title: Probing of exopolysaccharides with green fluorescence protein-labeled carbohydrate-binding module in Escherichia coli biofilms and flocs induced by bcsB overexpression.
abstract: Polysaccharides are major structural constituents to develop the three-dimensional architecture of Escherichia coli biofilms. In this study, confocal laser scanning microscopy was applied in combination with a fluorescent probe to analyze the location and arrangement of exopolysaccharide (EPSh) in microcolonies of E. coli K-12 derived strains, formed as biofilms on solid surfaces and flocs in the liquid phase. For this purpose, a novel fluorescent probe was constructed by conjugating a carbohydrate-binding module 3, from Paenibacillus curdlanolyticus, with the green fluorescence protein (GFP-CBM3). The GFP-CBM3 fused protein exhibited strong affinity to microcrystalline cellulose. Moreover, GFP-CBM3 specifically bound to cell-dense microcolonies in the E. coli biofilms, and to their flocs induced by bcsB overexpression. Therefore, the fused protein presents as a novel marker for EPSh produced by E. coli cells. Overexpression of bcsB was associated with abundant EPSh production and enhanced E. coli biofilm formation, which was similarly detectable by GFP-CBM3 probing.
authors.affiliation: Division of Chemical Engineering, Graduate School of Engineering Science, Osaka University, 1-3 Machikaneyama-cho, Toyonaka, Osaka 560-8531, Japan.
authors.forename: Minh Hong
authors.initials: MH
authors.lastname: Nguyen
authors.affiliation: Division of Chemical Engineering, Graduate School of Engineering Science, Osaka University, 1-3 Machikaneyama-cho, Toyonaka, Osaka 560-8531, Japan.
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Ojima
authors.affiliation: Applied Microbiology Laboratory, Graduate School of Bioresources, Mie University, 1577 Kurimamachiya-cho, Tsu, Mie 514-8507, Japan.
authors.forename: Makiko
authors.initials: M
authors.lastname: Sakka
authors.affiliation: Applied Microbiology Laboratory, Graduate School of Bioresources, Mie University, 1577 Kurimamachiya-cho, Tsu, Mie 514-8507, Japan.
authors.forename: Kazuo
authors.initials: K
authors.lastname: Sakka
authors.affiliation: Division of Chemical Engineering, Graduate School of Engineering Science, Osaka University, 1-3 Machikaneyama-cho, Toyonaka, Osaka 560-8531, Japan. Electronic address: taya@cheng.es.osaka-u.ac.jp.
authors.forename: Masahito
authors.initials: M
authors.lastname: Taya
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D018441:Biofilms; D002482:Cellulose / Q000737:chemistry; D048168:Escherichia coli K12 / Q000235:genetics / Q000378:metabolism*; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry*; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D018613:Microscopy, Confocal; D056507:Paenibacillus / Q000737:chemistry; D011135:Polysaccharides, Bacterial / Q000096:biosynthesis*; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D005456:Fluorescent Dyes; D011135:Polysaccharides, Bacterial; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; C055213:saccharide-binding proteins; D049452:Green Fluorescent Proteins; D002482:Cellulose; C109691:microcrystalline cellulose
keywords: Biofilm; Escherichia coli; Flocculation; Green fluorescence protein-labeled carbohydrate-binding module; bcsB