Imperial College London

Professor Martin Buck FRS

Faculty of Natural SciencesDepartment of Life Sciences

Professor of Molecular Microbiology
 
 
 
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Contact

 

+44 (0)20 7594 5442m.buck

 
 
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Location

 

448Sir Alexander Fleming BuildingSouth Kensington Campus

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Summary

 

Publications

Publication Type
Year
to

182 results found

Engl C, Jovanovic G, Brackston RD, Kotta-Loizou I, Buck Met al., 2020, The route to transcription initiation determines the mode of transcriptional bursting in E. coli, Nature Communications, Vol: 11, ISSN: 2041-1723

Transcription is fundamentally noisy, leading to significant heterogeneity across bacterial populations. Noise is often attributed to burstiness, but the underlying mechanisms and their dependence on the mode of promotor regulation remain unclear. Here, we measure E. coli single cell mRNA levels for two stress responses that depend on bacterial sigma factors with different mode of transcription initiation (σ70 and σ54). By fitting a stochastic model to the observed mRNA distributions, we show that the transition from low to high expression of the σ70-controlled stress response is regulated via the burst size, while that of the σ54-controlled stress response is regulated via the burst frequency. Therefore, transcription initiation involving σ54 differs from other bacterial systems, and yields bursting kinetics characteristic of eukaryotic systems.

Journal article

Gao F, Danson AE, Ye F, Jovanovic M, Buck M, Zhang Xet al., 2020, Bacterial enhancer binding proteins - AAA+ proteins in transcription activation, Biomolecules, Vol: 10, ISSN: 2218-273X

Bacterial enhancer-binding proteins (bEBPs) are specialised transcriptional activators. bEBPs are hexameric AAA+ ATPases and use ATPase activities to remodel RNA polymerase (RNAP) complexes that contain the major variant sigma factor, σ54 to convert the initial closed complex to the transcription competent open complex. Earlier crystal structures of AAA+ domains alone have led to proposals of how nucleotide-bound states are sensed and propagated to substrate interactions. Recently, the structure of the AAA+ domain of a bEBP bound to RNAP-σ54-promoter DNA was revealed. Together with structures of the closed complex, an intermediate state where DNA is partially loaded into the RNAP cleft and the open promoter complex, a mechanistic understanding of how bEBPs use ATP to activate transcription can now be proposed. This review summarises current structural models and the emerging understanding of how this special class of AAA+ proteins utilises ATPase activities to allow σ54-dependent transcription initiation.

Journal article

Ye F, Kotta-Loizou I, Jovanovic M, Liu X, Dryden DT, Buck M, Zhang Xet al., 2020, Structural basis of transcription inhibition by the DNA mimic protein Ocr of bacteriophage T7., eLife, Vol: 9, ISSN: 2050-084X

Bacteriophage T7 infects Escherichia coli and evades the host restriction/modification system. The Ocr protein of T7 was shown to exist as a dimer mimicking DNA and to bind to host restriction enzymes, thus preventing the degradation of the viral genome by the host. Here we report that Ocr can also inhibit host transcription by directly binding to bacterial RNA polymerase (RNAP) and competing with the recruitment of RNAP by sigma factors. Using cryo electron microscopy, we determined the structures of Ocr bound to RNAP. The structures show that an Ocr dimer binds to RNAP in the cleft, where key regions of sigma bind and where DNA resides during transcription synthesis, thus providing a structural basis for the transcription inhibition. Our results reveal the versatility of Ocr in interfering with host systems and suggest possible strategies that could be exploited in adopting DNA mimicry as a basis for forming novel antibiotics.

Journal article

Wang Z, Zhao S, Jiang S, Wang Y, Buck M, Matthews S, Liu Bet al., 2019, Resonance assignments of N-terminal receiver domain of sigma factor S regulator RssB from Escherichia coli, BIOMOLECULAR NMR ASSIGNMENTS, Vol: 13, Pages: 333-337, ISSN: 1874-2718

Journal article

Danson AE, Jovanovic M, Buck M, Zhang Xet al., 2019, Mechanisms of sigma(54)-Dependent Transcription Initiation and Regulation, JOURNAL OF MOLECULAR BIOLOGY, Vol: 431, Pages: 3960-3974, ISSN: 0022-2836

