Investigating the assembly and function of the enteropathogenic Escherichia coli type 3 secretion system

The full abstract for this thesis is available in the body of the thesis, and will be available when the embargo expires.Medicine, Faculty ofBiochemistry and Molecular Biology, Department ofGraduat

Mapping the DNA-Binding Motif of Scabin Toxin, a Guanine Modifying Enzyme from Streptomyces scabies

Scabin is a mono-ADP-ribosyltransferase toxin/enzyme and possible virulence factor produced by the agriculture pathogen, Streptomyces scabies. Recently, molecular dynamic approaches and MD simulations revealed its interaction with both NAD+ and DNA substrates. An Essential Dynamics Analysis identified a crab-claw-like mechanism, including coupled changes in the exposed motifs, and the Rβ1-RLa-NLc-STTβ2-WPN-WARTT-(QxE)ARTT sequence motif was proposed as a catalytic signature of the Pierisin family of DNA-acting toxins. A new fluorescence assay was devised to measure the kinetics for both RNA and DNA substrates. Several protein variants were prepared to probe the Scabin-NAD-DNA molecular model and to reveal the reaction mechanism for the transfer of ADP-ribose to the guanine base in the DNA substrate. The results revealed that there are several lysine and arginine residues in Scabin that are important for binding the DNA substrate; also, key residues such as Asn110 in the mechanism of ADP-ribose transfer to the guanine base were identified. The DNA-binding residues are shared with ScARP from Streptomyces coelicolor but are not conserved with Pierisin-1, suggesting that the modification of guanine bases by ADP-ribosyltransferases is divergent even in the Pierisin family

Dynamics of Scabin toxin. A proposal for the binding mode of the DNA substrate

<div><p>Scabin is a mono-ADP-ribosyltransferase enzyme and is a putative virulence factor produced by the plant pathogen, <i>Streptomyces scabies</i>. Previously, crystal structures of Scabin were solved in the presence and absence of substrate analogues and inhibitors. Herein, experimental (hydrogen-deuterium exchange), simulated (molecular dynamics), and theoretical (Gaussian Network Modeling) approaches were systematically applied to study the dynamics of apo-Scabin in the context of a Scabin·NAD⁺·DNA model. MD simulations revealed that the apo-Scabin solution conformation correlates well with the X-ray crystal structure, beyond the conformation of the exposed, mobile regions. In turn, the MD fluctuations correspond with the crystallographic B-factors, with the fluctuations derived from a Gaussian network model, and with the experimental H/D exchange rates. An Essential Dynamics Analysis identified the dynamic aspects of the toxin as a crab-claw-like mechanism of two topological domains, along with coupled deformations of exposed motifs. The “crab-claw” movement resembles the motion of C3-like toxins and emerges as a property of the central β scaffold of catalytic single domain toxins. The exposure and high mobility of the <i>cis</i> side motifs in the Scabin β-core suggest involvement in DNA substrate binding. A ternary Scabin·NAD⁺·DNA model was produced via an independent docking methodology, where the intermolecular interactions correspond to the region of high mobility identified by dynamics analyses and agree with binding and kinetic data reported for wild-type and Scabin variants. Based on data for the Pierisin-like toxin group, the sequence motif R_β1–R_La–N_Lc–STT_β2–W_PN–W_ARTT–(QxE)_ARTT emerges as a catalytic signature involved in the enzymatic activity of these DNA-acting toxins. However, these results also show that Scabin possesses a unique DNA-binding motif within the Pierisin-like toxin group.</p></div

Effect of Toxic Phthalate-Based Plasticizer on the Biodegradability of Polyhydroxyalkanoate

While new biodegradable materials are being rapidly introduced to address plastic pollution, their end-of-life impacts remain unclear. Biodegradable plastics typically comprise a biopolymer matrix with functional additives and/or solid fillers, which may be toxic. Here, using an established method for continuous biodegradation monitoring, we investigated the impact of a commonly used plasticizer, dibutyl phthalate (DBP), on the biodegradation of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) in soil. The presence of DBP delayed the initial stage of PHBV biodegradation but then accelerated subsequent rates of biodegradation. Furthermore, it led to significant increases in total bacterial and fungal biomass and altered the composition of microbial communities with significant increases in the relative abundances of Thauera (gammaproteobacterial) and Mucor circinelloides (fungal) populations. It is proposed, with evidence from biodegradation behavior and microbial analysis, that the presence of DBP likely stimulated a microbial community shift, introduced higher proportions of more readily degradable amorphous regions from the plasticizing effect, and facilitated access to the bulk polymer matrix for microorganisms or at least their associated enzymes. These effects in combination overcame the initial inhibition effect of the DBP and resulted in a net increase in the rate of biodegradation of PHBV

HDX mass spectra for pepsin digestion of the Scabin-DNA complex.

<p>Representative spectra of peptides obtained from pepsin digestion of non-deuterated apo-Scabin (top), labeled with a 4.1 s HDX pulse (middle) and complexed with DNA (bottom) labeled by a 4.1 s HDX pulse. Representative peptides are depicted that showed no change (left) or a decrease in uptake located in the PN-loop (middle) and β_6/7-turn (right). Percent deuterium uptake is indicated on each spectrum.</p

Scabin mobility in the Scabin· DNA complex.

<p>Normalized MSqF derived from a GNM (160 C_α nodes, γ = 1, cutoff = 10 Å) for the active conformation of Scabin in the ternary complex calculated without (blue) or with (red) the inclusion of the P-atoms of the DNA molecule as nodes. The difference (bound—apo) in relative mobility is plotted in black. Nodes corresponding to β-strands are highlighted in yellow.</p

Experimental and simulated mobility profiles of apo-Scabin.

<p>(<b>a</b>) Normalized MSqF from the Grand ensemble of conformations (blue) and normalized <i>B</i>-factors for the 5DAZ structure (black). Highlighted with yellow are the crystallographic <i>B</i>-factors of residues belonging to SS₁ and SS2 loops. (<b>b</b>) Normalized MSqF from the Grand ensemble of conformations by only using the set [5,159] of PC_G modes (black), and normalized MSqF from a GNM on the Trace_G conformation by using all, [1,159], GNM_G modes (blue). For the GNM, the (force constant took the value γ(<i>N</i>₁,<i>N</i>₂) = 1 for any <i>N</i>₁ and <i>N</i>₂ node of the network, except when one or both nodes belong to specific segments or residues (<i>e</i>.<i>g</i>., PN-loop, SS₁-loop, Cys42, etc.) as follows: γ(PN,<i>N</i>₂) = γ(ARTT,<i>N</i>₂) = 0.64, γ(SS₁,SS₁) = γ(SS₂,SS₂) = 1.07, and γ(C42,C72) = γ(C176,C190) = 3.57.</p