Publications
197 results found
Wang B, Buck M, 2014, Rapid engineering of versatile molecular logic gates using heterologous genetic transcriptional modules, Chemical Communications, Vol: 50, Pages: 11642-11644, ISSN: 1359-7345
We designed and constructed versatile modular genetic logic gates in bacterial cells. These function as digital logic 1-input Buffer gate, 2-input and 3-input AND gates with one inverted input and integrate multiple chemical input signals in customised logic manners. Such rapidly engineered devices serve to achieve increased sensing signal selectivity.
Darbari VC, Lawton E, Lu D, et al., 2014, Molecular basis of nucleotide-dependent substrate engagement and remodeling by an AAA plus activator, NUCLEIC ACIDS RESEARCH, Vol: 42, Pages: 9249-9261, ISSN: 0305-1048
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- Citations: 1
Schumacher J, Behrends V, Pan Z, et al., 2013, Nitrogen and Carbon Status Are Integrated at the Transcriptional Level by the Nitrogen Regulator NtrC <i>In Vivo</i>, MBIO, Vol: 4, ISSN: 2150-7511
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- Citations: 45
Wang B, Barahona M, Buck M, et al., 2013, Rewiring cell signalling through chimaeric regulatory protein engineering, BIOCHEMICAL SOCIETY TRANSACTIONS, Vol: 41, Pages: 1195-1200, ISSN: 0300-5127
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- Citations: 17
Zhang N, Simpson T, Lawton E, et al., 2013, A Key Hydrophobic Patch Identified in an AAA<SUP>+</SUP> Protein Essential for Its <i>In Trans</i> Inhibitory Regulation, JOURNAL OF MOLECULAR BIOLOGY, Vol: 425, Pages: 2656-2669, ISSN: 0022-2836
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- Citations: 9
Mehta P, Jovanovic G, Lenn T, et al., 2013, Single molecule imaging of localisations, dynamics and stoichiometry of a regulated enhancer-binding protein in live Escherichia coli cells, 9th European-Biophysical-Societies-Association Congress, Publisher: SPRINGER, Pages: S202-S202, ISSN: 0175-7571
Mehta P, Jovanovic G, Lenn T, et al., 2013, Dynamics and stoichiometry of a regulated enhancer-binding protein in live <i>Escherichia coli</i> cells, NATURE COMMUNICATIONS, Vol: 4, ISSN: 2041-1723
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- Citations: 23
Wiesler SC, Weinzierl RO, Buck M, 2013, An aromatic residue switch in enhancer-dependent bacterial RNA polymerase controls transcription intermediate complex activity, Nucleic Acids Res, ISSN: 1362-4962
The formation of the open promoter complex (RPo) in which the melted DNA containing the transcription start site is located at the RNA polymerase (RNAP) catalytic centre is an obligatory step in the transcription of DNA into RNA catalyzed by RNAP. In the RPo, an extensive network of interactions is established between DNA, RNAP and the sigma-factor and the formation of functional RPo occurs via a series of transcriptional intermediates (collectively 'RPi'). A single tryptophan is ideally positioned to directly engage with the flipped out base of the non-template strand at the +1 site. Evidence suggests that this tryptophan (i) is involved in either forward translocation or DNA scrunching and (ii) in sigma54-regulated promoters limits the transcription activity of at least one intermediate complex (RPi) before the formation of a fully functional RPo. Limiting RPi activity may be important in preventing the premature synthesis of abortive transcripts, suggesting its involvement in a general mechanism driving the RPi to RPo transition for transcription initiation.
