Epsilon/Zeta Toxin-Antitoxin Systems in Gram-negative Bacteria

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

Cooperative virulence can emerge via horizontal gene transfer but is stabilized by transmission

Intestinal inflammation fuels Salmonella Typhimurium ( S .Tm) transmission despite a fitness cost associated with the expression of virulence. Cheater mutants can emerge that profit from inflammation without enduring this cost. Intestinal virulence in S .Tm is therefore a cooperative trait, and its evolution a conundrum. Horizontal gene transfer (HGT) of cooperative alleles may facilitate the emergence of cooperative virulence, despite its instability. To test this hypothesis, we cloned hilD , coding for a master regulator of virulence, into a conjugative plasmid that is highly transferrable during intestinal colonization. We demonstrate that virulence can emerge by hilD transfer between avirulent strains in vivo . However, this was indeed unstable and hilD mutant cheaters arose within a few days. The timing of cheater emergence depended on the cost. We further show that stabilization of cooperative virulence in S .Tm is dependent on transmission dynamics, strengthened by population bottlenecks, leading cheaters to extinction and allowing cooperators to thrive

The expression of virulence increases outer-membrane permeability and sensitivity to envelope stress in Salmonella Typhimurium

Environmental cues modulate the expression of virulence in bacterial pathogens. However, while cues that upregulate virulence are often intuitive and mechanistically well understood, this is less so for cues that downregulate virulence. In this study, we noticed that upregulation of the HilD virulence regulon in Salmonella Typhimurium ( S .Tm) sensitized cells to membrane stress mediated by cholate, Tris/EDTA or heat. Further monitoring of membrane status and stress resistance of S .Tm cells in relation to virulence expression, revealed that co-expressed virulence factors embedded in the envelope (including the Type Three Secretion System 1 and the flagella) increased permeability, and stress sensitivity of the membrane. Importantly, pretreating the bacteria by sublethal stress inhibited virulence expression and restored stress resistance. As such, these results demonstrate a trade-off between virulence and stress resistance, which explains the downregulation of virulence expression in response to harsh environments in S .Tm

Nucleosome accessibility governed by the dimer/tetramer interface

Nucleosomes are multi-component macromolecular assemblies which present a formidable obstacle to enzymatic activities that require access to the DNA, e.g. DNA and RNA polymerases. The mechanism and pathway(s) by which nucleosomes disassemble to allow DNA access are not well understood. Here we present evidence from single molecule FRET experiments for a previously uncharacterized intermediate structural state before H2A–H2B dimer release, which is characterized by an increased distance between H2B and the nucleosomal dyad. This suggests that the first step in nucleosome disassembly is the opening of the (H3–H4)2 tetramer/(H2A–H2B) dimer interface, followed by H2A–H2B dimer release from the DNA and, lastly, (H3–H4)2 tetramer removal. We estimate that the open intermediate state is populated at 0.2–3% under physiological conditions. This finding could have significant in vivo implications for factor-mediated histone removal and exchange, as well as for regulating DNA accessibility to the transcription and replication machinery

Observation of Charge-Dependent Azimuthal Correlations in p-Pb Collisions and Its Implication for the Search for the Chiral Magnetic Effect

Peer reviewe

Measurement of the t(t)over-bar production cross section in pp collisions at root s=7 TeV in dilepton final states containing a tau

The top quark pair production cross section is measured in dilepton events with one electron or muon, and one hadronically decaying tau lepton from the decay t (t) over bar -> (l nu(l))((sic)(h)nu((sic)))b (b) over bar, (l = e, mu). The data sample corresponds to an integrated luminosity of 2.0 fb(-1) for the electron channel and 2.2 fb(-1) for the muon channel, collected by the CMS detector at the LHC. This is the first measurement of the t (t) over bar cross section explicitly including tau leptons in proton- proton collisions at root s = 7 TeV. The measured value sigma(t (t) over bar) = 143 +/- 14(stat) +/- 22(syst) +/- 3(lumi) pb is consistent with the standard model predictions

Measurement of the t-channel single top quark production cross section in pp collisions at √s = 7TeV

This is the pre-print version of the Published Article which can be accessed from the link below.Electroweak production of the top quark is measured for the first time in pp collisions at √s=7  TeV, using a data set collected with the CMS detector at the LHC and corresponding to an integrated luminosity of 36  pb-1. With an event selection optimized for t-channel production, two complementary analyses are performed. The first one exploits the special angular properties of the signal, together with background estimates from the data. The second approach uses a multivariate analysis technique to probe the compatibility with signal topology expected from electroweak top-quark production. The combined measurement of the cross section is 83.6±29.8(stat+syst)±3.3(lumi)  pb, consistent with the standard model expectation