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Abstract

Their genetic repertoire allows bacteria to adapt to constantly changing environments and to survive inhospitable conditions. Such genetic modules are toxin-antitoxin (TA) systems, which increase the survival of a population by inducing dormancy or cell death of a subpopulation of cells. They encode intracellular toxins, which have the potential to kill the cell, and are therefore tightly regulated by cognate antitoxins that inhibit the toxic activity. Toxicity is exerted by interference with essential cellular processes including replication, translation and homeostasis. Correspondingly, the toxins of the zeta family were found to poison cell wall synthesis by phosphorylation of the peptidoglycan precursor UDP-N-acetylglucosamine (UNAG). Zeta toxins are counteracted by small, proteinaceous epsilon antitoxins. These epsilon/zeta TA systems were first discovered as plasmid maintenance modules on the plasmids of Gram-positive streptococci and have long been thought to be confined to Gram-positive bacteria. This thesis provides the first biochemical and structural characterization of zeta toxin homologs from Gram-negative bacteria - EzeT from Escherichia coli and the plasmidic epsilon/zeta systems from Neisseria gonorrhoeae - showing that these proteins exhibit zeta-like kinase activity. Moreover, several differences to the hitherto described streptococcal epsilon/zeta systems provide evidence for the diversity of the epsilon/zeta TA family. The first part of this thesis describes the characterization of the elongated zeta homolog EzeT, which was found to combine toxin and antitoxin functionalities in one polypeptide chain. The protein consists of two domains, one of which was demonstrated to have UNAG kinase activity in vitro and in vivo. The enzymatic activity is abrogated in the presence of the N-terminal antitoxin domain and mutational analysis indicated that inhibition is performed by an epsilon-like mechanism. Furthermore, EzeT toxicity is temperature dependent, leading to a lytic phenotype and spherical morphology at ambient temperature. The presented novel type of toxin inhibition by a covalently linked antitoxin sets EzeT apart from other TA systems and necessitates the analysis of other elongated or orphan toxin homologs that have been identified in bacterial genomes. In the second part of this thesis, the X-ray crystal structure of the epsilon/zeta system ε1/ζ1 of N. gonorrhoeae is presented, together with biochemical studies that confirmed ζ1 as a UNAG kinase, which is inhibited by binding of ε1. Bioinformatic analysis of ζ1 showed that in the amino acid sequence the catalytically important Walker A motif, commonly found in P-loop kinases, is located closer to the C-terminus than in homologous proteins. Nevertheless, ζ1 adopts a typical zeta-like three-dimensional structure with a conserved architecture of the catalytic center. This is achieved by a rearrangement of the secondary structure elements and a corresponding topological rewiring, forming a fold unusual for P-loop kinases. However, nucleotide binding and hydrolysis are not impaired and have been observed spectroscopically and by determination of the ADP-bound complex structure. In addition to the zeta-like domain, ζ1 contains a C-terminal domain that comprises an OB-fold with similarity to DNA-binding modules. This domain is also present in two other gonococcal epsilon/zeta homologs, which are located on the same plasmid. As presented in this work, these systems, called ε2/ζ2 and ε3/ζ3, phosphorylate UNAG, but do not directly interact with ε1/ζ1. Furthermore, the X-ray crystal structure of the heterodimeric ε1/ζ1 complex revealed that inhibition is mediated by ε1 wrapping around the zeta core, indicating a different mode of inhibition compared to the streptococcal antitoxins. In conclusion, data presented in this thesis show that Gram-negative bacteria encode active UNAG kinases. The characterization of these systems reveals novel inhibition mechanisms by the antitoxins and highlights the structural diversity of zeta toxins.