Journal article

Gang S, Sharma S, Saraf M, Buck M, Schumacher Jet al., 2019, Analysis of Indole-3-acetic Acid (IAA) Production in Klebsiella by LC-MS/MS and the Salkowski Method, BIO-PROTOCOL, Vol: 9

Journal article

Danson A, Jovanovic M, Buck M, Zhang Xet al., 2019, Mechanisms of s54-dependent transcription initiation and regulation, Journal of Molecular Biology, ISSN: 0022-2836

Cellular RNA polymerase is a multi-subunit macromolecular assembly responsible for gene transcription, a highly regulated process conserved from bacteria to humans. In bacteria, sigma factors are employed to mediate gene-specific expression in response to a variety of environmental conditions. The major variant σ factor, σ54, has a specific role in stress responses. Unlike σ70-dependent transcription, which often can spontaneously proceed to initiation, σ54-dependent transcription requires an additional ATPase protein for activation. As a result, structures of a number of distinct functional states during the dynamic process of transcription initiation have been captured using the σ54 system with both x-ray crystallography and cryo electron microscopy, furthering our understanding of σ54-dependent transcription initiation and DNA opening. Comparisons with σ70 and eukaryotic polymerases reveal unique and common features during transcription initiation.

Journal article

Glyde R, Ye F, Jovanovic M, Kotta-Loizou I, Buck M, Zhang Xet al., 2018, Structures of bacterial RNA polymerase complexes reveal mechanisms of DNA loading and transcription initiation, Molecular Cell, Vol: 70, Pages: 1111-1120.e3, ISSN: 1097-2765

Gene transcription is carried out by multi-subunit RNA polymerases (RNAP).Transcription initiation is a dynamic multi-step process that involves the opening of the double stranded DNA to form a transcription bubble and delivery of the template strand deep into the RNAP for RNA synthesis. Applying cryo electron microscopy to a unique transcription system using 54 (N), the major bacterial variant sigma factor, we capture a new intermediate state at 4.1 Å where promoter DNA is caught at the entrance of the RNAP cleft. Combining with new structures of the open promotercomplex and an initial de novo transcribing complex at 3.4 and 3.7 Å respectively, our studies reveal the dynamics of DNA loading and mechanism of transcription bubble stabilisation that involves coordinated, large scale conformational changes of the universally conserved features within RNAP and DNA. In addition, our studies reveal a novel mechanism of strand separation by 54.

Journal article

Gang S, Sarah M, Waite C, Buck M, Schumacher Jet al., 2018, Mutualism between Klebsiella SGM 81 and Dianthus caryophyllus in modulating root plasticity and rhizospheric bacterial density, Plant and Soil, Vol: 424, Pages: 273-288, ISSN: 0032-079X

AimsDianthus caryophyllus is a commercially important ornamental flower. Plant growth promoting rhizobacteria are increasingly applied as bio-fertilisers and bio-fortifiers. We studied the effect of a rhizospheric isolate Klebsiella SGM 81 strain to promote D. caryophyllus growth under sterile and non-sterile conditions, to colonise its root system endophytically and its impact on the cultivatable microbial community. We identified the auxin indole-3-acetic acid (IAA) production of Klebsiella SGM 81 as major bacterial trait most likely to enhance growth of D. caryophyllus.MethodsipdC dependent IAA production of SGM 81 was quantified using LC-MS/MS and localised proximal to D. caryophyllus roots and correlated to root growth promotion and characteristic morphological changes. SGM 81 cells were localised on and within the plant root using 3D rendering confocal microscopy of gfp expressing SGM 81. Using Salkowski reagent IAA production was quantified and localised proximal to roots in situ. The effect of different bacterial titres on rhizosphere bacterial population was CFU enumerated on nutrient agar. The genome sequence of Klebsiella SGM 81 (accession number PRJEB21197) was determined to validate PGP traits and phylogenic relationships.ResultsInoculation of D. caryophyllus roots with Klebsiella SGM 81 drastically promoted plant growth when grown in agar and soil, concomitant with a burst in root hair formation, suggesting an increase in root auxin activity. We sequenced the Klebsiella SGM 81 genome, identified the presence of a canonical ipdC gene in Klebsiella SGM 81, confirmed bacterial production and secretion of IAA in batch culture using LC-MS/MS and localised plant dependent IAA production by SGM 81 proximal to roots. We found Klebsiella SGM 81 to be a rhizoplane and endophytic coloniser of D. caryophyllus roots in a dose dependent manner. We found no adverse effects of SGM 81 on the overall rhizospheric microbial population unless supplied to soil in very high