Galvao CW, Souza EM, Etto RM, et al., 2012, The RecX protein interacts with the RecA protein and modulates its activity in Herbaspirillum seropedicae, Brazilian Journal of Medical and Biological Research, Vol: 45, Pages: 1127-1134, ISSN: 0100-879X
DNA repair is crucial to the survival of all organisms. The bacterial RecA protein is a central component in the SOS response and in recombinational and SOS DNA repairs. The RecX protein has been characterized as a negative modulator of RecA activity in many bacteria. The recA and recX genes of Herbaspirillum seropedicae constitute a single operon, and evidence suggests that RecX participates in SOS repair. In the present study, we show that the H. seropedicae RecX protein (RecXHs) can interact with the H. seropedicae RecA protein (RecAHs) and that RecAHs possesses ATP binding, ATP hydrolyzing and DNA strand exchange activities. RecXHs inhibited 90% of the RecAHs DNA strand exchange activity even when present in a 50-fold lower molar concentration than RecAHs. RecAHs ATP binding was not affected by the addition of RecX, but the ATPase activity was reduced. When RecXHs was present before the formation of RecA filaments (RecA-ssDNA), inhibition of ATPase activity was substantially reduced and excess ssDNA also partially suppressed this inhibition. The results suggest that the RecXHs protein negatively modulates the RecAHs activities by protein-protein interactions and also by DNA-protein interactions.
Zhang N, Joly N, Buck M, 2012, A common feature from different subunits of a homomeric AAA plus protein contacts three spatially distinct transcription elements, NUCLEIC ACIDS RESEARCH, Vol: 40, Pages: 9139-9152, ISSN: 0305-1048
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- Citations: 12
Wang B, Barahona M, Buck M, 2012, A modular cell-based biosensor using engineered genetic logic circuits to detect and integrate multiple environmental signals, Biosensors and Bioelectronics, Vol: 40, Pages: 368-376, ISSN: 0956-5663
Cells perceive a wide variety of cellular and environmental signals, which are often processed combinatorially to generate particular phenotypic responses. Here, we employ both single and mixed cell type populations, pre-programmed with engineered modular cell signalling and sensing circuits, as processing units to detect and integrate multiple environmental signals. Based on an engineered modular genetic AND logic gate, we report the construction of a set of scalable synthetic microbe-based biosensors comprising exchangeable sensory, signal processing and actuation modules. These cellular biosensors were engineered using distinct signalling sensory modules to precisely identify various chemical signals, and combinations thereof, with a quantitative fluorescent output. The genetic logic gate used can function as a biological filter and an amplifier to enhance the sensing selectivity and sensitivity of cell-based biosensors. In particular, an Escherichia coli consortium-based biosensor has been constructed that can detect and integrate three environmental signals (arsenic, mercury and copper ion levels) via either its native two-component signal transduction pathways or synthetic signalling sensors derived from other bacteria in combination with a cell-cell communication module. We demonstrate how a modular cell-based biosensor can be engineered predictably using exchangeable synthetic gene circuit modules to sense and integrate multiple-input signals. This study illustrates some of the key practical design principles required for the future application of these biosensors in broad environmental and healthcare areas.
Joly N, Zhang N, Buck M, 2012, ATPase Site Architecture Is Required for Self-Assembly and Remodeling Activity of a Hexameric AAA plus Transcriptional Activator, MOLECULAR CELL, Vol: 47, Pages: 484-490, ISSN: 1097-2765
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- Citations: 19
Wang B, Buck M, 2012, Customizing cell signaling using engineered genetic logic circuits, Trends in Microbiology, Vol: 20, Pages: 376-384, ISSN: 0966-842X
Cells live in an ever-changing environment and continuously sense, process and react to environmental signals using their inherent signaling and gene regulatory networks. Recently, there have been great advances on rewiring the native cell signaling and gene networks to program cells to sense multiple noncognate signals and integrate them in a logical manner before initiating a desired response. Here, we summarize the current state-of-the-art of engineering synthetic genetic logic circuits to customize cellular signaling behaviors, and discuss their promising applications in biocomputing, environmental, biotechnological and biomedical areas as well as the remaining challenges in this growing field.
