Das DegS-DegU Zwei-Komponenten System und dessen Rolle bei der Regulation des osmotisch induzierbaren yqiHIK Operons aus Bacillus subtilis

Water availability is essential for the development of living cells. In the upper layers of soil, the natural habitat of the gram-positive soil bacterium B. subtilis, it is faced to various stresses that have an influence on the availability of water. Long dry periods lead to a hyperosmotic stress that finally could cause plasmolysis. To avoid this loss of water, B. subtilis is able to accumulate compatible solutes by either transporting them or synthesizing them and to protect itself from the negative impacts of high osmolality/salinity. In terms of the osmoadaptation the big question is how B. subtilis is able to sense osmotic stress. Two component systems (TCS) play a crucial role in sensing environmental stresses. Transcriptomic data of osmotically stressed B. subtilis cells revealed that only the DegS-DegU TCS is salt inducible (299). This system controls different processes which are characteristic for the transition from the exponential to the stationary phase and for cellular differentiation procedures. The DegS-DegU TCS was identified to be salt inducible, however the exact mechanism how it reacts to osmotic stress remained unclear (299). In the course of my study reporter gene studies confirmed the salt induction of the degSU operon. For the first time the positive feedback loop of DegU~P on its own promotor (degUP3) under salt stress conditions was shown. The expression of degUP3 is only activated upon high salt concentrations (≥ 0.8 M NaCl). Reporter gene studies of two proteins (RapG and PhrG) that regulate the TCS (236), were shown to be salt inducible. Phenotypic characterisations highlighted the role of the DegS-DegU TCS during the growth of B. subtilis. The deletion of the DegS-DegU TCS lead to a slightly longer lag phase under hyperosmotic conditions. Interestingly the hyperphosphorylation of DegU leads to a strong enhancement of growth under physiological and hyperosmotic conditions. This phenotype is not due to the de novo synthesis of the compatible solute proline. To analyse a putative role of DegS-DegU in the adaptation to high salinity, genes which are regulated by DegU~P were analysed in terms of their salt induction. Here I could show that half of the DegU~P regulon (197) is salt inducible, but this induction is independent of the DegS-DegU TCS. This was schown by reporter gene analysis of genes which belong both to the DegU~P regulon as well as to the salt modulon. These genes were still salt inducible, although the DegS-DegU TCS was deleted. In conjunction with this, the recently identified type VII secretion system (T7SS, yukE-yueD) was analysed in detail. Deletion of the T7SS revealed no phenotype under hyperosmotic conditions in B. subtilis 168 laboratory strain. Reporter gene analysis showed that the T7SS is salt inducible and regulated by the DegS-DegU TCS. Under physiological conditions DegU~P acts a transcriptional activator, but under hyperosmotic conditions DegU~P seems to be a repressor. Furthermore, the involvement of the biofilm regulators SinR, a repressor, and RemA, an activator, was proven. The main focus of my studies was the analysis of the regulation of the osmotically inducible yqiHIK operon and its regulation by the DegS-DegU two component system. The yqiHIK operon, which was shown to be highly salt inducible in different transcriptomic data (172, 230, 299), is additionally regulated by the DegS-DegU TCS (86). Reporter gene and Northern blot analysis showed that yqiHIK is transcribed in one operon, which is salt inducible. Furthermore, the analysis highlighted the role of DegU~P as a transcriptional activator. Truncations of a far upstream AT-rich region and site directed mutagenesis of putative DegU~P binding sites, highlighted a DegU~P binding site at a distance of 203 bp from the transcriptional start site. The N-acetylmuramoyl–L-alanine amidase YqiI was localized by Western blot analysis in the extracellular space and the glycerophosphodiester phosphodiesterase YqiK in the cytoplasm. This results enables me to suggest a physiological function for the yqiHIK encoded proteins. The characteristics of the yqiHIK operon were further used as a device to analyse the role of the DegS-DegU TCS during the osmoadaptation. The promoter activity of the SigA-type promoter of yqiHIK was increased by site directed mutagenesis and showed a salt induction which is independent of the TCS DegS-DegU. Additionally the yqiHIK operon was still salt inducible, although the putative osmosensor was membrane bound instead of cytoplasmically localized by a synthetically produced chimeric enzyme (KinC-DegS). This chimeric enzyme does not respond to osmotic stresses (193). Contrary to a prior hypothesis (279), I could show that DegS is not the globally-acting salt sensor in B. subtilis. The DegS-DegU TCS however contributes to the transcriptional control of a multitude of salt stress regulated genes in B. subtilis.