RelA-SpoT homolog enzymes as effectors of Toxin-Antitoxin systems

Doctoral theses defended at the University of Tartu, summary in Estonian.

Like all living organisms, bacteria sense the environment and respond to plethora of stresses by adjusting their physiology accordingly. One of the central bacterial stress pathways is the stringent response (SR). The stringent response mediates the bacterial adaptation to nutrient limitation as well as to in response to abiotic environmental stresses like heat shock. More than six decades ago alarmone nucleotides ppGpp and pppGpp – collectively referred to as (p)ppGpp – or the “magic spots” were discovered to be produced in Escherichia coli cells as a response to amino acids limitation. The first physiologial role of the SR to be characterised was inhibition of stable RNA (rRNA and tRNA) synthesis, coordinated with induction of expression of genes involved in amino acid biosynthesis and stress tolerance. However, decades of research have established that in addition to transcription, (p)ppGpp targets multiple other processes in the cell, such as translation, ribosome assembly, metabolism, and impact all the aspects of cell physiology including adaptation to nutrient limitation, antibiotic resistance and virulence. Another regulatory system in bacteria is based on the toxin – antitoxin (TA) systems. First representatives of toxin -antitoxin (TA) systems were discovered in the early 80s. The classical TA systems are bicistronic – i.e. comprised of two gene – operons, in which one gene encodes a protein toxin and the other encodes a protein or RNA antitoxin which neutralises the toxin, either directly or indirectly. Studies of TA systems have exploded in the last years, with numerous new TA families being discovered, their mechanisms of action being characterised, biological functions established and possible applications for biotechnology put forward. The most established functions include plasmid maintenance, defence against bacteriophages and regulation of cell physiology. In the current study new activities of the RSH family enzymes were described and the specificity of toxin neutralization by an PanA antitoxin family members described. Additionally, dimerization of ribosomes occurs during stress. This activity is useful for the cell survival but might decrease the activity of cell free translation systems in case lysates are used in biotechnology. Therefore, the effect of removing the proteins responsible for ribosome dimerization was investigated. It was found that enzymatic activities of RSH enzymes are not limited to production and degradation of (p)ppGpp. Members of toxSAS RSH PhRel2, FaRel2, PhRel and CapRel subfamilies catalyse pyrophosphorylation of tRNA 3'CCA end, and members of FaRel family catalyse synthesis of (pp)pApp. Members of SAH subfamily MESH1 and ATfaRel catalyse removal of the pyrophosphate from PP-tRNA and degradation of (pp)pApp. Antitoxins containing common PanA domain neutralize diverse toxin families. PanA-mediated toxin neutralisation is highly specific for the cognate toxin-antitoxin pair. Genetic elimination of ribosome dimerization factors in Firmicute bacterium B. subtilis (hfp) and yeast S. cerevisiae (stm1) strains is a promising strategy for producing more active in vitro translation lysates. Titration of Mg2+ and different reaction components are essential for achieving the optimal activity of the lysate.