O-PS) and the glycerol-phosphorylated cyclic β-(1,3)-glucans were identified in the culture supernatant of PA14, grown statically in minimal 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.ConclusionsWe 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.

In order to investigate the mechanism of benzo[a]pyrene uptake by a filamentous fungus Fusarium solani, a biochemical characterization of its concentrated culture filtrate has been conducted. The preparation contained approximately (w/w): 50% of total carbohydrate, 6.5% of uronic acid and 6% protein, as determined by colorimetric tests. Gel filtration and anion-exchange chromatographic profiles indicated that the main product of the culture filtrate was a glycoprotein, which contained mannose, glucose and galactose in an approximate molar ratio of 1.5:0.8:1. The polysaccharide fraction of the culture filtrate was prepared by treatment with proteinase K, followed by gel-filtration chromatography. Its chemical structure was studied by methylation analysis, gas-liquid chromatography-mass spectrometry (GC-MS) and Nuclear Magnetic Resonance spectroscopy (NMR). The major carbohydrate was a polymer of β-(1→6)-linked galactofuranose units fully branched at positions O-2 by single residues of -glucopyranose. The Fusarium concentrated culture filtrate increased 4-fold the BaP solubilization in comparison with its aqueous solubility and suggested that the carbohydrate present in this filtrate should probably be involved in this enhancement. Our findings point out the potential role of fungal glycoproteins in PAH microbial bioavaibility, an important step for PAH biodegradation.

No abstract