45 results found
Sharma S, Gang S, Schumacher J, et al., 2021, Genomic appraisal of Klebsiella PGPB isolated from soil to enhance the growth of barley, GENES & GENOMICS, Vol: 43, Pages: 869-883, ISSN: 1976-9571
Gang S, Sharma S, Saraf M, et al., 2021, Bacterial indole-3-acetic acid influences soil nitrogen acquisition in barley and chickpea, Plants, Vol: 10, ISSN: 2223-7747
Farming of barley and chickpea is nitrogen (N) fertilizer dependent. Using strategies that increase the nitrogen use efficiency (NUE) and its components, nitrogen uptake efficiency (NUpE) and nitrogen utilization efficiency (NUtE) would reduce the N fertilizer application in the soil and its adverse environmental effects. We evaluated the effects of three different strains of diazotroph Klebsiella (K.p. SSN1, K.q. SGM81, and K.o. M5a1) to understand the role of biological nitrogen fixation (BNF) and bacterial indole-3-acetic acid (IAA) on NUE of the plants. A field study revealed that K.p. SSN1 results in profound increment of root surface area by eightfold and threefold compared to uninoculated (control) in barley and chickpea, respectively. We measured significant increase in the plant tissue nitrogen, chlorophyll content, protein content, nitrate reductase activity, and nitrate concentration in the inoculated plants (p ≤ 0.05). Treated barley and chickpea exhibited higher NUE and the components compared to the control plants (K.p. SSN1 ≥ K.q. SGM81> K.o. M5a1). Specifically, K.q. SGM81 treatment in barley increased NUpE by 72%, while in chickpea, K.p. SSN1 increased it by 187%. The substantial improvement in the NUpE and NUE by the auxin producers K.p. SSN1 and K.q. SGM81 compared with non-auxin producer K.o. M5a1 was accompanied by an augmented root architecture suggesting larger contribution of IAA over marginal contribution of BNF in nitrogen acquisition from the soil.
Varghese F, Kabasakal BV, Cotton CA, et al., 2019, A low-potential terminal oxidase associated with the iron-only nitrogenase from the nitrogen-fixing bacterium Azotobacter vinelandii, Journal of Biological Chemistry, Vol: 294, Pages: 9367-9376, ISSN: 0021-9258
The biological route for nitrogen gas entering the biosphere is reduction to ammonia by the nitrogenase enzyme, which is inactivated by oxygen. Three types of nitrogenase exist, the least studied of which is the iron-only nitrogenase. The Anf3 protein in the bacterium Rhodobacter capsulatus is essential for diazotrophic (i.e. nitrogen-fixing) growth with the iron-only nitrogenase, but its enzymatic activity and function are unknown. Here, we biochemically and structurally characterize Anf3 from the model diazotrophic bacterium Azotobacter vinelandii. Determining the Anf3 crystal structure to atomic resolution, we observed that it is a dimeric flavocytochrome with an unusually close interaction between the heme and the flavin adenine dinucleotide cofactors. Measuring the reduction potentials by spectroelectrochemical redox titration, we observed values of -420 ± 10 mV and -330 ± 10 mV for the two FAD potentials and -340 ± 1 mV for the heme. We further show that Anf3 accepts electrons from spinach ferredoxin and that Anf3 consumes oxygen without generating superoxide or hydrogen peroxide. We predict that Anf3 protects the iron-only nitrogenase from oxygen inactivation by functioning as an oxidase in respiratory protection, with flavodoxin or ferredoxin as the physiological electron donors.
Gang S, Sharma S, Saraf M, et al., 2019, Analysis of Indole-3-acetic Acid (IAA) Production in Klebsiella by LC-MS/MS and the Salkowski Method, BIO-PROTOCOL, Vol: 9
Schumacher J, Waite C, Wang B, 2019, Synthetic transcription factors allowtuneable synthetic control of the complex bacterial nor regulon, EMBO: Creating is Understanding: Synthetic Biology Masters Complexity
Schumacher J, Waite C, 2018, In vivo absolute and relative Nif protein abundances of Klebsiella oxytoca, 13th European Nitrogen Fixation Conference
Gang S, Sarah M, Waite C, et 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
Liu Q, Schumacher J, Wan X, et al., 2017, Orthogonality and burdens of heterologous AND gate gene circuits in E. coli, ACS Synthetic Biology, Vol: 7, Pages: 553-564, ISSN: 2161-5063
Synthetic biology approaches commonly introduce heterologous gene networks into a host to predictably program cells, with the expectation of the synthetic network being orthogonal to the host background. However, introduced circuits may interfere with the host’s physiology, either indirectly by posing a metabolic burden and/or through unintended direct interactions between parts of the circuit with those of the host, affecting functionality. Here we used RNA-Seq transcriptome analysis to quantify the interactions between a representative heterologous AND gate circuit and the host Escherichia coli under various conditions including circuit designs and plasmid copy numbers. We show that the circuit plasmid copy number outweighs circuit composition for their effect on host gene expression with medium-copy number plasmid showing more prominent interference than its low-copy number counterpart. In contrast, the circuits have a stronger influence on the host growth with a metabolic load increasing with the copy number of the circuits. Notably, we show that variation of copy number, an increase from low to medium copy, caused different types of change observed in the behaviour of components in the AND gate circuit leading to the unbalance of the two gate-inputs and thus counterintuitive output attenuation. The study demonstrates the circuit plasmid copy number is a key factor that can dramatically affect the orthogonality, burden and functionality of the heterologous circuits in the host chassis. The results provide important guide for future efforts to design orthogonal and robust gene circuits with minimal unwanted interaction and burden to their host.
