Stoichiometry and perturbation studies of the LiaFSR system of Bacillus subtilis.

The response regulator/histidine kinase pair LiaRS of Bacillus subtilis, together with its membrane-bound inhibitor protein LiaF, constitutes an envelope stress-sensing module that is conserved in Firmicutes bacteria. LiaR positively autoregulates the expression of the liaIH-liaGFSR operon from a strictly LiaR-dependent promoter (P(liaI) ). A comprehensive perturbation analysis revealed that the functionality of the LiaFSR system is very susceptible to alterations of its protein composition and amounts. A genetic analysis indicates a LiaF:LiaS:LiaR ratio of 18:4:1. An excess of LiaS over LiaR was subsequently verified by quantitative Western analysis. This stoichiometry, which is crucial to maintain a functional Lia system, differs from any other two-component system studied to date, in which the response regulator is present in excess over the histidine kinase. Moreover, we demonstrate that LiaS is a bifunctional histidine kinase that acts as a phosphatase on LiaR in the absence of a suitable stimulus. An increased amount of LiaR - both in the presence and in the absence of LiaS - leads to a strong induction of P(liaI) activity due to phosphorylation of the response regulator by acetyl phosphate. Our data demonstrate that LiaRS, in contrast to other two-component systems, is non-robust with regard to perturbations of its stoichiometry.

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 (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.