SpoIIS components of Bacillus subtilis programmed cell death
Pamela Pavelčíková, Patrik Florek, Katarína
Muchová, and Imrich Barák
Institute of Molecular Biology, Slovak Academy of Science, 845 51 Bratislava, Slovakia
The spoIIS locus from Bacillus subtilis consists of two genes, spoIISA and spoIISB. The spoIISA gene encodes a 248-residue protein. It contains three transmembrane helices and the last two-thirds of the protein form negatively charged domain, localized in the cytoplasm. The SpoIISB is a small, 56-residue, basic, hydrophilic protein.
The spoIISB translation start codon overlaps the spoIISA translation stop codon , which together with our findings indicates, that these two genes constitute an operon.
The spoIIS locus is organized in a similar fashion to the addiction modules, which were found in many prokaryotic organisms and through these systems the programmed cell death is frequently mediated in bacteria .
Addiction modules consist of two genes, a labile antitoxin and a stable toxin. The product of the antitoxic protein antagonizes the toxic effect of the latter .
In contrast to spoIISA mutant, which efficiently sporulates, a null mutation of spoIISB leads to a sporulation block short after formation of asymmetric septum. Disruption of the whole spoIIS locus has also no effect on sporulation. When induced during vegetative growth in B. subtilis, expression of SpoIISA is toxic and causes cell lysis. So this revealed that SpoIISB protein neutralizes the toxic effect of SpoIISA .
In this work we showed that SpoIISA has a toxic effect also in Escherichia coli cells, when is present without SpoIISB. SpoIISA is responsible for the cell lysis during vegetative growth. In this system we tested different parts of the SpoIISA protein and it was found, that no just transmembrane or cytosolic domain, but only the whole SpoIISA is required for the toxicity. The effect of SpoIISA protein is then bactericidal, rather than bacteriostatic. We observed very quick decrease of amount of colony forming units in the cell population carrying sole spoIISA gene in comparison with cell population carrying spoIISA and spoIISB genes.
The aim of our research is to solve the structure of the complex formed by cytosolic part of SpoIISA together with SpoIISB protein. As we found, one copy of each spoIIS gene leads to expression a very heterogenic protein complexes caused by an abundance of SpoIISA. Thus, we constructed pET-Duet vector derivative containing one copy of the gene corresponding to His6-tag fused cytosolic part of SpoIISA with two copies of intact spoIISB. After expression we isolated and purified it using Ni2+ chelate affinity chromatography gaining stable homogenous protein complex, which was used for crystallization trials. In a few screening conditions we obtained promising crystals.
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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.