1,721,066 research outputs found
Mechanism of SigR inhibition by the anti-sigma factor RsrA from Streptomyces coelicolor
Get PDFThe extracytoplasmic function (ECF) sigma factor SigR (σR) is a bacterial transcription factor involved in the regulation of cytosolic redox homeostasis in Streptomyces coelicolor. SigR activity is regulated by its cognate anti-sigma factor RsrA. RsrA is a member of the zinc-anti-sigma (ZAS) family, which in its reduced state binds a single Zn2+ ion coordinated by 3 cysteine residues and a histidine. RsrA senses oxidative stress within the cytosol and responds by the formation of intramolecular disulphide bonds between its Zn-ligating cysteine residues. This results in the loss of the metal ion, a conformational rearrangement, and dissociation of SigR by an unknown mechanism. This releases SigR to bind RNA polymerase and direct the expression of a large regulon including that of the thioredoxin pathway, thereby re-establishing redox homeostasis. No structural information is available for the SigR-RsrA complex in Streptomyces coelicolor. Moreover, little is known of the mechanism by which RsrA associates with SigR and regulates gene transcription, or the role of zinc in SigR binding. In this work, a stopped-flow fluorescence-based assay was developed for investigating the kinetics of SigR-RsrA complex formation to aid our understanding of the role of the metal ion. Ultimately, trying to understand how oxidative stress causes dissociation of the Streptomyces coelicolor complex. Through tryptophan fluorescence emission quenching experiments, the SigR region 2 tryptophan (W88) and region 4 tryptophan (W159) were found to be sensitive to RsrA binding, likely denoting the binding sites for the anti-sigma factor. Pre-steady state experiments demonstrated that the metal ion has little effect on the rate of association (Kon 8.9 and 17.3x106 M-1s-1 at 35°C in the presence and absence of zinc, respectively) but a large effect on the dissociation rate constant (>500-fold faster in the absence of zinc at 35°C). Moreover, the kinetically derived Kds (0.27 nM and 62.4 nM with and without zinc respectively) agreed with the equilibrium constant for the complex derived through isothermal titration calorimetry (0.78 nM and 101.7 nM), validating the kinetic model for binding experiments. Zinc therefore has an important role in the structure of RsrA, affecting SigR-RsrA complex affinity, the off-rate for the complex and inhibition of SigR activity. Future work will exploit these assays to determine the role of zinc in redox sensing by RsrA
Force transduction through the bacterial cell envelope
No full textThe Tol-Pal system is a conserved machine in double-membraned bacteria that stabilises the Gram-negative bacterial outer membrane. In this thesis, I aim to elucidate the mechanism by which the Tol-Pal system dynamically stabilises this important protective layer. I examine how assembly of the trans-periplasmic motor-transducer complex recruits a membrane stabilising lipoprotein, Pal, to the division site. To contextualise the experimental aspects of this thesis, I first examine the physiological role of the Tol-Pal system, and its cellular function in E. coli. Through use of mutagenesis, fluorescence microscopy, and live cell assays, I demonstrate that system function can be modulated by altering the secondary structure of the periplasm spanning transducer protein, TolA. I also examine the cell-wide implications of reducing TolA function in such a manner, with respect to membrane stability and cell division (chapter three). Subsequent bioinformatic investigations into the phylogeny of the Tol-Pal system reveal its unexpectedly broad distribution throughout the kingdom of bacteria and hint at its evolutionary origin (chapter four). Further molecular insights were gleaned through in vivo crosslinking, where I probe the conformational rearrangements that occur within the TolQR motor in response to proton flux (chapter five). Additional modelling and bioinformatic data reveal the putative TolA binding site on the TolQR motor, and I demonstrate the biological relevance of a previously unknown interaction motif involved in TolA-TolR interactions and force transduction (chapter six). Finally, I describe the optimisation of electron cryotomography (cryo-ET) to structurally visualise the TolQR-TolA assembly in an energetically stalled state (chapter seven). Overall, this work reveals the function, distribution, conservation and organisation of bacterial Tol-Pal systems and their likely evolutionary origin. I also propose a model for the mechanism by which these protein machines use proton flux to stabilise the outer membrane in E. coli
Bacterial protein import mediated by an iron transporter
