brasilense (Burdman et al., 2000a), were present and participated in cell-to-cell aggregation and flocculation. The addition of 0.5 M arabinose to flocculating cultures of both mutant strains caused a significant reduction in the total amount of flocculation (Fig. 3a), suggesting that arabinose contributes to the structure and/or the stability of the flocs formed by these strains. In addition, we found that flocs selleck of the AB102 (ΔcheY1) strain were significantly more sensitive to the competitive addition of exogenous arabinose (Fig. 3a) than the flocs
of AB101. Similarly, high concentrations of glucose (0.5 M) reduced flocculation in both mutant strains and flocs formed by the AB102 (ΔcheY1) strain appeared to be more sensitive to the addition of glucose, with almost complete inhibition of flocculation after the addition of 0.5 M glucose (Fig. Depsipeptide clinical trial 3b). To further investigate differences in the extracellular matrix,
we used FITC-conjugated lentil lectin (LcH) (affinity for α-mannose and α-glucose) and lima bean lectin (LBL) (affinity for N-acetyl galactosamine) to probe for specific carbohydrates present on or around the cell surface. Wild-type cells did not show any significant binding of either lectin after 24 h of growth as determined by fluorescence imaging and statistical analysis (Fig. 4; Table S1). Both OSBPL9 lectins were found to stain AB101 (ΔcheA1) cells and the surrounding material (Fig. 4b and h). In comparison with AB101, AB102 (ΔcheY1) cells displayed reduced staining by both lectins (Fig. 4c and i). When normalized to the fluorescence signal of Syto61 that stains all cells (Fig. 4d–f and j–l), the lectin fluorescence signal detected for AB102 (ΔcheY1) floc structures was significantly (P=0.05) reduced for both lectins with respect to AB101 (Table S1). The lipopolysaccharides profiles of the mutant and wild-type strains grown under flocculating and nonflocculating conditions
were compared. Under conditions of growth in rich medium (TY), all strains had similar lipopolysaccharides profiles (Fig. 5). Differences in lipopolysaccharides profiles were detected between the strains as early as 24 h of incubation in flocculation medium, which corresponds to the time at which both mutant strains, but not the wild-type strain, flocculate. Under these conditions and compared with the lipopolysaccharides profile of the wild-type strain, a low-molecular-weight band (arrow 2, Fig. 5) is absent from the profile of both mutant strains while another low-molecular-weight band (arrow 3, Fig. 5) is significantly reduced. A higher molecular weight band (Fig. 5, arrow 1) is also clearly visible for all strains, but more abundant in the lipopolysaccharides profile of both mutant strains at 24 h.