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The microorganism Bacillus subtilis lives in a challenging habitat, i.e. the upper layers of the soil. Due to its ability to differentiate between several developmental programs (e.g. Biofilm formation, sporulation) the organism is able to adapt to various environmental stress conditions as well as nutrient limitation. Water availability is a crucial factor for all microorganisms. A key feature during the acclimatization process of salt stresses B. subtilis cells is the accumulation of large amounts of compatible solutes. Based on a genome-wide profiling study of osmotically stressed cells a plenty of salt induced genes could be identified. However, the majority of these gene products are uncharacterized so far.
Interestingly, among the osmotically induced genes there are several cell wall modifying proteins. Members of this group of osmotically induced genes are for example the genes yqiH and yqiI. The yqiH gene codes for a putative lipoprotein of unknown function whereas the gene yqiI is predicted to code for an N-acetylmuramoyl-L-alanine amidase. These enzymes belong to the group of cell wall hydrolases (autolysins) which are involved in several important cellular processes. The hydrolytic activity of the YqiI protein was confirmed in this study by the in vitro zymogram technique.
Furthermore, drastic but transient, changes in the morphology of salt challenged B. subtilis cells could be observed during the acclimatization process. In sum, the transcription profiling study as well as the cross morphological deformations lets me hypothesize that modifications of the cell wall could be a new and so far unexplored facet in the osmo adaptation process in B. subtilis. To address a possible impact of the encoded YqiH and YqiI proteins on this process, a physiological characterization as well as a genetic analysis of the transcription of the yqiHIK operon was performed in this PhD thesis.
RT-PCR based analyses revealed a co-transcription of the yqiHI genes with a third gene, yqiK. The analysis of a yqiHIK deletion mutant showed a delayed growth phenotype under normal and hyper osmotic growth conditions. This growth defect was exclusively due to the lack of the amidase YqiI. In addition, I could show that the YqiHIK proteins were not critically involved in the reshaping of the cell wall under hyperosmotic conditions. Reporter gene studies revealed an expression of the yqiHIK gene cluster only when the B. subtilis cell was exposed to a significant level of osmotic stress (> 0.7 M NaCl). Primer extension analyses lead to the identification of a SigA-type promoter that is responsible for osmotic induced transcription of the yqiHIK gene cluster which was furthermore subject of a detailed mutagenesis study. A deletion analysis of the yqiHIK regulatory region was performed that showed an AT-rich DNA segment that is critical for activity and osmotic regulation of the yqiHIK operon. Additionally, the AT-rich region was identified as a target for the response regulator DegU from B. subtilis. As a key aspect of this thesis, it could be demonstrated that the two component system DegS/DegU is crucial for the regulation of the expression of the yqiHIK genes at high salinity growth conditions. This result pointed to, how the B. subtilis cell is able to recognize differences in the external NaCl concentration.
Beside the salt stress induction of the yqiHIK operon, two additional regulatory aspects of the yqiHIK gene cluster were discovered. On the one hand it was found that the expression of this transcriptional unit was initiated directly after the cells have entered the sporulation pathway. The SinR protein contributes to this regulatory facet and is also involved in preventing the yqiHIK expression under normal physiological growth conditions. On the other hand an increase of the yqiHIK transcription could be observed in the so called “death zone”, after the cells have passed through the stationary growth phase. An involvement of the SigA promoter mediating osmotic control of the yqiHIK expression could be excluded with respect to both of the above described regulatory aspects.
In conclusion, the YqiHIK proteins seem to be involved in three different physiological processes: (1) Adaptation to hyperosmotic growth conditions, (2) sporulation and (3) peptidoglycan recycling. The expression of the yqiHIK operon is at least regulated by two differently controlled promoters. Furthermore, two transcriptional regulators could be discovered that manage the expression of the yqiHIK operon. The complex pattern of the transcription of the yqiHIK gene cluster is an informative example of how bacteria can recognize changes in their environment and adjust to these changes by selectively triggering gene expression.