The increased attachment of AB101 and AB102 may be partly dependent on changes in cell surface-exposed polysaccharides that are modulated in Che1-dependent manner (Bible et al., 2008; Edwards et al., 2011). To directly evaluate the contribution of specific sugar-binding molecules on promoting attachment and biofilm formation, glass surfaces were treated with LcH or WGA lectins, prior to incubation with A. brasilense cells. AFM imaging indicated that the lectin treatment increased attachment for all strains, with the most significant increase in attachment seen for the AB101, AB102, and AB103 strains on LcH-treated glass surfaces (Fig. 2). The increased attachment was comparable for all strains on WGA-treated glass surfaces (Fig. 2). Although the ability of cells to attach to lectin-treated glass surfaces varied greatly between the strains, no distinctive visible extracellular structure(s), such as flagella, pili or specific patterns in the EPS (exopolysaccharide) matrices, could be attributed to this difference (Fig. S3). This does not account for expression variation in outer membrane proteins (OMPs), polysaccharides, or other adhesions beyond the resolution capabilities of the AFM scans (Fig. S3). Next, confocal microscopy was used to analyze attachment of cells to lectin-treated glass (Fig. 3). Prior to imaging, the lectin-treated surfaces on which cells attached were gently and briefly washed to ensure that only primary attachment to the surface was accounted for and to reduce possible confounding interpretations resulting from secondary attachment events (e.g. to other cells). Under these conditions, the attachment pattern of the Che1 mutant strains on lectin-treated surfaces were similar to that observed by AFM with attachment to LcH-treated glass surfaces, but not WGA treated-glass surface, directly correlating with the flocculation phenotypes of the strains: strains that flocculate more than wild type (AB101, AB102, and AB103) also attached to LcH-treated glass surfaces more (Table 3). Given that cells did not attach to glass in the absence of lectins, the surface attachment detected here is likely via interaction between cell surface exposed sugar residues and the lectins. The two lectins tested mediated different patterns of attachment for the che1 strains tested, suggesting distinct surface-exposed sugar residues between the strains, an observation consistent with similar conclusions reached previously (Edwards et al., 2011). The observation that LcH-dependent attachment correlated with the increased flocculation behavior of some of the che1 mutant strains provides further support to the notion that Che1-dependent changes in cell-to-cell aggregation and flocculation involves remodeling of the extracellular matrix, some of which is shown here to promote surface attachment. Regardless of the exact effects that Che1 signaling has on cell surface changes which are currently investigated in our laboratory, the data obtained here show that attachment of A. brasilense is increased by nitrogen limitation and further suggests that it depends on sugar-exposed residues that have lectin-binding properties, in agreement with the proposition made previously by Mora et al. (2008). Increasing attachment of A. brasilense to root surfaces may thus ultimately depends on fine-tuning metabolic activities, including limiting nitrogen availability that is shown here as a key modulator of attachment to surfaces.