CRYSTALLIZATION OF KILLER PROTEIN SPOIISA AND ITS ANTIDOTE SPOIISB FROM BACILLUS SUBTILIS
Patrik Florek1, Katarína Muchová1, Richard J. Lewis2 and Imrich Barák1*
2Laboratory of Molecular Biophysics, The Rex Richard Building, University of Oxford, South Parks Road, Oxford OX1 3QU, UK
The B. subtilis spoIISA gene encodes a 248-residue protein containing three predicted transmembrane domains  with the last two-thirds of protein being located in the cytoplasm. The spoIISB gene codes for a hydrophilic 56-residue protein. None of these proteins shares any sequentional similarity to a protein of known function, providing no clue to their function and evolutionary origin. The spoIISB translation start codon overlaps the spoIISA translation stop codon what is a strong indication that the two genes constitute an operon .
A null mutation in spoIISB leads to the strong sporulation defect, whereas disruption of either spoIISA or whole spoIIS locus has no effect on sporulation. Altogether, this facts indicates that a) SpoIISA prevents normal progression of the sporulation process; b)SpoIISB neutralizes the action of SpoIISA; and c) spoIIS locus does not play essential role in sporulation process. The strain carrying the spoIISB null allele does not exhibit any obvious defect during exponential growth. This immunity of exponentially growing cells to the absence of SpoIISB most likely reflects the existence of a threshold concentration below which SpoIISA does not significantly impair cell viability, since the induced expression of additional spoIISA gene copy led to rapid drop in optipcal density of exponential phase cell .
Since it has structural features of an integral membrane protein, SpoIISA could act as a holin and allow some endolysin to gain access to the peptidoglycan . Local solubilization of the cell wall would lead to membrane disruption and consequently to the large plasmolysis zones which were observed by electron microscopy . However, SpoIISA does not show any similarity to known holins and is significantly larger than holins identified so far . It is therefore quite possible that the cytoplasmic membrane itself is the target of the toxic action of SpoIISA.
In our work we over-expressed cytosolic part of SpoIISA His-tag fusion protein together with intact SpoIISB protein in Escherichia coli. The both proteins were purified using single step metal chelate affinity chromatography, and therefore isolated proteins formed stable complex, which indicates their specific interactions. The gel filtration and electrophoresis experiments showed that the most abundant form of the complex is oligomer consisting of two SpoIISA and two SpoIISB molecules. This observation confirms the results gain using genetic complementation experiments, which predicted that SpoIIA acts as an oligomer . The purified SpoIISA-SpoIISB protein complex was used for crystallization trials.
Work in authors laboratories is supported by grant 2/1004/22 from the Slovak Academy of Sciences and Wellcome Trust Project and Collaborative Research Initiative Grants (056247/Z/98/Z and 066732/Z/01/Z, respectively).
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