Planktonic bacteria were washed off and adherent bacteria were fixed and stained with DAPI. The adherence of TT01pam (B) is presented as a percentage of the data determined for the corresponding parental strain TT01rif (A). Bacterial counts were performed at 60× magnification and the data represent the mean values of 12 fields from triplicate experiments (± St.Dev) (C). To study in more detail the role of Pam in attachment and its adhesive properties, we used surface plasmon resonance (SPR) to measure binding to an abiotic gold surface. First, we used washed cells
Selleck CUDC-907 in an attempt to assess the role of Pam when it is bound to the EPS surrounding the bacterium: TT01pam showed increased binding to the surface compared to the parental TT01rif (Fig. 6A),
indicating that the presence of the protein reduces adhesion to the surface in these conditions. Similarly, in Pam-expressing E. coli we observed a decrease in adhesion compared to E. coli selleck screening library control (Fig. 6B). Using SPR to assess the effect of Pam secreted into the medium, we analyzed the supernatants of cultures. In this case we found the opposite effect: when Pam, either from TT01rif or recombinant E. coli cultures, was secreted in the supernatant we observed a greater change in SPR angle, indicating that in the presence of Pam more material bound to the gold surface than from the supernatant of cells lacking Pam, TT01pam and control E. coli (Figs. 6C and 6D). We checked that this effect was due specifically to the presence of Pam in the supernatant by blocking Pam binding with addition of the anti-Pam antibody (X. Muñoz-Berbel, M. Sanchez-Contreras and A. T. A. Jenkins, unpublished data). These
Cilengitide mw results suggest that secreted Pam binds Y-27632 in vivo to surfaces, while when Pam is bound to the cell surface it makes these cells less able to attach. Figure 6 Surface plasmon resonance analysis of Pam-mediated adhesion on gold-coated glass probes. (A and B) Presence of the protein on the cell surface (washed cells) showed decreased adhesion to untreated gold surfaces in both TT01rif and E. coli pBADpam (+Pam), when compared with the correspondent strains lacking Pam, TT01pam and E. coli pBAD respectively (-Pam). (C and D) Supernatants from cultures expressing pam, TT01rif and E. coli pBADpam (+Pam), showed more adhesion than those lacking the protein TT01pam and E. coli pBAD (-Pam), indicating the ability of free Pam to adhere to surfaces. Structural studies of Pam In order to better understand the physicochemical properties that confer on Pam the ability to bind EPS and influence cell attachment, we investigated the structural properties of the protein by circular dichroism (CD) spectroscopy and differential scanning calorimetry (DSC). CD spectra at near-UV and far-UV wavelengths were obtained for purified heterologously produced Pam. Weak spectra were recorded in the near-UV, but a strong signal was obtained between 182 nm and 240 nm in the far-UV range.