This confirms that any difference in the dispersal assay is

caused by effects of NO and NOS on active dispersal of vegetative biofilm cells and not on germination of spores. Interestingly, the addition of exogenously supplied NO with the chemical NO donor SNAP to the nos mutant and L-NAME-inhibited wild-type cells did not restore dispersal to wild-type levels. We used NO microsensors to measure whether R406 the Selleck P5091 extracellular NO concentrations established by the NO donor during the dispersal assay were sufficient to complement for the loss of NOS synthesis. We found that addition of 300 μM SNAP to the dispersal drop resulted in an NO concentration between 150 to 200 nM (Figure 6). NO was consumed within the biofilm resulting in NO concentrations around the lower detection limit (~ 30 nM). Apparent NO consumption did not depend on the ability of B. subtilis to synthesize NO with NOS. NO concentrations

within SCH727965 biofilms not exposed to the NO donor were also around the lower detection limit and could not be quantified with confidence. Thus, we could not discern if similar extracellular concentrations of NO were present during the different treatments in the biofilm microenvironment. Figure 6 Nitric oxide microprofiles measured during the dispersal assay. The y-axis shows the biofilm depth with 0 (dashed line) denoting the surface of the biofilm. Positive values are inside the spot colony biofilm and negative values are above the biofilm in the MSgg medium drop. MSgg medium was supplemented with 300 μM of the NO donor SNAP (closed symbols) or supplied without supplementation of SNAP (open symbols). Wild-type B. subtilis

3610 was incubated with a drop of MSgg (A) without further supplementation and (B) further supplemented with 100 μM NOS inhibitor L-NAME. (C) shows B. subtilis 3610 Δnos supplied with MSgg without further supplementation. Error bars depict the standard deviation (N = 10) between repeated measurements at the same position in the sample reflecting the precision of the measurement. Taken together the results show that the addition of the NO donor during the dispersal experiment potentially provided a sufficient flux of extracellular NO to complement the deficiency for NO synthesis. The apparent failure of complementation suggests that NOS-derived NO is not an intercellular signalling molecule for the maintenance of these cells in the biofilm. Rather, it mediates its effect on dispersal at defined intracellular concentrations, which cannot be restored by the exogenous addition of NO. Defined intracellular NO concentrations would be particularly important if NOS-mediated signalling proceeds via redox-based modifications of enzymes [3] or when it is used for biosynthesis of other signalling molecules [8]. Our results suggest that one of these two mechanisms might act within B. subtilis cells to facilitate the maintenance of cells in the biofilm. Kolodkin-Gal et al. [29] described the disassembly of B.