caa a22 4500 605506469 CHVBK 20210128100627.0 cr unu---uuuuu 210128e20150601xx s 000 0 eng 10.1007/s00253-015-6570-9 doi (NATIONALLICENCE)springer-10.1007/s00253-015-6570-9 Epicocconone, a sensitive and specific fluorescent dye for in situ quantification of extracellular proteins within bacterial biofilms [Elektronische Daten] [I. Randrianjatovo, E. Girbal-Neuhauser, C-E. Marcato-Romain] Biofilms are ecosystems of closely associated bacteria encapsulated in an extracellular matrix mainly composed of polysaccharides and proteins. A novel approach was developed for in situ quantification of extracellular proteins (ePNs) in various bacterial biofilms using epicocconone, a natural, fluorescent compound that binds amine residues of proteins. Six commercial proteins were tested for their reaction with epicocconone, and bovine serum albumin (BSA) was selected for assay optimization. The optimized protocol, performed as a microassay, allowed protein amounts as low as 0.7μg to as high as 50μg per well to be detected. Addition of monosaccharides or polysaccharides (glucose, dextran or alginate) to the standard BSA solutions (0 to 250μgml−1) showed little or no sugar interference up to 2000μgml−1, thus providing an assessment of the specificity of epicocconone for proteins. The optimized protocol was then applied to three different biofilms, and in situ quantification of ePN showed contrasted protein amounts of 22.1 ± 3.1, 38.3 ± 7.1 and 0.3 ± 0.1μg equivalent BSA of proteins for 48-h biofilms of Pseudomonas aeruginosa, Bacillus licheniformis and Weissella confusa, respectively. Possible interference due to global matrix compounds on the in situ quantification of proteins was also investigated by applying the standard addition method (SAM). Low error percentages were obtained, indicating a correct quantification of both the ePN and the added proteins. For the first time, a specific and sensitive assay has been developed for in situ determination of ePN produced by bacterial cells. This advance should lead to an accurate, rapid tool for further protein labelling and microscopic observation of the extracellular matrix of biofilms. Springer-Verlag Berlin Heidelberg, 2015 Extracellular proteins nationallicence Epicocconone nationallicence Biofilm nationallicence In situ nationallicence Quantification nationallicence Standard addition method nationallicence Randrianjatovo I. Université de Toulouse, Laboratoire de Biotechnologies Alimentaire et Environnementale, EA 4565 (LBAE), Institut Universitaire de Technologie, Université Paul Sabatier, 24 rue d'Embaquès, F-32000, Auch, France aut Girbal-Neuhauser E. Université de Toulouse, Laboratoire de Biotechnologies Alimentaire et Environnementale, EA 4565 (LBAE), Institut Universitaire de Technologie, Université Paul Sabatier, 24 rue d'Embaquès, F-32000, Auch, France aut Marcato-Romain C-E Université de Toulouse, Laboratoire de Biotechnologies Alimentaire et Environnementale, EA 4565 (LBAE), Institut Universitaire de Technologie, Université Paul Sabatier, 24 rue d'Embaquès, F-32000, Auch, France aut Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/11(2015-06-01), 4835-4844 0175-7598 99:11<4835 2015 99 253 https://doi.org/10.1007/s00253-015-6570-9 text/html Onlinezugriff via DOI BK010053 XK010053 XK010000 Metadata rights reserved Springer special CC-BY-NC licence nationallicence 1 research-article jats NATIONALLICENCE NATIONALLICENCE NL-springer NATIONALLICENCE 856 40 https://doi.org/10.1007/s00253-015-6570-9 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Randrianjatovo I. Université de Toulouse, Laboratoire de Biotechnologies Alimentaire et Environnementale, EA 4565 (LBAE), Institut Universitaire de Technologie, Université Paul Sabatier, 24 rue d'Embaquès, F-32000, Auch, France aut NATIONALLICENCE 700 1- Girbal-Neuhauser E. Université de Toulouse, Laboratoire de Biotechnologies Alimentaire et Environnementale, EA 4565 (LBAE), Institut Universitaire de Technologie, Université Paul Sabatier, 24 rue d'Embaquès, F-32000, Auch, France aut NATIONALLICENCE 700 1- Marcato-Romain C-E Université de Toulouse, Laboratoire de Biotechnologies Alimentaire et Environnementale, EA 4565 (LBAE), Institut Universitaire de Technologie, Université Paul Sabatier, 24 rue d'Embaquès, F-32000, Auch, France aut NATIONALLICENCE 773 0- Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/11(2015-06-01), 4835-4844 0175-7598 99:11<4835 2015 99 253