Publications
197 results found
Bonato P, Alves LR, Osaki JH, et 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.
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.
Engl C, Schafer J, Kotta-Loizou I, et 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.
Schafer J, Jovanovic G, Kotta-Loizou I, et 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].
Zhang N, Jovanovic G, McDonald C, et 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.
Schafer J, Jovanovic G, Kotta-Loizou I, et 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%.
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.
Jovanovic G, Mehta P, McDonald C, et 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.
Zhang N, Schaefer J, Sharma A, et 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.
McDonald C, Jovanovic G, Ces O, et 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.
Yang Y, Darbari VC, Zhang N, et 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.
Schaefer J, Engl C, Zhang N, et 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.
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.
Mehta P, Jovanovic G, Ying L, et 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
Wang B, Barahona M, Buck M, 2015, Amplification of small molecule-inducible gene expression via tuning of intracellular receptor densities, NUCLEIC ACIDS RESEARCH, Vol: 43, Pages: 1955-1964, ISSN: 0305-1048
Jovanovic G, Sheng X, Ale A, et al., 2015, Phosphorelay of non-orthodox two component systems functions through a bi-molecular mechanism <i>in vivo</i>: the case of ArcB, MOLECULAR BIOSYSTEMS, Vol: 11, Pages: 1348-1359, ISSN: 1742-206X
- Author Web Link
- Cite
- Citations: 5
Buck M, Engl C, Joly N, et al., 2015, In vitro and in vivo methodologies for studying the Sigma 54-dependent transcription., Methods Mol Biol, Vol: 1276, Pages: 53-79
Here we describe approaches and methods to assaying in vitro the major variant bacterial sigma factor, Sigma 54 (σ(54)), in a purified system. We include the complete transcription system, binding interactions between σ54 and its activators, as well as the self-assembly and the critical ATPase activity of the cognate activators which serve to remodel the closed promoter complexes. We also present in vivo methodologies that are used to study the impact of physiological processes, metabolic states, global signalling networks, and cellular architecture on the control of σ(54)-dependent gene expression.
Jovanovic G, Mehta P, Ying L, et al., 2014, Anionic lipids and the cytoskeletal proteins MreB and RodZ define the spatio-temporal distribution and function of membrane stress controller PspA in <i>Escherichia coli</i>, MICROBIOLOGY-SGM, Vol: 160, Pages: 2374-2386, ISSN: 1350-0872
- Author Web Link
- Open Access Link
- Cite
- Citations: 15
Brown DR, Barton G, Pan Z, et al., 2014, Combinatorial stress responses: direct coupling of two major stress responses in Escherichia coli, Microbial Cell, Vol: 1, Pages: 315-317, ISSN: 2311-2638
Nitrogen is an essential element for all life, and this isno different for the bacterial cell. Numerous cellularmacromolecules contain nitrogen, including proteins,nucleic acids and cell wall components. In Escherichiacoli and related bacteria, the nitrogen stress (Ntr) responseallows cells to rapidly sense and adapt to nitrogenlimitation by scavenging for alternative nitrogensources through the transcriptional activation oftransport systems and catabolic and biosynthetic operonsby the global transcriptional regulator NtrC. Nitrogen-starvedbacterial cells also synthesize the(p)ppGpp effector molecules of a second global bacterialstress response - the stringent response. Recently,we showed that the transcription of relA, the genewhich encodes the major (p)ppGpp synthetase in E.coli, is activated by NtrC during nitrogen starvation.Our results revealed that in E. coli and related bacteria,NtrC functions in combinatorial stress and serves tocouple two major stress responses, the Ntr responseand stringent response.
Wang B, Barahona M, Buck M, 2014, Engineering modular and tunable genetic amplifiers for scaling transcriptional signals in cascaded gene networks, Nucleic Acids Research
Synthetic biology aims to control and reprogram signal processing pathways within living cells so as to realize repurposed, beneficial applications. Here we report the design and construction of a set of modular and gain-tunable genetic amplifiers in Escherichia coli capable of amplifying a transcriptional signal with wide tunable-gain control in cascaded gene networks. The devices are engineered using orthogonal genetic components (hrpRS, hrpV and PhrpL) from the hrp (hypersensitive response and pathogenicity) gene regulatory network in Pseudomonas syringae. The amplifiers can linearly scale up to 21-fold the transcriptional input with a large output dynamic range, yet not introducing significant time delay or significant noise during signal amplification. The set of genetic amplifiers achieves different gains and input dynamic ranges by varying the expression levels of the underlying ligand-free activator proteins in the device. As their electronic counterparts, these engineered transcriptional amplifiers can act as fundamental building blocks in the design of biological systems by predictably and dynamically modulating transcriptional signal flows to implement advanced intra- and extra-cellular control functions.