Zhang N, Buck M, 2012, Formation of MgF3--dependent complexes between an AAA+ ATPase and <sigma>54, FEBS Open Bio
Burrows PC, Wiesler SC, Pan Z, et al., 2012, Bacterial Virulence Gene Expression Contributed by the Alternative sigma factor sigma54, Pages: 27-58
Joly N, Zhang N, Buck M, et al., 2012, Coupling AAA protein function to regulated gene expression, BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH, Vol: 1823, Pages: 108-116, ISSN: 0167-4889
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- Citations: 28
Wiesler SC, Burrows PC, Buck M, 2012, A dual switch controls bacterial enhancer-dependent transcription, Nucleic Acids Res, Vol: 40, Pages: 10878-10892, ISSN: 1362-4962
Bacterial RNA polymerases (RNAPs) are targets for antibiotics. Myxopyronin binds to the RNAP switch regions to block structural rearrangements needed for formation of open promoter complexes. Bacterial RNAPs containing the major variant sigma(54) factor are activated by enhancer-binding proteins (bEBPs) and transcribe genes whose products are needed in pathogenicity and stress responses. We show that (i) enhancer-dependent RNAPs help Escherichia coli to survive in the presence of myxopyronin, (ii) enhancer-dependent RNAPs partially resist inhibition by myxopyronin and (iii) ATP hydrolysis catalysed by bEBPs is obligatory for functional interaction of the RNAP switch regions with the transcription start site. We demonstrate that enhancer-dependent promoters contain two barriers to full DNA opening, allowing tight regulation of transcription initiation. bEBPs engage in a dual switch to (i) allow propagation of nucleated DNA melting from an upstream DNA fork junction and (ii) complete the formation of the transcription bubble and downstream DNA fork junction at the RNA synthesis start site, resulting in switch region-dependent RNAP clamp closure and open promoter complex formation.
Wang B, Kitney RI, Joly N, et al., 2011, Engineering modular and orthogonal genetic logic gates for robust digital-like synthetic biology, Nature Communications, Vol: 2:508
Modular and orthogonal genetic logic gates are essential for building robust biologically based digital devices to customize cell signalling in synthetic biology. Here we constructed an orthogonal AND gate in Escherichia coli using a novel hetero-regulation module from Pseudomonas syringae. The device comprises two co-activating genes hrpR and hrpS controlled by separate promoter inputs, and a σ54-dependent hrpL promoter driving the output. The hrpL promoter is activated only when both genes are expressed, generating digital-like AND integration behaviour. The AND gate is demonstrated to be modular by applying new regulated promoters to the inputs, and connecting the output to a NOT gate module to produce a combinatorial NAND gate. The circuits were assembled using a parts-based engineering approach of quantitative characterization, modelling, followed by construction and testing. The results show that new genetic logic devices can be engineered predictably from novel native orthogonal biological control elements using quantitatively in-context characterized parts.
Toni T, Jovanovic G, Huvet M, et al., 2011, From Qualitative Data to Quantitative Models: Analysis of the Phage Shock Protein Response in Escherichia coli, BMC Systems Biology, Vol: 5, ISSN: 1752-0509
Background: Bacteria have evolved a rich set of mechanisms for sensing and adapting to adverse conditions intheir environment. These are crucial for their survival, which requires them to react to extracellular stresses such asheat shock, ethanol treatment or phage infection. Here we focus on studying the phage shock protein (Psp) stressresponse in Escherichia coli induced by a phage infection or other damage to the bacterial membrane. This systemhas not yet been theoretically modelled or analysed in silico.Results: We develop a model of the Psp response system, and illustrate how such models can be constructed andanalyzed in light of available sparse and qualitative information in order to generate novel biological hypothesesabout their dynamical behaviour. We analyze this model using tools from Petri-net theory and study its dynamicalrange that is consistent with currently available knowledge by conditioning model parameters on the availabledata in an approximate Bayesian computation (ABC) framework. Within this ABC approach we analyze stochasticand deterministic dynamics. This analysis allows us to identify different types of behaviour and these mechanisticinsights can in turn be used to design new, more detailed and time-resolved experiments.Conclusions: We have developed the first mechanistic model of the Psp response in E. coli. This model allows usto predict the possible qualitative stochastic and deterministic dynamic behaviours of key molecular players in thestress response. Our inferential approach can be applied to stress response and signalling systems more generally:in the ABC framework we can condition mathematical models on qualitative data in order to delimit e.g.parameter ranges or the qualitative system dynamics in light of available end-point or qualitative information.
