Identification, isolation, and structural studies of extracellular polysaccharides produced by Caulobacter crescentus

Caulobacters are adherent prosthecate bacteria that are members of bacterial biofouling communities in many environments. Investigation of the cell surface carbohydrates produced by two strains of the freshwater Caulobacter crescentus, CB2A and CB15A, revealed a hitherto undetected extracellular polysaccharide (EPS) or capsule. Isolation and characterization of the EPS fractions showed that each strain produced a unique neutral EPS which could not be readily removed from the cell surface by washing. Monosaccharide analysis showed that the main CB2A EPS contained D-glucose, D-gulose, and D-fucose in a ratio of 3:1:1, whereas the CB15A EPS fraction contained D-galactose, D-glucose, D-mannose, and D-fucose in approximately equal amounts. Methylation analysis of the main CB2A EPS showed the presence of terminal glucose and gulose groups, 3-linked fucosyl, and two 3,4-linked glucosyl units, thus confirming the pentasaccharide repeating unit indicated by 1H nuclear magnetic resonance analysis. Similar studies of the CB15A EPS revealed a tetrasaccharide repeating unit consisting of terminal galactose, 4-linked fucosyl, 3-linked glucosyl, and 3,4-linked mannosyl residues. EPS was not detectable by thin-section electron microscopy techniques, including some methods designed to preserve or enhance capsules, nor was the EPS readily detected on the cell surface by scanning electron microscopy when conventional fixation techniques were used; however, a structure consistent with EPS was revealed when samples were prepared by cryofixation and freeze-substitution methods.