Figure 2.

B. subtilis cell differentiation and mutually exclusive regulation in a multicellular community. (A,B) Colony (A) and pellicle (B) biofilms formed on a solid agar medium or at the air–liquid interface by B. subtilis. (C) A cartoon demonstrating the coexistence of multiple differentiated cell types in a B. subtilis biofilm. A fruiting body is an aerial projection rising from the surface of the biofilm. Spores are preferentially located at the tip of the fruiting bodies. (D) A scanning electron microscopy image of the fruiting body structure. Scale bar, 5 μm. (E) A bright-field image of free spores associated with the tip of the fruiting body from a thin-section sample of a biofilm (images are provided by Angelini L). Arrows point to phase-bright spores. Scale bar, 25 μm. (F) Differentiation of two mutually exclusive cell types, motile and sessile chained cells, in a population. Motile cells are producing GFP under the control of the promoter for the motility gene hag while chained cells are producing mKate2 under the control of the promoter for the biofilm matrix gene tapA. Scale bar, 5 μm. (G) The epigenetic switch controlling the two mutually exclusive cell types in (F) consists of primarily three regulatory proteins, SinR, SinI, and SlrR. SinR is a biofilm repressor, Sin I is an antagonist protein of SinR whose gene is activated by Spo0A~P (0A~P). SlrR is another SinR counteracting protein. SlrR and SinR form a double negative feedback loop in which SinR represses the gene slrR while the SlrR protein antagonizes SinR, similar to SinI. A heterocomplex of SinR•SlrR represses motility and cell separation genes [24]. (H) A temporal lineage of B. subtilis cell development. 0A~P levels are a key determinant of cell differentiation. Intermediate levels of 0A~P drive the transition of motile cells to chains of cells also expressing matrix genes and shut down motility genes indirectly through the SinI-SinR-SlrR epigenetic switch [24]. High levels of 0A~P activate hundreds of genes involved in sporulation, thus leading some matrix producers to become sporulating cells while simultaneously turning off matrix production. (I) The regulatory region of sinI contains both an activator and multiple operators of 0A~P, whose sequences imperfectly match the consensus 0A~P binding site. This allows both activation of sinI by intermediate levels of 0A~P and repression of sinI when the 0A~P concentration reaches high levels [35].