Phosphorylated DegU Manipulates Cell Fate Differentiation in the Bacillus subtilis Biofilm

Cell differentiation is ubiquitous and facilitates division of labor and development. Bacteria are capable of multicellular behaviors that benefit the bacterial community as a whole. A striking example of bacterial differentiation occurs throughout the formation of a biofilm. During Bacillus subtilis biofilm formation, a subpopulation of cells differentiates into a specialized population that synthesizes the exopolysaccharide and the TasA amyloid components of the extracellular matrix. The differentiation process is indirectly controlled by the transcription factor Spo0A that facilitates transcription of the eps and tapA (tasA) operons. DegU is a transcription factor involved in regulating biofilm formation. Here, using a combination of genetics and live single-cell cytological techniques, we define the mechanism of biofilm inhibition at high levels of phosphorylated DegU (DegU∼P) by showing that transcription from the eps and tapA promoter regions is inhibited. Data demonstrating that this is not a direct regulatory event are presented. We demonstrate that DegU∼P controls the frequency with which cells activate transcription from the operons needed for matrix biosynthesis in favor of an off state. Subsequent experimental analysis led us to conclude that DegU∼P functions to increase the level of Spo0A∼P, driving cell fate differentiation toward the terminal developmental process of sporulation.

BslA is a self-assembling bacterial hydrophobin that coats the Bacillus subtilis biofilm

Biofilms represent the predominant mode of microbial growth in the natural environment. Bacillus subtilis is a ubiquitous Gram-positive soil bacterium that functions as an effective plant growth-promoting agent. The biofilm matrix is composed of an exopolysaccharide and an amyloid fiber-forming protein, TasA, and assembles with the aid of a small secreted protein, BslA. Here we show that natively synthesized and secreted BslA forms surface layers around the biofilm. Biophysical analysis demonstrates that BslA can self-assemble at interfaces, forming an elastic film. Molecular function is revealed from analysis of the crystal structure of BslA, which consists of an Ig-type fold with the addition of an unusual, extremely hydrophobic "cap" region. A combination of in vivo biofilm formation and in vitro biophysical analysis demonstrates that the central hydrophobic residues of the cap are essential to allow a hydrophobic, nonwetting biofilm to form as they control the surface activity of the BslA protein. The hydrophobic cap exhibits physiochemical properties remarkably similar to the hydrophobic surface found in fungal hydrophobins; thus, BslA is a structurally defined bacterial hydrophobin. We suggest that biofilms formed by other species of bacteria may have evolved similar mechanisms to provide protection to the resident bacterial community.