Abstract
Biofilms are a multicellular way of life, where bacterial cells are close together and embedded in a hydrated macromolecular matrix which offers a number of advantages to the cells. Extracellular polysaccharides play an important role in matrix setup and maintenance. A water-insoluble polysaccharide was isolated and purified from the biofilm produced by Burkholderia cenocepacia strain H111, a cystic fibrosis pathogen. Its composition and glycosidic linkages were determined using Gas–Liquid Chromatography–Mass Spectrometry (GLC–MS) on appropriate carbohydrate derivatives while its complete structure was unraveled by 1D and 2D NMR spectroscopy in deuterated sodium hydroxide (NaOD) aqueous solutions. All the collected data demonstrated the following repeating unit for the water-insoluble B. cenocepacia biofilm polysaccharide: [3)-α-d-Galp-(1→3)-α-d-Glcp-(1→3)-α-d-Galp-(1→3)-α-d-Manp-(1→]n Molecular modelling was used, coupled with NMR Nuclear Overhauser Effect (NOE) data, to obtain information about local structural motifs which could give hints about the polysaccharide insolubility. Both modelling and NMR data pointed at restricted dynamics of local conformations which were ascribed to the presence of inter-residue hydrogen bonds and to steric restrictions. In addition, the good correlation between NOE data and calculated interatomic distances by molecular dynamics simulations validated potential energy functions used for calculations.
Highlights
Burkholderia cenocepacia belongs to the Burkholderia Cepacia Complex (BCC), a group of 22 closely related species that are commonly found in the environment, and in many cases have been isolated from cystic fibrosis (CF) patients
The Epol H111-INS was isolated from biofilm produced by the B. cenocepacia ∆bcsB/pBerA strain [7], which was derived from B. cenocepacia H111, a cystic fibrosis clinical isolate [9]
In the B. cenocepacia ∆bcsB/pBerA strain, the berA gene is present on the multicopy plasmid pBerA, and the BerA protein is overproduced, which results in overproduction of the Burkholderia cenocepacia exopolysaccharide (Bep) exopolysaccharide
Summary
Burkholderia cenocepacia belongs to the Burkholderia Cepacia Complex (BCC), a group of 22 closely related species that are commonly found in the environment, and in many cases have been isolated from cystic fibrosis (CF) patients. Its products were hypothesized to be involved in the biosynthesis of a major exopolysaccharide that provides structural stability to the biofilms formed by B. cenocepacia. With the aim of elucidating the structure of the exopolysaccharides in B. cenocepacia H111 biofilms, a strain of B. cenocepacia H111 overproducing BerA and lacking cellulose production was used to form biofilms, and a water-insoluble exopolysaccharide, named Epol H111-INS, was extracted directly from the biofilms. The Epol H111-INS composition and glycosidic linkages were determined using GasLiquid Chromatography–Mass Spectrometry (GLC–MS) on appropriate carbohydrate derivatives while its complete structure was elucidated by use of 1 D and 2D NMR spectroscopy recorded in deuterated sodium hydroxide (NaOD) aqueous solutions, since the base produces a limited amount of ionized hydroxyl groups which prevent polymer aggregation and promotes solubilization in water. The results of the modelling analysis were compared with the NMR NOE data as a test of the accuracy of the MM energy functions
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