PROTEIN-PROTEIN INTERACTIONS DURING EARLY SPORULATION EVENTS IN BACILLUS SUBTILIS
K. Šurdová, Z. Chromíková, I. Barák
Institute of Molecular Biology, Slovak Academy of Sciences, 845 51 Bratislava
Sporulation is an excellent example of prokaryotic differentiation. During the sporulation, B. subtilis goes through asymmetric division. Thus, bacterium gives rise to two diverse cells with different fates: the smaller prespore which develops into highly resistant spore, and the larger ‘mother’ cell which is predetermined to abolish. Different gene expression profiles are provided by sequential activation of compartment-specific sigma factors.
sF is the first compartment-specific sigma factor. However, it is expressed well before the asymmetric septation, but becomes active only in the prespore . Being the first one in a cascade of compartment-specific s factors, the activation of sF is crucial for normal progression in sporulation. Proteins participating in the spatial and temporal control of sF activity are also expressed in the predivisional sporangium and include SpoIIE, SpoIIAA and SpoIIAB. SpoIIAB is an anti-sigma factor that interacts with sF and retains it in an inactive form . This inhibition is perturbed by SpoIIAA protein. When phosphorylated, SpoIIAA is not able to interact with SpoIIAB. To release sF from an inhibitory complex, SpoIIE phosphatase dephosphorylates SpoIIAA~P which, in turn, activates sF [3,4].
SpoIIE is a transmembrane protein with dual function. Its phosphatase activity accounts for sF activation, while the central domain of SpoIIE is involved in asymmetric septum formation . This is accomplished via the interaction with its binding partner FtsZ, which is the most important underlying protein for cell division and forms tubulin-like protofilaments. SpoIIE switches on a cascade of reactions that permit cell to continue in sporulation. This activity is restricted purely to the prespore compartment by its phosphatase function. Being a key regulatory protein, the question is, how the action of SpoIIE is regulated.
Contradictory results account for not unified theory of sF activation and the mechanism of its timing and spatial control. Many biochemical studies have been executed, but there still remain uncertainties.
For elucidation, two approaches are to be employed to study protein-protein interactions: AFM and SPR. Both procedures are preceded by the isolation of proteins, using heterologous E. coli expression systems and a variety of purification steps. Thus, purification of proteins was our first principal goal. We also report the improvements in SpoIIE isolation and purification steps that emerge to be beneficial not only for biochemical but also for crystallographic studies.
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The work in author’s laboratory is supported by grant 2/1004/21 from the Slovak Academy of Sciences and by grant APVT-51-027804 from Ministry of Education of Slovak Republic.