33 results found
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
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 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.
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
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
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
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
Wigneshweraraj S, Burrows P, Bose D, et al., 2008, THE MECHANISTIC BASIS OF nif GENE ACTIVATION, Joint Conference of the 15th International Congress on Nitrogen Fixation/12 International Conference of the African-Association-for-Biological-Nitrogen-Fixation, Publisher: SPRINGER, Pages: 339-+, ISSN: 0924-1949
Schumacher J, Joly N, Rappas M, et al., 2007, Sensor I threonine of the AAA plus ATPase transcriptional activator PspF is involved in coupling nucleoticle triphosphate hydrolysis to the restructuring of sigma(54)-RNA polymerase, JOURNAL OF BIOLOGICAL CHEMISTRY, Vol: 282, Pages: 9825-9833, ISSN: 0021-9258
Joly N, Schumacher J, Buck M, 2006, Heterogeneous nucleotide occupancy stimulates functionality of phage shock protein F, an AAA plus transcriptional activator, JOURNAL OF BIOLOGICAL CHEMISTRY, Vol: 281, Pages: 34997-35007, ISSN: 0021-9258
Schumacher J, Joly N, Rappas M, et al., 2006, Structures and organisation of AAA plus enhancer binding proteins in transcriptional activation, JOURNAL OF STRUCTURAL BIOLOGY, Vol: 156, Pages: 190-199, ISSN: 1047-8477
Rappas M, Schumacher J, Niwa H, et al., 2006, Structural basis of the nucleotide driven conformational changes in the AAA(+) domain of transcription activator PspF, JOURNAL OF MOLECULAR BIOLOGY, Vol: 357, Pages: 481-492, ISSN: 0022-2836
Rappas M, Schumacher J, Beuron F, et al., 2005, Structural insights into the activity of enhancer-binding proteins, SCIENCE, Vol: 307, Pages: 1972-1975, ISSN: 0036-8075
Wigneshweraraj SR, Burrows PC, Bordes P, et al., 2005, The structural basis for nif gene activation, 14th International Nitrogen Fixation Congress, Publisher: SPRINGER, Pages: 59-63
Wigneshweplaraj SR, Burrows PC, Bordes P, et al., 2005, The second paradigm for activation of transcription, PROGRESS IN NUCLEIC ACID RESEARCH AND MOLECULAR BIOLOGY, VOL 79, Vol: 79, Pages: 339-369, ISSN: 0079-6603
Schumacher J, Zhang XD, Jones S, et al., 2004, ATP-dependent transcriptional activation by bacterial PspF AAA plus protein, JOURNAL OF MOLECULAR BIOLOGY, Vol: 338, Pages: 863-875, ISSN: 0022-2836
Cannon WV, Schumacher J, Buck M, 2004, Nucleotide-dependent interactions between a fork junction-RNA polymerase complex and an AAA plus transcriptional activator protein, NUCLEIC ACIDS RESEARCH, Vol: 32, Pages: 4596-4608, ISSN: 0305-1048
Bordes P, Wigneshweraraj SR, Schumacher J, et al., 2003, The ATP hydrolyzing transcription activator phage shock protein F of Escherichia coli: Identifying a surface that binds sigma(54), PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, Vol: 100, Pages: 2278-2283, ISSN: 0027-8424
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