Joly N, Buck M, 2011, Single Chain Forms of the Enhancer Binding Protein PspF Provide Insights into Geometric Requirements for Gene Activation, JOURNAL OF BIOLOGICAL CHEMISTRY, Vol: 286
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- Citations: 8
Jovanovic M, James EH, Burrows PC, et al., 2011, Regulation of the co-evolved HrpR and HrpS AAA plus proteins required for <i>Pseudomonas syringae</i> pathogenicity, NATURE COMMUNICATIONS, Vol: 2, ISSN: 2041-1723
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- Citations: 84
Buck CEATBMBJTDMGJM, 2011, Dissipation of Proton Motive Force is not Sufficient to Inducethe Phage Shock Protein Response in Escherichia coli, Curr Microbiol
Bartos A, Wang B, Buck M, et al., 2011, Toxic metal detection in foodstuff. Synthetic biology approach used to create biosensors., Annual Meeting of the American-Society-for-Cell-Biology (ASCB), Publisher: AMER SOC CELL BIOLOGY, ISSN: 1059-1524
Lenn T, Gkekas CN, Bernard L, et al., 2011, Measuring the stoichiometry of functional PspA complexes in living bacterial cells by single molecule photobleaching, CHEMICAL COMMUNICATIONS, Vol: 47, Pages: 400-402, ISSN: 1359-7345
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- Citations: 20
Jovanovic M, Burrows PC, Bose D, et al., 2011, Activity map of the Escherichia coli RNA polymerase bridge helix, J Biol Chem, Vol: 286, Pages: 14469-14479, ISSN: 1083-351X
Transcription, the synthesis of RNA from a DNA template, is performed by multisubunit RNA polymerases (RNAPs) in all cellular organisms. The bridge helix (BH) is a distinct feature of all multisubunit RNAPs and makes direct interactions with several active site-associated mobile features implicated in the nucleotide addition cycle and RNA and DNA binding. Because the BH has been captured in both kinked and straight conformations in different crystals structures of RNAP, recently supported by molecular dynamics studies, it has been proposed that cycling between these conformations is an integral part of the nucleotide addition cycle. To further evaluate the role of the BH, we conducted systematic alanine scanning mutagenesis of the Escherichia coli RNAP BH to determine its contributions to activities required for transcription. Combining our data with an atomic model of E. coli RNAP, we suggest that alterations in the interactions between the BH and (i) the trigger loop, (ii) fork loop 2, and (iii) switch 2 can help explain the observed changes in RNAP functionality associated with some of the BH variants. Additionally, we show that extensive defects in E. coli RNAP functionality depend upon a single previously not studied lysine residue (Lys-781) that is strictly conserved in all bacteria. It appears that direct interactions made by the BH with other conserved features of RNAP are lost in some of the E. coli alanine substitution variants, which we infer results in conformational changes in RNAP that modify RNAP functionality.
Buck M, Hoover TR, 2010, An ATPase R-Finger Leaves Its Print on Transcriptional Activation, STRUCTURE, Vol: 18, Pages: 1391-1392, ISSN: 0969-2126
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- Citations: 2
Jovanovic G, Engl C, Mayhew AJ, et al., 2010, Properties of the phage-shock-protein (Psp) regulatory complex that govern signal transduction and induction of the Psp response in <i>Escherichia coli</i>, MICROBIOLOGY-SGM, Vol: 156, Pages: 2920-2932, ISSN: 1350-0872
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- Citations: 29
Joly N, Engl C, Jovanovic G, et al., 2010, Managing membrane stress: the phage shock protein (Psp) response, from molecular mechanisms to physiology, FEMS MICROBIOLOGY REVIEWS, Vol: 34, Pages: 797-827, ISSN: 0168-6445
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- Citations: 167
Burrows PC, Joly N, Buck M, 2010, A prehydrolysis state of an AAA plus ATPase supports transcription activation of an enhancer-dependent RNA polymerase, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, Vol: 107, Pages: 9376-9381, ISSN: 0027-8424
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- Citations: 26
Joly N, Buck M, 2010, Engineered Interfaces of an AAA<SUP>+</SUP> ATPase Reveal a New Nucleotide-dependent Coordination Mechanism, JOURNAL OF BIOLOGICAL CHEMISTRY, Vol: 285, Pages: 15178-15186
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- Citations: 17
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