Bacterial programmed cell death – genetical, biochemical, and structural insight

Patrik Florek, Pamela Pavelčíková, Katarína Muchová, and Imrich Barák

Institute of Molecular Biology, Slovak Academy of Sciences, Bratislava

Programmed cell death in bacteria, as it analogue in eukaryotes, is defined as active process, which during the development or due to environmental signals leads to death of a particular cell.

The examples of programmed cell lysis during the developmental processes are mother cell lysis of sporulating Bacilli, fruiting body formation of Myxococcus bacteria, and colony rearrangement of Streptomyces species. On the other hand, genes responsible for programmed cell death as a consequence of effect of various harmful agents from an environment have been found in wide range of prokaryotes. It has been shown that a presence of these agents starts a self-destruction program of seriously affected cells, rather than killing the cells by the agents themselves. The act of suicide is in, these cases, usually performed by a stable toxin protein, effect of which is otherwise neutralized by its unstable antidote. Transcription of both genes coding for toxin and its antitoxin, which can be protein or untranslated antisense RNA, is frequently regulated from one common promoter. Such systems have been first identified stabilizing plasmid DNAs bearing corresponding genes. However, presently there are known several toxin-antitoxin families, cassettes of which are present on chromosomes of variety of bacteria.

There are only few identified cellular targets of toxin, but in all instances the toxicity directs either the cytoplasmic membrane integrity, or the replication and translation machineries, which are all factors crucial for cell viability. In few cases the mechanisms of toxicity is known even at the molecular level, shading more light to our understanding of processes affected by the toxin.

Out of dozen main groups of toxin-antitoxin systems, molecular structure of five toxin and three antitoxin proteins are already known. In some cases, these structures bring many interesting information about the way how a toxin affects function of its cellular target, or how its activity is regulated through the binding of the antidote. However, in majority of these systems, structures of toxin-antitoxin and toxin-target complexes have to be solved. This is still sometimes complicated by the fact that the cellular targets of several toxins are not identified yet. Instability of antitoxin proteins are frequently due to their degradation by specific proteases. Thus it makes them an interesting subjects for biochemical and structural studies.

In our work we focused on genetical, biochemical and structural studies of components of SpoIIS toxin-antitoxin system from Bacillus subtilis. SpoIISA toxic protein comprises two domains – N-terminal membrane spanning and C-terminal cytosolic parts. Our results indicate that neither of these two domains alone is sufficient for toxic effect. However, the oligomerization driven by cytosolic parts is needed for the toxicity of SpoIISA. This oligomerization seems to be blocked by tight binding of basic SpoIISB antitoxin to negatively charged C-terminal part of SpoIISA. To further appreciate the mechanisms of this regulation we attempted to crystallize both proteins and their complex.

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.