Functional and biochemical analysis of truncated SpoIISA toxin in Bacillus subtilis


J. Makroczyová1, P. Florek1, S. Rešetárová1, A. J. Wilkinson2 and I. Barák1


1Institute of Molecular Biology, Slovak Academy of Sciences, Dúbravská cesta 21, 845 51 Bratislava, Slovakia

2Department of Chemistry, University of York, Heslington, York YO1 5OD, UK


Toxin-antitoxin were found to be encoded on vast number of bacterial plasmids and genomes and are thought to be associated with the maintenance of extrachromosomal DNA, bacteriostasis and possibly altruistic cell death. Chromosomally encoded SpoIISA-SpoIISB system appears to define a new family of toxin-antitoxin modules that is conserved among Bacilli.

SpoIISA proteic toxin from B. subtilis was shown to affect cell mebrane of either mother cell during sporulation process or, when artificially over-expressed in vegetatively growing cells. This 248-amino residue protein was predicted to be composed of two domains – a membrane spanning domain consisting of three helices and a negatively charged cytosolic domain (CSpoIISA), which binds small, positively charged SpoIISB protein [1].

Recently solved crystal structure of CSpoIISA2:SpoIISB2 heterotetrameric complex [2] revealed that the toxin portion of the complex constitutes so called GAF (cGMP phospho- diesterase, adenylate cyclase, FhlA) domain, a fold which is widespread in proteins of diverse function including cell signalling proteins, transcription factors and proteins involved in light detection in bacteria, fungi and plants [3]. The GAF domain has three tiers, a basal layer of two or more α-helices, a middle tier of four or more strands that form a mixed β-pleated sheet and a distal layer of more variable structure made up of segments connecting the strands of the β-sheet. Many GAF domains are dimers formed by the packing of α-helices from the basal layer to form a four helix bundle. In case of SpoIISA, the two protomers of its cytosolic domain form extensive hydrophobic interactions through an intermolecular 4-helix bundle formed by the first and the last CSpoIISA alpha helices [2].

Since alternative prediction of the SpoIISA topology suggested that the last CSpoIISA α-helix might swap from intermolecular interface and become incorporated into cytoplasmic membrane, in this work we assessed the toxicity of the C-terminally truncated SpoIISA lacking the last alpha helix and the ability of SpoIISB antitoxin to neutralize the eventual lethal activity in B. subtilis cells. Moreover, we examined the effect of this C-terminal SpoIISA deletion on the CSpoIISA dimerization and the CSpoIISA-SpoIISB complex formation in vitro.

1.  E. Adler, I. Barák, P. Stragier, J. Bacteriol., 183, (2001), 3574.

2.   P. Florek, V. M. Levdikov, E. Blagova, A. A. Lebedev, R. Škrabana, S. Rešetárová, P. Pavelčíková, I. Barák, A. J. Wilkinson, (2010), doi:10.1074/jbc.M110.172429.

3.   L. Aravind, C. P. Ponting,, Trends Biochem. Sci. 22, (1997), 458.


This work was supported by Grant 2/0016/10 from Slovak Academy of Sciences, by grants from the Slovak Research and Development Agency under the contract No. APVT-51-0278 and No. LPP-0218-06, by Grant from European Science Foundation ESF-EC-0106, and by Grant 082829/Z/07/Z from The Wellcome Trust.