Selective Pressure for Biofilm Formation in Bacillus subtilis: Differential Effect of Mutations in the Master Regulator SinR on Bistability.

Biofilm formation by requires the expression of genes encoding enzymes for extracellular polysaccharide synthesis and for an amyloid-like protein. The master regulator SinR represses all the corresponding genes, and repression of these key biofilm genes is lifted when SinR interacts with its cognate antagonist proteins. The YmdB phosphodiesterase is a recently discovered factor that is involved in the control of SinR activity: cells lacking YmdB exhibit hyperactive SinR and are unable to relieve the repression of the biofilm genes. In this study, we have examined the dynamics of gene expression patterns in wild-type and mutant cells by microfluidic analysis coupled to time-lapse microscopy. Our results confirm the bistable expression pattern for motility and biofilm genes in the wild-type strain and the loss of biofilm gene expression in the mutant. Moreover, we demonstrated dynamic behavior in subpopulations of the wild-type strain that is characterized by switches in sets of the expressed genes. In order to gain further insights into the role of YmdB, we isolated a set of spontaneous suppressor mutants derived from mutants that had regained the ability to form complex colonies and biofilms. Interestingly, all of the mutations affected SinR. In some mutants, large genomic regions encompassing were deleted, whereas others had alleles encoding SinR variants. Functional and biochemical studies with these SinR variants revealed how these proteins allowed biofilm gene expression in the mutant strains. Many bacteria are able to choose between two mutually exclusive lifestyles: biofilm formation and motility. In the model bacterium , this choice is made by each individual cell rather than at the population level. The transcriptional repressor SinR is the master regulator in this decision-making process. The regulation of SinR activity involves complex control of its own expression and of its interaction with antagonist proteins. We show that the YmdB phosphodiesterase is required to allow the expression of SinR-repressed genes in a subpopulation of cells and that such subpopulations can switch between different SinR activity states. Suppressor analyses revealed that mutants readily acquire mutations affecting SinR, thus restoring biofilm formation. These findings suggest that cells experience selective pressure to form the extracellular matrix that is characteristic of biofilms and that YmdB is required for the homeostasis of SinR and/or its antagonists.