Bacteria adapt their physiology to environmental changes, through coordinated changes of gene transcription patterns and post translational modifications that entail metabolic adaptations. My group investigates the biochemical and cell signaling events underpinning such cellular adaptations in plant-microbe environments. In particular, we study plant associative nitrogen fixing bacteria such as Klebsiella oxytoca as plant nitrogen bio-fertilisers and Pseudomonas syringae as plant pathogen model. Apart from conventional in vitro and in vitro methods, we use synthetic and systems (-omics) biology approaches to tackle current fundamental challenges associated with the complexity of plant-microbe systems and apply this knowledge for systems metabolic engineering.
Bacterial EBPs activate the alternative sigma 54 RNA polymerase involved in bacterial stress adaptations and pathogenesis. Prominent EBPs are NtrC, NifA, PspF and the unusual EBP pair HrpRS, involved in nitrogen limitation, phage shock and plant pathogenesis, respectively. EBPs comprise an AAA ATPase that couple the energy from ATP hydrolysis to restructure the closed σ54 RNA polymerase-promoter complex into a transcriptionally competent open complex. The activity of the AAA domains is controlled by in cis domains, as is the case for NtrC, or by additional proteins in trans, as is the case for HrpRS or the phage shock protein PspF. Promoter specificity of EBPs relies on the C-terminal HTH domain(s) that bind to upstream enhancer elements. In order to function EBPs and probably most if not all AAA proteins, their AAA domains form oligomeric ring structures and ATP hydrolysis between the subunits is interdependent, probably to ensure coordinated motions of the complex to realise their function. Studying the biochemistry and structure of EBPs allow us to better understand the molecular mechanisms of how ATP hydrolysis in converted into physical work by these nano-machines and how they can be switched on and off.
Figure 1: Hexameric ring structure of EBP AAA domains. adopted from: Rappas, M., Schumacher, J., Beuron, F., Niwa, H., Bordes, P., Wigneshweraraj, S., Keetch, C. A., Robinson, C. V., Buck, M., and Zhang, X. (2005) Structural insights into the activity of enhancer-binding proteins, Science 307, 1972-1975.
Synthetic Transcription Factors
Bacteria usually have many EBPs that respond to different environmental or intracellular signals, switching on distinct σ54 RNA dependent promoters. The modular domain organisation of EBPs enabled to design a number of chimeric EBPs in order to rewire the signal input/ transcription output functionalities of these systems. Because of the functional interdependence of domains in multi-domain proteins through allosteric interactions, recombining non cognate domains commonly requires domain-domain interface design, a challenge we address using bio-informatics, structural and functional knowledge. It is thus possible to engineer synthetic EBPs that enable to trigger transcription of targeted genes using non-cognate inducing signals. Such synthetic proteins can provide proof of principle of for the rational design of EBPs with novel desired functionalities. These synthetic proteins are also valuable tools in studying system behaviour. For instance they can be used for temporal transcription control of targeted genes, uncoupled from the native feedback loops, to identify control level hierarchies and network cross talk.
EBPs and their regulatory systems
Two regulatory systems that involve EBPs are being studied, the aforementioned ntr regulatory system of Escherichia coli involving NtrC and the hypersensitive response and pathogenicity system of Pseudomonas syringae. Apart from more conventional genetic and biochemical approaches, multiple-reaction-monitoring (MRM) MS is applied to gain a more quantitative insights into the systems behaviour of these regulatory networks. This approach allows us to measure how the quantities of regulatory proteins change over time under varying conditions and monitor what post-translational modifications occur, as well as determining to what extend targeted proteins are expressed. For instance, the nitrogen status in the cell is sensed at various levels, largely through the key regulatory proteins PII and GlnK through uridylilation, which impact on NtrC activity to activate the glnk and other genes, and also act on the central nitrogen assimilatory enzyme glutamine synthetase by controlling its adenylation state. MRM-MS allows unravelling the time resolved protein adaptation response to a changing environment and we model such adaptation processes in collaboration with Profs. Michael Stumpf and Mauricio Barahona. The EBP pair HrpR-HrpS is unusual in that these close paralogues, are strictly co-dependent, raising questions about their potential benefit. HrpR-HrpS control genes that are required for plant infection and appear to be a defining feature of P. syringae pathogens (Fig 2). Pseudomonas syringae are important pathogens of agriculturally important crops, including tomato, rice, bean and kiwi. We employ MRM-MS to investigate how regulatory proteins upstream of the HrpR-HrpS regulatory cascade change under infectious conditions to better understand what triggers the infectious cycle in vitro and in planta.
