Envelope glycosylation determined by lectins in microscopy sections of Acinetobacter venetianus induced by diesel fuel

It was suggested in a previous study that cells of Acinetobacter venetianus VE-C3 adhere to diesel fuel by synthesizing a capsular polysaccharide containing glucose and/or mannose. To study the fine structure of cells and localization of bacterial polysaccharide in the presence of diesel fuel, two lectins were used: ConA, an agglutinin from Canavalia ensiformis specific for mannose and/or glucose residues, and PNA, an agglutinin from Arachis hypogaea, for terminal galactose residues. The lectins were conjugated with electron dense ferritin for transmission electron microscopy (TEM) and with fluorescein isothiocyanate (FITC) for scanning confocal laser microscopy (SCLM). Samples were prepared by freeze substitution, which allows glycosylation to be determined in situ in thin sections of specimens. The distribution of glycosylation was imaged with and without treatment of specimens with their specific hapten (glucose and galactose). The glycosylation activity produced a polysaccharide capsule. Emulsified diesel fuel nanodroplets were observed at the cell envelope perimeter. Fine structure of vesicles consisted of polysaccharide and diesel fuel nanodroplets. Lectin blotting analysis showed ConA-positive glycoprotein with an apparent molecular mass of 22 kDa in the outer membrane. Its production was induced by diesel fuel. This glycoprotein was probably responsible for bioemulsifying activity at the cell envelope. Several other glycoproteins were positive for PNA lectin, the main constituent migrating with an apparent molecular weight of 17.8 kDa. However, they were all constitutive and probably involved in cell biofilm formation at the oil surface.