The surface associated communities of microorganism in biofilms are encased in a matrix of extracellular polymeric substances (EPS). The EPS is made up of mainly polysaccharides, proteins, nucleic acids and lipids and plays an important role in maintaining the integrity of the biofilm (1). Although the general composition of the EPS is known, it can be highly variable among strains and among different growth conditions for the same strain. Due to the large variety of biopolymers in nature and the difficulty in their analysis, EPS has been called ‘the dark matter of biofilms’ (2). In order to develop a comprehensive understanding of the matrix of the biofilm, EPS was extracted from four Pseudomonas spp., mCherry-expressing Pseudomonas fluorescens, GFP-expressing Pseudomonas putida and the wild types of Pseudomonas fluorescens and Pseudomonas putida. The extractions were carried out on biofilms grown on glass slides using the cation exchange resin (CER) method. Colorimetric methods were used to quantify the sugars and proteins present in the EPS. These colorimetric assays showed that there was a larger amount of proteins present compared to sugars. The proteins present in all four biofilms of Pseudomonas spp. were identified by LC-MS/MS while NMR and HPLC were used to identify the sugars present. The knowledge gained by these results have the potential to aid in the development of biofilm eradication methods through the targeting of specific components of the EPS. 1. Starkey, M., et al. (2004), American Society of Microbiology: 174–191.2. Flemming, H.-C. and J. Wingender (2010). Nature Reviews Microbiology 8 : 623.

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