Schumacher J, 2014, Differential secretome analysis of Pseudomonas syringae pv tomato using gel-free MS proteomics, Frontiers in Plant Science, Vol: 5
Jovanovic M, Lawton E, Schumacher J, et al., 2014, Interplay among Pseudomonas syringae HrpR, HrpS and HrpV proteins for regulation of the type III secretion system, FEMS MICROBIOLOGY LETTERS, Vol: 356, Pages: 201-211, ISSN: 0378-1097
Pseudomonas syringae pv. tomato DC3000, a plant pathogenic gram-negative bacterium, employs the type III secretion system (T3SS) to cause disease in tomato and Arabidopsis and to induce the hypersensitive response in nonhost plants. The expression of T3SS is regulated by the HrpL extracytoplasmic sigma factor. Expression of HrpL is controlled by transcriptional activators HrpR and HrpS and negative regulator HrpV. In this study, we analysed the organization of HrpRS and HrpV regulatory proteins and interplay between them. We identified one key residue I26 in HrpS required for repression by HrpV. Substitution of I26 in HrpS abolishes its interaction with HrpV and impairs interactions between HrpS and HrpR and the self-association of HrpS. We show that HrpS self-associates and can associate simultaneously with HrpR and HrpV. We now propose that HrpS has a central role in the assembly of the regulatory HrpRSV complex. Deletion analysis of HrpR and HrpS proteins showed that C-terminal parts of HrpR and HrpS confer determinants indispensable for their self-assembly.
Schumacher J, Wang B, Bonatto AC, et al., 2014, Synthetic transcription factors allow regulon wide control and shifting the nitrogen/carbon balance in bacteria, NEW BIOTECHNOLOGY, Vol: 31, Pages: S22-S22, ISSN: 1871-6784
- Author Web Link
- Cite
- Citations: 1
Brown DR, Barton G, Pan Z, et al., 2014, Nitrogen stress response and stringent response are coupled in Escherichia coli, Nature Communications, Vol: 5, ISSN: 2041-1723
Assimilation of nitrogen is an essential process in bacteria. The nitrogen regulation stress response is an adaptive mechanism used by nitrogen-starved Escherichia coli to scavenge for alternative nitrogen sources and requires the global transcriptional regulator NtrC. In addition, nitrogen-starved E. coli cells synthesize a signal molecule, guanosine tetraphosphate (ppGpp), which serves as an effector molecule of many processes including transcription to initiate global physiological changes, collectively termed the stringent response. The regulatory mechanisms leading to elevated ppGpp levels during nutritional stresses remain elusive. Here, we show that transcription of relA, a key gene responsible for the synthesis of ppGpp, is activated by NtrC during nitrogen starvation. The results reveal that NtrC couples these two major bacterial stress responses to manage conditions of nitrogen limitation, and provide novel mechanistic insights into how a specific nutritional stress leads to elevating ppGpp levels in bacteria.
Wallrodt I, Jelsbak L, Thomsen LE, et al., 2014, Removal of the phage-shock protein PspB causes reduction of virulence in <i>Salmonella enterica</i> serovar Typhimurium independently of NRAMP1, JOURNAL OF MEDICAL MICROBIOLOGY, Vol: 63, Pages: 788-795, ISSN: 0022-2615
- Author Web Link
- Cite
- Citations: 12
Engl C, Waite CJ, McKenna JF, et al., 2014, Chp8, a Diguanylate Cyclase from <i>Pseudomonas syringae</i> pv. Tomato DC3000, Suppresses the Pathogen-Associated Molecular Pattern Flagellin, Increases Extracellular Polysaccharides, and Promotes Plant Immune Evasion, MBIO, Vol: 5, ISSN: 2150-7511
- Author Web Link
- Cite
- Citations: 30
Lawton E, Jovanovic M, Joly N, et al., 2014, Determination of the Self-Association Residues within a Homomeric and a Heteromeric AAA plus Enhancer Binding Protein, JOURNAL OF MOLECULAR BIOLOGY, Vol: 426, Pages: 1692-1710, ISSN: 0022-2836
- Author Web Link
- Cite
- Citations: 5
Jovanovic G, Mehta P, McDonald C, et al., 2014, The N-Terminal Amphipathic Helices Determine Regulatory and Effector Functions of Phage Shock Protein A (PspA) in <i>Escherichia coli</i>, JOURNAL OF MOLECULAR BIOLOGY, Vol: 426, Pages: 1498-1511, ISSN: 0022-2836
- Author Web Link
- Cite
- Citations: 31
Sharma A, Leach RN, Gell C, et al., 2014, Domain movements of the enhancer-dependent sigma factor drive DNA delivery into the RNA polymerase active site: insights from single molecule studies, NUCLEIC ACIDS RESEARCH, Vol: 42, Pages: 5177-5190, ISSN: 0305-1048
- Author Web Link
- Open Access Link
- Cite
- Citations: 18
Zhang N, Gordiyenko Y, Joly N, et al., 2014, Subunit Dynamics and Nucleotide-Dependent Asymmetry of an AAA<SUP>+</SUP> Transcription Complex, JOURNAL OF MOLECULAR BIOLOGY, Vol: 426, Pages: 71-83, ISSN: 0022-2836
- Author Web Link
- Cite
- Citations: 10
This data is extracted from the Web of Science and reproduced under a licence from Thomson Reuters. You may not copy or re-distribute this data in whole or in part without the written consent of the Science business of Thomson Reuters.