Journal article

Jovanovic M, Waite C, James E, Synn N, Simpson T, Kotta-Loizou I, Buck Met al., 2017, Functional Characterization of Key Residues in Regulatory Proteins HrpG and HrpV of Pseudomonas syringae pv. tomato DC3000, Molecular Plant-Microbe Interactions, Vol: 30, Pages: 656-665, ISSN: 0894-0282

The plant pathogen Pseudomonas syringae pv. tomato DC3000 uses a type III secretion system (T3SS) to transfer effector proteins into the host. The expression of T3SS proteins is controlled by the HrpL σ factor. Transcription of hrpL is σ54-dependent and bacterial enhancer-binding proteins HrpR and HrpS coactivate the hrpL promoter. The HrpV protein imposes negative control upon HrpR and HrpS through direct interaction with HrpS. HrpG interacts with HrpV and relieves such negative control. The sequence alignments across Hrp group I-type plant pathogens revealed conserved HrpV and HrpG amino acids. To establish structure–function relationships in HrpV and HrpG, either truncated or alanine substitution mutants were constructed. Key functional residues in HrpV and HrpG are found within their C-terminal regions. In HrpG, L101 and L105 are indispensable for the ability of HrpG to directly interact with HrpV and suppress HrpV-dependent negative regulation of HrpR and HrpS. In HrpV, L108 and G110 are major determinants for interactions with HrpS and HrpG. We propose that mutually exclusive binding of HrpS and HrpG to the same binding site of HrpV governs a transition from negative control to activation of the HrpRS complex leading to HrpL expression and pathogenicity of P. syringae.

Journal article

Glyde R, Ye F, Darbari V, Zhang N, Buck M, Zhang Xet al., 2017, Structures of RNA polymerase closed and intermediate complexes revealmechanisms of DNA opening and transcription initiation, Molecular Cell, Vol: 67, Pages: 106-116, ISSN: 1097-4164

Gene transcription is carried out by RNA polymerases (RNAPs). For transcription to occur, the closed promoter complex (RPc), where DNA is double stranded, must isomerize into an open promoter complex (RPo), where the DNA is melted out into a transcription bubble and the single-stranded template DNA is delivered to the RNAP active site. Using a bacterial RNAP containing the alternative σ54 factor and cryoelectron microscopy, we determined structures of RPc and the activator-bound intermediate complex en route to RPo at 3.8 and 5.8 Å. Our structures show how RNAP-σ54 interacts with promoter DNA to initiate the DNA distortions required for transcription bubble formation, and how the activator interacts with RPc, leading to significant conformational changes in RNAP and σ54 that promote RPo formation. We propose that DNA melting is an active process initiated in RPc and that the RNAP conformations of intermediates are significantly different from that of RPc and RPo.

Journal article

Gosztolai A, Schumacher J, Behrends V, Bundy JG, Heydenreich F, Bennett MH, Buck M, Barahona Met al., 2017, GlnK facilitates the dynamic regulation of bacterial nitrogen assimilation, Biophysical Journal, Vol: 112, Pages: 2219-2230, ISSN: 1542-0086

Ammonium assimilation in Escherichia coli is regulated by two paralogous proteins (GlnB and GlnK), which orchestrate interactions with regulators of gene expression, transport proteins, and metabolic pathways. Yet how they conjointly modulate the activity of glutamine synthetase, the key enzyme for nitrogen assimilation, is poorly understood. We combine experiments and theory to study the dynamic roles of GlnB and GlnK during nitrogen starvation and upshift. We measure time-resolved in vivo concentrations of metabolites, total and posttranslationally modified proteins, and develop a concise biochemical model of GlnB and GlnK that incorporates competition for active and allosteric sites, as well as functional sequestration of GlnK. The model predicts the responses of glutamine synthetase, GlnB, and GlnK under time-varying external ammonium level in the wild-type and two genetic knock-outs. Our results show that GlnK is tightly regulated under nitrogen-rich conditions, yet it is expressed during ammonium run-out and starvation. This suggests a role for GlnK as a buffer of nitrogen shock after starvation, and provides a further functional link between nitrogen and carbon metabolisms.

