Figure 5

Influence of NO and NO synthase on (A) dispersal and (B) germination of B. subtilis 3610. (A) The dispersal assay was conducted with 3610 wild-type (white bars) and 3610Δnos (gray bars). Colonies grew for 4 d on MSgg agar and were mounted with a drop of 100 μL MSgg medium. The NOS inhibitor L-NAME and the NO scavenger c-PTIO were supplemented to agar and drop, while the NO donor SNAP was only supplemented to the drop. Vegetative cells that dispersed within 2 h into the drop liquid were quantified with flow cytometry. Error bars indicate standard error (N = 10). (B) The germination assay was conducted in a separate experiment, employing a similar set-up and the same treatments as for the dispersal assay. MSgg medium (including supplements) was mixed with B. subtilis spores, placed as a 100 μL drop on a sterile polystyrene surface and incubated for 2 h. Spores only (open bars in panel B) and total cells (hatched bars in panel B) were determined by plating and counting the colony forming units (cfu). The results are normalized to the spore concentration. Error bars indicate standard deviation (N = 5). The results show 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.