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.