SpaK/SpaR two-component system characterized by a structure-driven domain-fusion method and in vitro phosphorylation studies.

Here we introduce a quantitative structure-driven computational domain-fusion method, which we used to predict the structures of proteins believed to be involved in regulation of the subtilin pathway in Bacillus subtilis, and used to predict a protein-protein complex formed by interaction between the proteins. Homology modeling of SpaK and SpaR yielded preliminary structural models based on a best template for SpaK comprising a dimer of a histidine kinase, and for SpaR a response regulator protein. Our LGA code was used to identify multi-domain proteins with structure homology to both modeled structures, yielding a set of domain-fusion templates then used to model a hypothetical SpaK/SpaR complex. The models were used to identify putative functional residues and residues at the protein-protein interface, and bioinformatics was used to compare functionally and structurally relevant residues in corresponding positions among proteins with structural homology to the templates. Models of the complex were evaluated in light of known properties of the functional residues within two-component systems involving His-Asp phosphorelays. Based on this analysis, a phosphotransferase complexed with a beryllofluoride was selected as the optimal template for modeling a SpaK/SpaR complex conformation. In vitro phosphorylation studies performed using wild type and site-directed SpaK mutant proteins validated the predictions derived from application of the structure-driven domain-fusion method: SpaK was phosphorylated in the presence of (32)P-ATP and the phosphate moiety was subsequently transferred to SpaR, supporting the hypothesis that SpaK and SpaR function as sensor and response regulator, respectively, in a two-component signal transduction system, and furthermore suggesting that the structure-driven domain-fusion approach correctly predicted a physical interaction between SpaK and SpaR. Our domain-fusion algorithm leverages quantitative structure information and provides a tool for generation of hypotheses regarding protein function, which can then be tested using empirical methods

Production of membrane histidine kinases and structural studies on Thiosulfate dehydrogenase

This thesis is organized in three chapters. Chapter 1 comprises a brief introduction on the methodology used throughout the work herein presented. Chapter 2 describes the production of membrane histidine kinases from Staphylococcus aureus and Clostridium difficile and chapter 3 the structural studies of a thiosulfate dehydrogenase from Campylobacter jejuni. The experimental work was performed in the Membrane Protein Crystallography Laboratory at Instituto de Tecnologia Química e Biológica, and also included a visit to SOLEIL synchrotron, Paris, France, to collect X-ray diffraction data

The LiaFSR three-component System of Bacillus subtilis

Soil bacteria are exposed to constant changes in temperature, moisture, and oxygen content. Additionally, they have to encounter different antimicrobial substances, which are produced by competing bacteria. Those agents often target the bacterial cell envelope, which is an essential structure composed of the cell wall and cell membrane. In order to counteract such life-threatening conditions, bacteria developed signal transducing systems to monitor their environment and to respond signal-specifically to any stress conditions, mostly by differential gene expression. Different principles of signal transducing systems have been evolved: one-component systems (1CSs), two-component systems (2CSs), and extracytoplasmic function (ECF) sigma factors. Bacillus subtilis is a soil bacterium, which counteracts cell envelope stress by four different 2CSs (LiaSR, BceRS, PsdRS, and YxdJK) and at least three different ECF sigma factors (σX, σM, and σW). In the course of the present thesis, the LiaSR 2CS was investigated in detail. The LiaSR 2CS of B. subtilis is a cell envelope stress-sensing system that shows a high dynamic range of induction in response to cell wall antibiotics like bacitracin. It provides no resistance against its inducer molecules. Rather, it is a damage-sensing system that maintains the cell envelope integrity under stress conditions. The membrane-anchored histidine kinase (HK) LiaS and its cognate response regulator (RR) LiaR work together with a third protein, LiaF, which was identified as the inhibitor of the 2CS. Upon induction, the target promoter PliaI is induced by phosphorylated LiaR, leading to the expression of the liaIH-liaGFSR locus, with liaIH as being the most induced genes. In the first part of this thesis, the mechanisms of stimulus perception and signal transduction of the LiaFSR system were analyzed. Therefore, the native stoichiometry of the proteins LiaF, LiaS, and LiaR were determined genetically and biochemically with a resulting ratio of 18 to 4 to 1. We found out that maintaining this specific stoichiometry is crucial for the functionality of the LiaFSR system and thus a proper response to cell envelope stress. Changing the relative protein ratios by the overproduction of either LiaS or LiaR leads to a constitutive activation of the promoter PliaI. These data suggest a non-robust behavior of the LiaFSR system regarding perturbations of its stoichiometry, which stands in contrast to quantitative analyses of other well-known 2CSs. Furthermore, a HK-independent phosphorylation of the RR LiaR was observed. This happened in each case if the amount of LiaR exceeded those of LiaS, irrespective of the presence or absence of a stimulus. By using growth media supplied with different carbon sources, acetyl phosphate was identified as being the phosphoryl group-donor for LiaR under these conditions. Moreover, by performing a mutagenesis experiment, we obtained genetic evidence that LiaS is a bifunctional HK offering both a kinase and a phosphatase activity. In the second part of this thesis, the liaI promoter was used to generate a protein expression toolbox for the use in B. subtilis, referred to as the LIKE (from the German “Lia-kontrollierte Expression”) system. PliaI is a perfect candidate for driving recombinant protein expression. It is tightly regulated under non-inducing conditions showing no significant basal expression levels. Depending on the inducer molecule concentration, it is induced up to 1000-fold reaching a maximum already 30 minutes after addition of the inducer. Two expression vectors, an integrative and a replicative one, were constructed consisting of an alternative liaI promoter, which was optimized to enhance promoter strength. Additionally, different B. subtilis expression hosts were generated that possess liaIH deletions to prevent undesired protein production. The feasibility of the LIKE system was evaluated by using gfp and ydfG as reporter genes and bacitracin as inducer molecule. As a result, both proteins were successfully overproduced