Get PDFMultidrug resistant bacteria (MDR) have the potential to push back society to the pre-antibiotic era. Although discovered before penicillin, the inexorable rise in antibiotic resistance has revitalised interest in bacteriocins as treatments for bacterial infections. Bacteriocins are protein antibiotics principal to competition amongst pathogens and commensals, but the mechanisms by which they translocate across the Gram-negative cell envelope are poorly understood. The work presented in this thesis demonstrates how the endonuclease bacteriocin pyocin S2 (pyoS2) exploits the iron transporter FpvAI to translocate across the outer membrane (OM) of Pseudomonas aeruginosa. FpvAI is a 22-strand β-barrel and virulence factor in P. aeruginosa that transports iron into the cell in the form of a small siderophore, ferripyoverdine (Fe-Pvd). Uptake of Fe-Pvd requires the proton motive force (PMF), which is transduced to the ligand-bound receptor by TonB1 and its partner proteins ExbB-ExbD in the inner membrane (IM). The crystal structure of the high affinity complex (Kd = 240 pM) formed between the N-terminal domain of pyoS2 (pyoS2NTD) and FpvAI is presented, which shows pyoS2NTD mimics Fe-Pvd, and induces the same conformational changes in the receptor. Fluorescently-labelled pyoS2NTD was actively imported into P. aeruginosa PAO1 cells and this import was dependent on the PMF, TonB1 and a TonB1-box motif at the N-terminus of pyoS2NTD. Finally, photo-activated crosslinking of stalled translocation intermediates demonstrated pyoS2NTD translocates through the FpvAI β-barrel lumen by a process analogous to that of Fe-Pvd. Following binding to FpvAI, translocation begins by the unfolding of a force-labile portion of the plug domain, opening a narrow channel through FpvAI. This enables pyoS2 to deliver its own TonB1-box to the periplasm where contact with TonB1 activates its import through the same channel, most likely as an unfolded polypeptide. Hence, this study demonstrates that bacteria possess a rudimentary protein import system that exploits energised nutrient transporters in the OM.</p
How the protein antibiotic pyocin S5 kills Pseudomonas aeruginosa
Get PDFAntimicrobial resistance is a deadly threat that is on the rise globally. Failure of small molecule antibiotic drug discovery programs and an increasing understanding of the dangers of antibiotic-induced dysbiosis of the microbiome have led to interest in bacteriocins as narrow-spectrum antibiotics. Pyocin S5 (PyoS5), a protein antibiotic against Pseudomonas aeruginosa, is the most potent colicin-like bacteriocin known but its structure and molecular mechanism remain poorly understood. This thesis presents the crystal structure of PyoS5, an elongated molecule with three structured domains and an unresolved N-terminal region, which enabled the functional, biochemical and biophysical characterization of this molecule. With its central domain, PyoS5 binds to common polysaccharide antigen (CPA), a lipopolysaccharide (LPS) antigen which acts as a receptor and accumulates the antibiotic on the cell surface. This is followed by binding to the outer membrane protein FptA, a TonB-dependent transporter (TBDT), by the N-terminal domain. A chimeric protein of Escherichia coli TonB and P. aeruginosa TonB1 allowed us to reconstruct the PyoS5 import process in E. coli, an approach that revealed that FptA acts as the translocator and that TonB1 energizes the import process by directly interacting with the N-terminal region of PyoS5. This import process and the structure of PyoS5 show strong similarity with those of PyoS2, which suggests that they are common to several pyocins targeting different TBDTs and delivering different cytotoxic domains into P. aeruginosa.</p
Colicin translocation through the E. coli cell envelope
Get PDFGram-negative bacteria have evolved mechanisms to kill other microbial species to compete for space and resources. One mechanism exploited by Escherichia coli is the production of
bacteriocins called colicins, which target closely-related sensitive E. coli strains. Colicins bind with high affinity to outer membrane proteins on the surface of E. coli, and following contact with proteins in the periplasm and inner membrane, translocate a cytotoxic domain to kill the cell. Killing displays first-order kinetics which has been suggested to mean a single molecule is
sufficient to kill a cell, although this has yet to be demonstrated.
The mechanism by which a folded colicin can cross two membranes to kill cells is still unknown, due to the lack of tools available. The aim of this research was to develop fluorescence-based microscopy tools to visualise colicin entry into E. coli and probe the mechanism of translocation in vivo.
