Control of bistable gene expression in Bacillus subtilis by the transcription factor SinR and the phosphodiesterase YmdB

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Cells of the Gram-positive soil bacterium Bacillus subtilis are capable of making a choice between different lifestyles, such as the explorative lifestyle that is characterized by motility, the sessile lifestyle (biofilm formation), the expression of genetic competence or sporulation. While the latter lifestyles are characteristic for post-exponential cells, growing cell have to choose between motility and biofilm formation; and the two lifestyles are mutually exclusive. The SinR protein is a transcription factor that controls both lifestyles as a master regulator. Its activity is controlled by inhibitory interactions with its antagonists SinI and SlrR, the expression of SlrR in turn is repressed in a negative feedback loop by SinR. This regulatory system meets the criteria for a bistable switch. We have discovered that a novel phosphodiesterase, YmdB, is required for bistable gene expression and heterogeneity within the population of B. subtilis. With this project, we want to unravel the molecular link between the YmdB and bistable gene expression of biofilm and motility genes in B. subtilis. The project is based on the following hypothesis: The results we have so far are compatible with the idea that YmdB is a phosphodiesterase with an RNase activity. One of the substrates of YmdB activity must be implicated in the translation or stability of the SinR protein. The increased amounts of SinR in the ymdB mutant result in an imbalance in the concentrations of SinR and its antagonists, SinI and SlrR. This is accompanied by permanent repression of biofilm genes and constitutive expression of motility genes of the SigD regulon. To study the effect of YmdB on the bistable switch that is made up of SinR and its antagonist proteins we will determine the absolute quantities of these proteins in populations and in specific sub-populations that have been sorted according to their mode of biofilm and motility gene expression. Moreover, we will study a SinR mutant protein that restores bistable gene expression to the ymdB mutant by biochemical and structural analysis. The data obtained from both approaches will feed into the modelling of the bistable switch. To study the molecular mechanism by which YmdB affects bistable gene expression, we will first determine the stabilities of the SinR protein and of the sinR mRNA in the wild type and the ymdB mutant. Moreover, RNA-Seq data will be evaluated to get insights into the target of YmdB at single nucleotide resolution. From the RNA-Seq and the proteome analysis, we expect to be able to identify the direct target of YmdB. Finally, we aim at studying the molecular details of the interaction between YmdB and its target molecule. We are confident that the proposed program will allow us to identify the molecular mechanism for the implication of YmdB in phenotypic heterogeneity and in the switch between biofilm formation and motility in B. subtilis.