Figure 2: Phylogenetic tree of HrpR/HrpS, indicating that the dual system is specific to Pseudomonas syringae plant pathogens compared to other pathogens with singly acting HrpS, adopted from: Jovanovic, M., James, E. H., Burrows, P. C., Rego, F. G., Buck, M., and Schumacher, J. (2011) Regulation of the co-evolved HrpR and HrpS AAA proteins required for Pseudomonas syringae pathogenicity, Nat Commun 2, 177.
Gang S, Saraf M, Waite C, et al. Mutualism between Klebsiella SGM 81 and Dianthus caryophyllus in modulating root plasticity and rhizospheric bacterial density. Plant & Soil 2017;. in pres
Waite C, Schumacher J, Jovanovic M, et al. Negative Autogenous Control of the Master Type III Secretion System Regulator HrpL in Pseudomonas syringae. mBio 2017;8(1) doi: 10.1128/mBio.02273-16 [published Online First: 2017/01/26]
Gosztolai A, Schumacher J, Behrends V, et al. GlnK Facilitates the Dynamic Regulation of Bacterial Nitrogen Assimilation. Biophys J 2017;112(10):2219-30. doi: 10.1016/j.bpj.2017.04.012 [published Online First: 2017/05/26]
Schumacher J. Native and synthetic gene regulation during adaptation to nitrogen limitation stress. In: Bruijn FJD, ed. Environmental Control of Gene Expression and Adaptation in Bacteria. 1st ed: Blackwell/ Wiley 2016:24.
Bonato P, Alves LR, Osaki JH, et al. The NtrY-NtrX two-component system is involved in controlling nitrate assimilation in Herbaspirillum seropedicae strain SmR1. The FEBS journal 2016;283(21):3919-30. doi: 10.1111/febs.13897 [published Online First: 2016/09/17]
Komorowski, Schumacher J, Behrends V, et al. Analog nitrogen sensing in Escherichia coli enables high fidelity information processing2015. doi: https://doi.org/10.1101/015792. In preparation for Proteomics.
Buck M, Engl C, Joly N, et al. In vitro and in vivo methodologies for studying the Sigma 54-dependent transcription. Methods Mol Biol 2015;1276:53-79. doi: 10.1007/978-1-4939-2392-2_4 [published Online First: 2015/02/11]
Wang BB, M.; Buck, M.; Schumacher, J. Synthetic transcription factors allow regulon wide control and shifting the Nitrogen/Carbon balance in bacteria. New biotechnology 2014;31:S22-S22.
Paloma Bonato; Lysangela R. Alves; Juliana H. Osaki; Liu Un Rigo; Fabio O. Pedrosa; Emanuel M. Souza; Nan Zhang, Jörg Schumacher, Martin Buck; Roseli Wassem; Leda S. Chubatsu. (2016). The NtrY/NtrX two-component system controls nitrate metabolism in Herbaspirillum seropedicae SmR1. The FEBS Journal 283(21):3919-3930.
Schumacher, J. $ (2016) Native and synthetic gene regulation during adaptation to nitrogen limitation stress, In Environmental Control of Gene Expression and Adaptation in Bacteria. (Bruijn, F. J. D., Ed.) 1st ed., p 24, Blackwell/ Wiley. ISBN: 9781119004882
Buck, M., Engl, C., Joly, N., Jovanovic, G., Jovanovic, M., Lawton, E., McDonald, C., Schumacher, J., Waite, C., and Zhang, N. (2015) In vitro and in vivo methodologies for studying the sigma 54-dependent transcription, Methods Mol Biol 1276, 53-79.
