Insights into the biofilm formation of Bacillus subtilis

Bacillus subtilis forms highly structured biofilms to protect itself against harmful environments, and to have an advantage in the competition with other species. The cells of B. subtilis that secrete the biofilm matrix, express genes involved in synthesis of exopolysaccharides (EPS) and the protein components of the matrix. The regulation of the transition from matrix production to motility is governed by the epigenetic SlrR-SinR switch. The deletion of the phosphodiesterase YmdB leads to total lack of biofilm formation, the repression of the SinR regulon, and to enhanced expression of the σD regulon. However, the molecular mechanisms by which YmdB is involved in the regulation of these developmental states still need to be elucidated, which was the aim of this thesis. We used different approaches to characterize the function of YmdB. We could show that the deletion of ymdB results in increased amounts of the master regulator SinR in the cell, leading to permanent repression of matrix genes. In addition, we investigated possible mechanisms by which YmdB could regulate SinR amounts in the cell. YmdB might function via impeding the access of the ribosome to its initiation site of the sinR transcript. Another approach to characterize the function of YmdB, was the identification of RNA interaction partners of the potential RNA-binding protein. Genes encoding for the potential interacting RNAs were tested for their impact on biofilm formation. The selected targets differently affected the biofilm formation but did not restore biofilm formation in the ymdB mutant nor did they result in loss of biofilm formation in the wild type. YmdB probably does not act via interaction with one specific RNA. Furthermore, the ymdB mutant forms quickly suppressor mutants, which harbor mainly mutations in SinR. We biochemically characterized several mutations for their impact on DNA- and protein interaction as well as on oligomerization state of SinR. Hereby, we could underline the importance of different residues of the protein for DNA binding, interaction with its antagonist SinI and the formation of the SinR tetramer. Aside from that, we documented the dynamics of gene expression patterns in wild type and ymdB mutant cells by microfluidic analysis coupled to time-lapse fluorescence microscopy. Our results confirm the bistable character for motility and matrix genes expression, as well as the quick introduction of suppressor mutations in the ymdB mutant, restoring matrix gene expression. Additionally, we analyzed the effect of the deletion of the RNA-binding protein SpoVG on biofilm formation and could detect an extended spreading of the macrocolony especially in combination with the deletion of the master regulator of biofilm formation, SinR. Additionally, SpoVG interacts with many RNAs, which indicates that SpoVG might has a global function as RNA-binding protein in B. subtilis. This work focused on the regulation and characterization of biofilm formation. We could specify the effect of YmdB on the homeostasis of the epigenetic SlrR-SinR switch for motility and biofilm formation, as well as the role of SpoVG as RNA-binding protein in B. subtilis.