Journal article

Waite C, Schumacher J, Jovanovic M, Bennett M, Buck Met al., 2017, Evading plant immunity: feedback control of the T3SS in Pseudomonas syringae., Microbial Cell, Vol: 4, Pages: 137-139, ISSN: 2311-2638

Microbes are responsible for over 10% of the global yield losses in staple crops such as wheat, rice, and maize. Understanding the decision-making strategies that enable bacterial plant pathogens to evade the host immune system and cause disease is essential for managing their ever growing threat to food security. Many utilise the needle-like type III secretion system (T3SS) to suppress plant immunity, by injecting effector proteins that inhibit eukaryotic signalling pathways into the host cell cytoplasm. Plants can in turn evolve resistance to specific pathogens via recognition and blocking of the T3SS effectors, so leading to an ongoing co-evolutionary 'arms race' between pathogen and host pairs. The extracytoplasmic function sigma factor HrpL co-ordinates the expression of the T3SS regulon in the leaf-dwelling Pseudomonas syringae and similar pathogens. Recently, we showed that association of HrpL with a target promoter directly adjacent to the hrpL gene imposes negative autogenous control on its own expression level due to overlapping regulatory elements. Our results suggest that by down-regulating T3SS function, this fine-tuning mechanism enables P. syringae to minimise effector-mediated elicitation of plant immunity.

Journal article

Waite CJ, Schumacher J, Jovanovic M, Bennett M, Buck Met al., 2017, Negative autogenous control of the master type III secretion system regulator HrpL in Pseudomonas syringae, mBio, Vol: 8, ISSN: 2150-7511

The type III secretion system (T3SS) is a principal virulence determi-nant of the model bacterial plant pathogenPseudomonas syringae. T3SS effectorproteins inhibit plant defense signaling pathways in susceptible hosts and elicitevolved immunity in resistant plants. The extracytoplasmic function sigma factorHrpL coordinates the expression of most T3SS genes. Transcription ofhrpLis depen-dent on sigma-54 and the codependent enhancer binding proteins HrpR and HrpSforhrpLpromoter activation.hrpLis oriented adjacently to and divergently from theHrpL-dependent genehrpJ, sharing an intergenic upstream regulatory region. Weshow that association of the RNA polymerase (RNAP)-HrpL complex with thehrpJpromoter element imposes negative autogenous control onhrpLtranscription inP. syringaepv.tomatoDC3000. ThehrpLpromoter was upregulated in a ΔhrpLmu-tant and was repressed by plasmid-bornehrpL. In a minimalEscherichia coliback-ground, the activity of HrpL was sufficient to achieve repression of reconstitutedhrpLtranscription. This repression was relieved if both the HrpL DNA-binding func-tion and thehrp-box sequence of thehrpJpromoter were compromised, implyingdependence upon thehrpJpromoter. DNA-bound RNAP-HrpL entirely occluded theHrpRS and partially occluded the integration host factor (IHF) recognition elementsof thehrpLpromoterin vitro, implicating inhibition of DNA binding by these factorsas a cause of negative autogenous control. A modest increase in the HrpL concen-tration caused hypersecretion of the HrpA1 pilus protein but intracellular accumula-tion of later T3SS substrates. We argue that negative feedback on HrpL activity fine-tunes expression of the T3SS regulon to minimize the elicitation of plant defenses.

Journal article

Buck M, Mcdonald C, Jovanovic G, Wallace BA, Ces Oet al., 2016, Structure and function of PspA and Vipp1 N-terminal peptides:Insights into the membrane stress sensing and mitigation, BBA Biomembranes, Vol: 1859, Pages: 28-39, ISSN: 0005-2736

