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

The LIKE system, a novel protein expression toolbox for Bacillus subtilis based on the liaI promoter

Background: Bacillus subtilis is a very important Gram-positive model organism of high biotechnological relevance, which is widely used as a host for the production of both secreted and cytoplasmic proteins. We developed a novel and efficient expression system, based on the liaI promoter (P-liaI) from B. subtilis, which is under control of the LiaRS antibiotic-inducible two-component system. In the absence of a stimulus, this promoter is kept tightly inactive. Upon induction by cell wall antibiotics, it shows an over 100-fold increase in activity within 10 min. Results: Based on these traits of P-liaI, we developed a novel LiaRS-controlled gene expression system for B. subtilis (the "LIKE" system). Two expression vectors, the integrative pLIKE-int and the replicative pLIKE-rep, were constructed. To enhance the performance of the P-liaI-derived system, site-directed mutagenesis was employed to optimize the ribosome binding site and alter its spacing to the initiation codon used for the translational fusion. The impact of these genetic modifications on protein production yield was measured using GFP as a model protein. Moreover, a number of tailored B. subtilis expression strains containing different markerless chromosomal deletions of the liaIH region were constructed to circumvent undesired protein production, enhance the positive autoregulation of the LiaRS system and thereby increase target gene expression strength from the P-liaI promoter. Conclusions: The LIKE protein expression system is a novel protein expression system, which offers a number of advantages over existing systems. Its major advantages are (i) a tightly switched-off promoter during exponential growth in the absence of a stimulus, (ii) a concentration-dependent activation of P-liaI in the presence of suitable inducers, (iii) a very fast but transient response with a very high dynamic range of over 100-fold (up to 1,000-fold) induction, (iv) a choice from a range of well-defined, commercially available, and affordable inducers and (v) the convenient conversion of LIKE-derived inducible expression strains into strong constitutive protein production factories

The LIKE system, a novel protein expression toolbox for <it>Bacillus subtilis</it> based on the <it>liaI</it> promoter

Abstract Background Bacillus subtilis is a very important Gram-positive model organism of high biotechnological relevance, which is widely used as a host for the production of both secreted and cytoplasmic proteins. We developed a novel and efficient expression system, based on the liaI promoter (PliaI) from B. subtilis, which is under control of the LiaRS antibiotic-inducible two-component system. In the absence of a stimulus, this promoter is kept tightly inactive. Upon induction by cell wall antibiotics, it shows an over 100-fold increase in activity within 10 min. Results Based on these traits of PliaI, we developed a novel LiaRS-controlled gene expression system for B. subtilis (the “LIKE" system). Two expression vectors, the integrative pLIKE-int and the replicative pLIKE-rep, were constructed. To enhance the performance of the PliaI-derived system, site-directed mutagenesis was employed to optimize the ribosome binding site and alter its spacing to the initiation codon used for the translational fusion. The impact of these genetic modifications on protein production yield was measured using GFP as a model protein. Moreover, a number of tailored B. subtilis expression strains containing different markerless chromosomal deletions of the liaIH region were constructed to circumvent undesired protein production, enhance the positive autoregulation of the LiaRS system and thereby increase target gene expression strength from the PliaI promoter. Conclusions The LIKE protein expression system is a novel protein expression system, which offers a number of advantages over existing systems. Its major advantages are (i) a tightly switched-off promoter during exponential growth in the absence of a stimulus, (ii) a concentration-dependent activation of PliaI in the presence of suitable inducers, (iii) a very fast but transient response with a very high dynamic range of over 100-fold (up to 1,000-fold) induction, (iv) a choice from a range of well-defined, commercially available, and affordable inducers and (v) the convenient conversion of LIKE-derived inducible expression strains into strong constitutive protein production factories.</p

der Ludwig‐Maximilians‐Universität München vorgelegt von

Hiermit versichere ich an Eides statt, dass die vorliegende Dissertation von mir selbständig und ohne unerlaubte Hilfe angefertigt wurde. Zudem wurden keine anderen als die angegebenen Quellen verwendet. Außerdem versichere ich, dass die Dissertation keiner anderen Prüfungskommission vorgelegt wurde und ich mich nicht anderweitig einer Doktorprüfung ohne Erfolg unterzoge