ColE9 was labelled with organic fluorophores within its cytotoxic domain and retained its catalytic activity in vitro, however its in vivo activity was reduced. Microscopy was optimised
using a ColE9 active site mutant and the AlexaFluor-647 dye, to generate a fluorescent probe capable of translocating into but not killing cells.
Translocation of ColE9*AF647 across the outer membrane (OM) was dependent on the PMF, in particular the electrical potential. Translocation of single-molecules of ColE9*AF647 to the
cytoplasm was visualised for the first time in vivo; ~4-5 molecules were observed per cell, in 30 minutes at 37 °C. Two populations of ColE9*AF647 molecules were present; a mobile fraction with a diffusion coefficient similar to cytoplasmic GFP (~4-10 µm2/s) and an immobile fraction, the origins of which are unknown. Finally, the rate-limiting step for ColE9 translocation across the OM was shown to be passage through OmpF or OmpC, which could be modulated by the fluorophore used. The present work demonstrates that Tol-dependent colicins translocate across the OM in an energy-dependent manner and must be completely unfolded in order to translocate through OmpF or OmpC.</p
Mining bacterial genomes for novel antimicrobials
Get PDFBacteria have evolved an arsenal of antimicrobial peptides and proteins to compete with
each other. Bacteriocins are diffusible toxins released into the environment and differ
from contact-dependent inhibitors (CDI) or type six secretions systems (T6SS) which
require cell-cell contact. Protein bacteriocins are potent narrow-spectrum toxins with
a modular domain organisation, making them ideal candidates for developing future
antimicrobials. Though progress is being made to exploit protein bacteriocins, current
limitations in our understanding of protein bacteriocin biology limits their possible use
as antimicrobials. Firstly, due to high sequence diversity and similarity to other competition
systems our understanding of the diversity and prevalence of protein bacteriocins
in bacteria is poor. Secondly, high levels of resistance have been reported with
tested under lab conditions. Finally, how protein bacteriocins translocate into the cell
is poorly understood. The aim of this study is to answer a sequence of questions relating
to these central limitations in our knowledge of protein bacteriocins. A bioinformatic
pipeline was developed which exploits Hidden Markov Models to unambiguously
identify nuclease bacteriocins and revealed a diverse family of proteins present in >2000
strains throughout the Enterobacteriaceae family and Pseudomonas spp. A novel grouping
scheme is devised for NBs based on conserved structural organisation. Investigation of
non-speci1c colicin resistance mechanism revealed an O-antigen dependent resistance
involving density dependent blocking of the receptor proteins in the outer membrane.
This resistance mechanism was sensitive to changes in environment and could be overcome
by additives. Finally, a conserved motif, the âDPYâ motif, was identi1ed across the
nuclease bacteriocin family and effectors of the T6SS. While the role of the DPY motif is
still unclear, it is associated with toxins which cross the inner membrane.</p
Structural and biophysical study of the KlebC import mechanism
Get PDFThe cell envelope of Gram-negative bacteria hosts many different protein machineries. These protein machineries are used by bacteria to perform different functions, such as cell division and nutrient acquisition. TolC is a trimeric protein that anchors at the OM and extends into the periplasm. TolC is used as a channel forexporting protein toxins and xenobiotics out of the cell but is also hijacked by bacteriocins as an import channel. This unique double-sided function of TolC has drawn interest to understand its roles in different biological processes and apply them to combat increasingly serious global antimicrobial crisis. The exploitation of TolC by a group B bacteriocin, KlebC, was studied using biochemical, biophysical and structural methods. It was previously discovered that the Ton system, known for its role in nutrient acquisition, is required to uptake KlebC into cells. Although the interactions between different group B bacteriocins and their outer membrane receptors have been elucidated, there has been no published studies on the interaction between group B bacteriocins and periplasmic TonB. This study has successfully demonstrated binding between KlebC and TonB, by analytical size exclusion chromatography and isothermal titration calorimetry. A binding affinity of 66 μM was measured between KlebC and TonB. The structure of the complex waspredicted by Alphafold 2.0 and validated by mutational approaches. To fully understand the uptake mechanism of KlebC, the ternary KlebC translocon (KlebC-TolC-TonB) was assembled in nanodiscs. Grids, containing the KlebC translocon, wereoptimised for single-particle cryogenic electron microscopy data collection and analysis for future structure determination of the complex
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
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