The phage shock protein (Psp) response maintains integrity ofthe innermembrane (IM) in responsetoextracytoplasmic stress conditionsand is widely distributed amongstenterobacteria. Its central componentPspA, a member of the IM30 peripheral membrane protein family, acts asa major effector of the systemthrough its direct association with the IM. Under non-stress conditions PspA also negatively regulates its own expression via direct interaction with the AAA+ ATPase PspF. PspA hasa counterpart in cyanobacteria calledVipp1, which is implicated in protection of the thylakoid membranes. PspA’s and Vipp1’s conservedN-terminal regions contain a putative amphipathic helix a (AHa) required for membranebinding.Anadjacent amphipathic helix b (AHb) in PspAis required for imposing negative control uponPspF.Here, purified peptides derived from the putative AH regions of PspA and Vipp1were used to directly probe their effectorand regulatory functions.We observed direct membrane-binding of AHaderived peptides and an accompanying change in secondary structure from unstructuredto alpha-helical establishing them as bonafidemembrane-sensing AH’s. The peptide-binding specificitiesand theireffects on membrane stability depend onmembrane anionic lipid content and stored curvature elastic stress,in agreement withfull length PspA and Vipp1 proteinfunctionalities. AHbof PspA inhibited the ATPase activity of PspF demonstratingits direct regulatory role. These findings provide new insight into the membrane binding and function of PspA and Vipp1and establish that synthetic peptides can be used to probe the structure-function of the IM30 protein family.

Journal article

Zhang N, Darbari VC, Glyde R, Zhang X, Buck Met al., 2016, The bacterial enhancer-dependent RNA polymerase, Biochemical Journal, Vol: 473, Pages: 3741-3753, ISSN: 1470-8728

Transcription initiation is highly regulated in bacterial cells, allowing adaptive gene regulation in response to environment cues. One class of promoter specificity factor called sigma54 enables such adaptive gene expression through its ability to lock the RNA polymerase down into a state unable to melt out promoter DNA for transcription initiation. Promoter DNA opening then occurs through the action of specialized transcription control proteins called bacterial enhancer-binding proteins (bEBPs) that remodel the sigma54 factor within the closed promoter complexes. The remodelling of sigma54 occurs through an ATP-binding and hydrolysis reaction carried out by the bEBPs. The regulation of bEBP self-assembly into typically homomeric hexamers allows regulated gene expression since the self-assembly is required for bEBP ATPase activity and its direct engagement with the sigma54 factor during the remodelling reaction. Crystallographic studies have now established that in the closed promoter complex, the sigma54 factor occupies the bacterial RNA polymerase in ways that will physically impede promoter DNA opening and the loading of melted out promoter DNA into the DNA-binding clefts of the RNA polymerase. Large-scale structural re-organizations of sigma54 require contact of the bEBP with an amino-terminal glutamine and leucine-rich sequence of sigma54, and lead to domain movements within the core RNA polymerase necessary for making open promoter complexes and synthesizing the nascent RNA transcript.

Journal article

Bonato P, Alves LR, Osaki JH, Rigo LU, Pedrosa FO, Souza EM, Zhang N, Schumacher J, Buck M, Wassem R, Chubatsu LSet al., 2016, The NtrY/NtrX two-component system is involved in controlling nitrate assimilation in Herbaspirillum seropedicae strain SmR1., FEBS Journal, Vol: 283, Pages: 3919-3930, ISSN: 1742-4658

Herbaspirillum seropedicae is a diazotrophic β-Proteobacterium found endophytically associated with gramineae (Poaceae or graminaceous plants) such as rice, sorghum and sugar cane. In this work we show that nitrate-dependent growth in this organism is regulated by the master nitrogen regulatory two-component system NtrB/NtrC, and by NtrY/NtrX which functions to specifically regulate nitrate metabolism. NtrY is a histidine kinase sensor protein predicted to be associated with the membrane and NtrX is the response regulator partner. The ntrYntrX genes are widely distributed in Proteobacteria. In α-Proteobacteria they are frequently located downstream from ntrBC, whereas in β-Proteobacteria these genes are located downstream from genes encoding a RNA methyltransferase and a proline-rich protein with unknown function. The α-Proteobacteria NtrX protein has an AAA+ domain, absent in those from β-Proteobacteria. An ntrY mutant of H. seropedicae showed wild type fixing nitrogen phenotype, but the nitrate dependent growth was abolished. Gene fusion assays indicated that NtrY is involved in the expression of genes coding for the assimilatory nitrate reductase as well as the nitrate-responsive two-component system NarX/NarL (narK and narX promoters, respectively). The purified NtrX protein was capable of binding the narK and narX promoters, and the binding site at the narX promoter for the NtrX protein was determined by DNA footprinting. In silico analyses revealed similar sequences in other promoter regions of H. seropedicae that are related to nitrate assimilation, supporting the role of the NtrY/NtrX system in regulating nitrate metabolism in H. seropedicae. This article is protected by copyright. All rights reserved.

