Toxin-antitoxin (TA) systems are genetic elements encoding a toxin and a compatible antitoxin, and are commonly present in bacteria and archaea. They consist of a toxin, that inhibits cell growth, and an antitoxin, that disables the action of the toxin. The most common and best characterised TA systems are type II systems, in which both the toxin and antitoxin are proteins which form a tight complex.
Microcystis aeruginosa is a cyanobacterium that inhabits diverse ecological niches and thrives very well in the environment. This is made possible by various genetic elements, including toxin-antitoxin systems. One pair of type II TA systems in M. aeruginosa is especially interesting, because in the genome of strain PCC 7806 it is located in close proximity to the gene that codes for one of the six orthocaspases present in this strain, MaOCA-Ia (formerly MaOC1). The putative TA pair consists of a toxin similar to those of the RelE/ParE family of toxins, as well as the cognate antitoxin. MaOCA-Ia codes for a proteolytically active protease, that preferentially cleaves after clusters of basic amino acid residues. Such sequences are also found in antitoxin 1067, with two regions containing two and three arginine clusters, respectively.
As a part of this thesis, we were interested in whether MaOCA-Ia cleaves antitoxin 1067 after these two regions. As this could not be previously confirmed with antibodies, we attempted to demonstrate cleavage by preparing mutant antitoxins. Therefore, mutations had to be intoduced into the DNA sequence of the wild-type antitoxin 1067. After expression of the mutant forms and their isolation, we were able to analyse the cleavage sites. In the context of this thesis, we prepared at the DNA level three different mutant versions of antitoxin 1067 by inverse polymerase chain reaction (PCR). The correctness of the sequences with the introduced mutations was verified by sequencing and by bioinformatic analysis of the sequences.
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