Gosztolai A, Schumacher J, Behrends V, et 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.
Waite C, Schumacher J, Jovanovic M, et 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.
Schumacher J, 2017, Proceedings of the 20th International Congress on Nitrogen Fixation, 20th International Congress on Nitrogen Fixation
Waite CJ, Schumacher J, Jovanovic M, et 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.
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.
Bruijn FJD, 2016, Stress and Environmental Regulation of Gene Expression and Adaptation in Bacteria, 2 Volume Set, Publisher: John Wiley & Sons, ISBN: 9781119004882
Covering the full breadth of current stress and environmental control of gene expression studies and expanding it towards future advances in the field, these two volumes are a one-stop reference for (non) medical molecular geneticists ...
Schumacher J, 2016, Synthetic Rebalancing of Nitrogen Fixation and Nitrogen Assimilation in Diazotrophs, 12th European Nitrogen Fixation Conference
Schumacher J, 2016, Systems metabolic engineering to rebalance nitrogen fixation and nitrogen assimilation in diazotrophs, BioSynSys
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.
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
Schumacher J, Behrends V, Pan Z, et al., 2013, Nitrogen and Carbon Status Are Integrated at the Transcriptional Level by the Nitrogen Regulator NtrC In Vivo, MBIO, Vol: 4, ISSN: 2150-7511
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
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.
Jovanovic M, James EH, Burrows PC, et al., 2011, Regulation of the co-evolved HrpR and HrpS AAA plus proteins required for Pseudomonas syringae pathogenicity, NATURE COMMUNICATIONS, Vol: 2, ISSN: 2041-1723
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
Burrows PC, Schumacher J, Amartey S, et al., 2009, Functional roles of the pre-sensor I insertion sequence in an AAA plus bacterial enhancer binding protein, Molecular Microbiology, Vol: 73, Pages: 519-533, ISSN: 0950-382X
Molecular machines belonging to the AAA+ superfamily of ATPases use NTP hydrolysis to remodel their versatile substrates. The presence of an insertion sequence defines the major phylogenetic pre‐sensor I insertion (pre‐SIi) AAA+ superclade. In the bacterial σ54‐dependent enhancer binding protein phage shock protein F (PspF) the pre‐SIi loop adopts different conformations depending on the nucleotide‐bound state. Single amino acid substitutions within the dynamic pre‐SIi loop of PspF drastically change the ATP hydrolysis parameters, indicating a structural link to the distant hydrolysis site. We used a site‐specific protein–DNA proximity assay to measure the contribution of the pre‐SIi loop in σ54‐dependent transcription and demonstrate that the pre‐SIi loop is a major structural feature mediating nucleotide state‐dependent differential engagement with Eσ54. We suggest that much, if not all, of the action of the pre‐SIi loop is mediated through the L1 loop and relies on a conserved molecular switch, identified in a crystal structure of one pre‐SIi variant and in accordance with the high covariance between some pre‐SIi residues and distinct residues outside the pre‐SIi sequence.
Burrows PC, Wigneshweraraj S, Bose D, et al., 2008, Visualizing the organization and reorganization of transcription complexes for gene expression, BIOCHEMICAL SOCIETY TRANSACTIONS, Vol: 36, Pages: 776-779, ISSN: 0300-5127
Schumacher J, Joly N, Claeys-Bouuaert IL, et al., 2008, Mechanism of homotropic control to coordinate hydrolysis in a hexameric AAA plus ring ATPase, JOURNAL OF MOLECULAR BIOLOGY, Vol: 381, Pages: 1-12, ISSN: 0022-2836
Wigneshweraraj S, Bose D, Burrows PC, et al., 2008, Modus operandi of the bacterial RNA polymerase containing the sigma(54) promoter-specificity factor, MOLECULAR MICROBIOLOGY, Vol: 68, Pages: 538-546, ISSN: 0950-382X
Bose D, Joly N, Pape T, et al., 2008, Dissecting the ATP hydrolysis pathway of bacterial enhancer-binding proteins, BIOCHEMICAL SOCIETY TRANSACTIONS, Vol: 36, Pages: 83-88, ISSN: 0300-5127
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