Journal article

Bradley RW, Buck M, Wang B, 2016, Recognizing and engineering digital-like logic gates and switches in gene regulatory networks, Current Opinion in Microbiology, Vol: 33, Pages: 74-82, ISSN: 1879-0364

A central aim of synthetic biology is to build organisms that can perform useful activities in response to specified conditions. The digital computing paradigm which has proved so successful in electrical engineering is being mapped to synthetic biological systems to allow them to make such decisions. However, stochastic molecular processes have graded input-output functions, thus, bioengineers must select those with desirable characteristics and refine their transfer functions to build logic gates with digital-like switching behaviour. Recent efforts in genome mining and the development of programmable RNA-based switches, especially CRISPRi, have greatly increased the number of parts available to synthetic biologists. Improvements to the digital characteristics of these parts are required to enable robust predictable design of deeply layered logic circuits.

Journal article

Engl C, Schafer J, Kotta-Loizou I, Buck Met al., 2016, Cellular and molecular phenotypes depending upon the RNA repair system RtcAB of Escherichia coli, Nucleic Acids Research, Vol: 44, Pages: 9933-9941, ISSN: 1362-4962

RNA ligases function pervasively across the three kingdoms of life for RNA repair, splicing and can be stress induced. The RtcB protein (also HSPC117, C22orf28, FAAP and D10Wsu52e) is one such conserved ligase, involved in tRNA and mRNA splicing. However, its physiological role is poorly described, especially in bacteria. We now show in Escherichia coli bacteria that the RtcR activated rtcAB genes function for ribosome homeostasis involving rRNA stability. Expression of rtcAB is activated by agents and genetic lesions which impair the translation apparatus or may cause oxidative damage in the cell. Rtc helps the cell to survive challenges to the translation apparatus, including ribosome targeting antibiotics. Further, loss of Rtc causes profound changes in chemotaxis and motility. Together, our data suggest that the Rtc system is part of a previously unrecognized adaptive response linking ribosome homeostasis with basic cell physiology and behaviour.

Journal article

Schafer J, Jovanovic G, Kotta-Loizou I, Buck Met al., 2016, A data comparison between a traditional and the single-step β-galactosidase assay, Data in Brief, Vol: 8, Pages: 350-352, ISSN: 2352-3409

This article describes reproducibility of a single-step automated β-galactosidase, and the equivalence of its data to the traditional assay ("Experiments in Molecular Genetics" [1]). This was done via a pairwise comparison of both methods using strains with Miller Unit [MU] values ranging from 0 to over 2000. The data presented in this article is associated with the research article entitled "A single-step method for mid to high throughput β-galactosidase assays in Escherichia coli using a microplate reader" [2].

Journal article

Zhang N, Jovanovic G, McDonald C, Ces O, Zhang X, Buck Met al., 2016, Transcription regulation and membrane stress management in enterobacterial pathogens, Advances in Experimental Medicine and Biology, Vol: 915, Pages: 207-230, ISSN: 0065-2598

Transcription regulation in a temporal and conditional manner underpins the lifecycle of enterobacterial pathogens. Upon exposure to a wide array of environmental cues, these pathogens modulate their gene expression via the RNA polymerase and associated sigma factors. Different sigma factors, either involved in general 'house-keeping' or specific responses, guide the RNA polymerase to their cognate promoter DNAs. The major alternative sigma54 factor when activated helps pathogens manage stresses and proliferate in their ecological niches. In this chapter, we review the function and regulation of the sigma54-dependent Phage shock protein (Psp) system-a major stress response when Gram-negative pathogens encounter damages to their inner membranes. We discuss the recent development on mechanisms of gene regulation, signal transduction and stress mitigation in light of different biophysical and biochemical approaches.

Journal article

Schafer J, Jovanovic G, Kotta-Loizou I, Buck Met al., 2016, Single-step method for β-galactosidase assays in Escherichia coli using a 96-well microplate reader, Analytical Biochemistry, Vol: 503, Pages: 56-57, ISSN: 1096-0309

Historically, the lacZ gene is one of the most universally used reporters of gene expression in molecular biology. Its activity can be quantified using an artificial substrate, o-nitrophenyl-ß-d-galactopyranoside (ONPG). However, the traditional method for measuring LacZ activity (first described by J. H. Miller in 1972) can be challenging for a large number of samples, is prone to variability, and involves hazardous compounds for lysis (e.g., chloroform, toluene). Here we describe a single-step assay using a 96-well microplate reader with a proven alternative cell permeabilization method. This modified protocol reduces handling time by 90%.

Journal article

Bradley RW, Buck M, Wang B, 2015, Tools and principles for microbial gene circuit engineering., Journal of Molecular Biology, Vol: 428, Pages: 862-888, ISSN: 1089-8638

Synthetic biologists aim to construct novel genetic circuits with useful applications through rational design and forward engineering. Given the complexity of signal processing that occurs in natural biological systems, engineered microbes have the potential to perform a wide range of desirable tasks that require sophisticated computation and control. Realising this goal will require accurate predictive design of complex synthetic gene circuits and accompanying large sets of quality modular and orthogonal genetic parts. Here we present a current overview of the versatile components and tools available for engineering gene circuits in microbes, including recently developed RNA-based tools that possess large dynamic ranges and can be easily programmed. We introduce design principles that enable robust and scalable circuit performance such as insulating a gene circuit against unwanted interactions with its context, and we describe efficient strategies for rapidly identifying and correcting causes of failure and fine-tuning circuit characteristics.

Journal article

Jovanovic G, Mehta P, McDonald C, Buck Met al., 2015, Promoter Order Strategy and Bacterial PspF Regulon Evolution, Evolutionary Biology: Biodiversification from Genotype to Phenotype, Editors: Pontarotti, Publisher: Springer, Pages: 263-283, ISBN: 978-3-319-19932-0

This book presents 20 selected contributions to the 18th Evolutionary Biology Meeting, which took place in September 2014 in Marseille.

Book chapter

Zhang N, Schaefer J, Sharma A, Rayner L, Zhang X, Tuma R, Stockley P, Buck Met al., 2015, Mutations in RNA Polymerase Bridge Helix and Switch Regions Affect Active-Site Networks and Transcript-Assisted Hydrolysis, Journal of Molecular Biology, Vol: 427, Pages: 3516-3526, ISSN: 1089-8638

In bacterial RNA polymerase (RNAP), the bridge helix and switch regions form an intricate network with the catalytic active centre and the main channel. These interactions are important for catalysis, hydrolysis and clamp domain movement. By targeting conserved residues in Escherichia coli RNAP, we are able to show that functions of these regions are differentially required during σ70-dependent and the contrasting σ54-dependent transcription activations and thus potentially underlie the key mechanistic differences between the two transcription paradigms. We further demonstrate that the transcription factor DksA directly regulates σ54-dependent activation both positively and negatively. This finding is consistent with the observed impacts of DksA on σ70-dependent promoters. DksA does not seem to significantly affect RNAP binding to a pre-melted promoter DNA but affects extensively activity at the stage of initial RNA synthesis on σ54-regulated promoters. Strikingly, removal of the σ54 Region I is sufficient to invert the action of DksA (from stimulation to inhibition or vice versa) at two test promoters. The RNAP mutants we generated also show a strong propensity to backtrack. These mutants increase the rate of transcript-hydrolysis cleavage to a level comparable to that seen in the Thermus aquaticus RNAP even in the absence of a non-complementary nucleotide. These novel phenotypes imply an important function of the bridge helix and switch regions as an anti-backtracking ratchet and an RNA hydrolysis regulator.

Journal article

McDonald C, Jovanovic G, Ces O, Buck Met al., 2015, Membrane Stored Curvature Elastic Stress Modulates Recruitment of Maintenance Proteins PspA and Vipp1, mBio, Vol: 6, Pages: e01188-15-e01188-15, ISSN: 2161-2129

Phage shock protein A (PspA), which is responsible for maintaining inner membrane integrity under stress in enterobacteria, and vesicle-inducting protein in plastids 1 (Vipp1), which functions for membrane maintenance and thylakoid biogenesis in cyanobacteria and plants, are similar peripheral membrane-binding proteins. Their homologous N-terminal amphipathic helices are required for membrane binding; however, the membrane features recognized and required for expressing their functionalities have remained largely uncharacterized. Rigorously controlled, in vitro methodologies with lipid vesicles and purified proteins were used in this study and provided the first biochemical and biophysical characterizations of membrane binding by PspA and Vipp1. Both proteins are found to sense stored curvature elastic (SCE) stress and anionic lipids within the membrane. PspA has an enhanced sensitivity for SCE stress and a higher affinity for the membrane than Vipp1. These variations in binding may be crucial for some of the proteins’ differing roles in vivo. Assays probing the transcriptional regulatory function of PspA in the presence of vesicles showed that a relief of transcription inhibition occurs in an SCE stress-specific manner. This in vitro recapitulation of membrane stress-dependent transcription control suggests that the Psp response may be mounted in vivo when a cell’s inner membrane experiences increased SCE stress.

Journal article

Yang Y, Darbari VC, Zhang N, Lu D, Glyde R, Wang Y-P, Winkelman JT, Gourse RL, Murakami KS, Buck M, Zhang Xet al., 2015, Structures of the RNA polymerase-sigma(54) reveal new and conserved regulatory strategies, Science, Vol: 349, Pages: 882-885, ISSN: 0036-8075

Transcription by RNA polymerase (RNAP) in bacteria requires specific promoter recognition by σ factors. The major variant σ factor (σ54) initially forms a transcriptionally silent complex requiring specialized adenosine triphosphate–dependent activators for initiation. Our crystal structure of the 450-kilodalton RNAP-σ54 holoenzyme at 3.8 angstroms reveals molecular details of σ54 and its interactions with RNAP. The structure explains how σ54 targets different regions in RNAP to exert its inhibitory function. Although σ54 and the major σ factor, σ70, have similar functional domains and contact similar regions of RNAP, unanticipated differences are observed in their domain arrangement and interactions with RNAP, explaining their distinct properties. Furthermore, we observe evolutionarily conserved regulatory hotspots in RNAPs that can be targeted by a diverse range of mechanisms to fine tune transcription.

Journal article

Schaefer J, Engl C, Zhang N, Lawton E, Buck Met al., 2015, Genome wide interactions of wild-type and activator bypass forms of σ54., Nucleic Acids Research, Vol: 43, Pages: 7280-7291, ISSN: 1362-4962

Enhancer-dependent transcription involving the promoter specificity factor σ(54) is widely distributed amongst bacteria and commonly associated with cell envelope function. For transcription initiation, σ(54)-RNA polymerase yields open promoter complexes through its remodelling by cognate AAA+ ATPase activators. Since activators can be bypassed in vitro, bypass transcription in vivo could be a source of emergent gene expression along evolutionary pathways yielding new control networks and transcription patterns. At a single test promoter in vivo bypass transcription was not observed. We now use genome-wide transcription profiling, genome-wide mutagenesis and gene over-expression strategies in Escherichia coli, to (i) scope the range of bypass transcription in vivo and (ii) identify genes which might alter bypass transcription in vivo. We find little evidence for pervasive bypass transcription in vivo with only a small subset of σ(54) promoters functioning without activators. Results also suggest no one gene limits bypass transcription in vivo, arguing bypass transcription is strongly kept in check. Promoter sequences subject to repression by σ(54) were evident, indicating loss of rpoN (encoding σ(54)) rather than creating rpoN bypass alleles would be one evolutionary route for new gene expression patterns. Finally, cold-shock promoters showed unusual σ(54)-dependence in vivo not readily correlated with conventional σ(54) binding-sites.

Journal article

Zhang N, Buck M, 2015, A Perspective on the Enhancer Dependent Bacterial RNA Polymerase, Biomolecules, Vol: 5, Pages: 1012-1019, ISSN: 2218-273X

Here we review recent findings and offer a perspective on how the major variant RNA polymerase of bacteria, which contains the sigma54 factor, functions for regulated gene expression. We consider what gaps exist in our understanding of its genetic, biochemical and biophysical functioning and how they might be addressed.

Journal article

Mehta P, Jovanovic G, Ying L, Buck Met al., 2015, Is the cellular and molecular machinery docile in the stationary phase of Escherichia coli?, Biochemical Society Transactions, Vol: 43, Pages: 168-171, ISSN: 1470-8752

Journal article

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