Wang, B. B., M.; Buck, M.; Schumacher, J.$ (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.
Jovanovic, M., Lawton, E., Schumacher, J., and Buck, M. (2014). Interplay among Pseudomonas syringae HrpR, HrpS and HrpV proteins for regulation of the type III secretion system. FEMS Microbiol Lett 356, 201-211.
Schumacher, J. $ , Waite, C.J., Bennett, M.H., Perez, M.F., Shethi, K., and Buck, M. (2014). Differential secretome analysis of Pseudomonas syringae pv tomato using gel-free MS proteomics. Frontiers in plant science 5, 242.
Wang, B., Barahona, M., Buck, M., and Schumacher, J. $ (2013) Rewiring cell signalling through chimaeric regulatory protein engineering, Biochem Soc Trans 41, 1195-1200.
Schumacher, J. $ , Behrends, V., Pan, Z., Brown, D. R., Heydenreich, F., Lewis, M. R., Bennett, M. H., Razzaghi, B., Komorowski, M., Barahona, M., Stumpf, M. P., Wigneshweraraj, S., Bundy, J. G., and Buck, M. (2013) Nitrogen and carbon status are integrated at the transcriptional level by the nitrogen regulator NtrC in vivo, mBio 4, e00881-00813.
Rainey P and Zhang X: F1000Prime Recommendation of [Schumacher J et al., MBio 2013, 4(6):e00881-13]. In F1000Prime, 14 Jan 2014; DOI: 10.3410/f.718181806.793489422.
Dixon R: F1000Prime Recommendation of [Schumacher J et al., MBio 2013, 4(6):e00881-13]. In F1000Prime, 07 Apr 2014; DOI: 10.3410/f.718181806.793489438.
Galvao, C. W., Souza, E. M., Etto, R. M., Pedrosa, F. O., Chubatsu, L. S., Yates, M. G., Schumacher, J., Buck, M., and Steffens, M. B. (2012) The RecX protein interacts with the RecA protein and modulates its activity in Herbaspirillum seropedicae, Brazilian journal of medical and biological research, 45, 1127-1134.
Jovanovic, M., James, E. H., Burrows, P. C., Rego, F. G. M., Buck, M., and Schumacher, J. $ (2010) Regulation of the co-evolved HrpR and HrpS AAA proteins required for Pseudomonas syringae pathogenicity Nature Commun.2, 177.
Burrows, P. C., Schumacher, J. $ *, Amartey, S., Ghosh, T., Burgis, T. A., Zhang, X., Nixon, B. T., and Buck, M. $ (2009) Functional roles of the pre-sensor I insertion sequence in an AAA bacterial enhancer binding protein, Mol Microbiol. 73, 519-533.
Wigneshweraraj, S., Bose, D., Burrows, P. C., Joly, N., Schumacher, J., Rappas, M., Pape, T., Zhang, X., Stockley, P., Severinov, K., and Buck, M. (2008) Modus operandi of the bacterial RNA polymerase containing the sigma54 promoter-specificity factor, Mol Microbiol 68, 538-546.
Schumacher, J$ ., Joly, N., Claeys-Bouuaert, I. L., Aziz, S. A., Rappas, M., Zhang, X., and Buck$ , M. (2008) Mechanism of homotropic control to coordinate hydrolysis in a hexameric AAA ring ATPase, J Mol Biol 381, 1-12.
Burrows, P. C., Wigneshweraraj, S., Bose, D., Joly, N., Schumacher, J., Rappas, M., Pape, T., Stockley, P. G., Zhang, X., and Buck, M. (2008) Visualizing the organization and reorganization of transcription complexes for gene expression, Biochem Soc Trans 36, 776-779.
Bose, D., Joly, N., Pape, T., Rappas, M., Schumacher, J., Buck, M., and Zhang, X. (2008) Dissecting the ATP hydrolysis pathway of bacterial enhancer-binding proteins, Biochem Soc Trans 36, 83-88.
Schumacher, J$ ., Joly, N., Rappas, M., Bradley, D., Wigneshweraraj, S. R., Zhang, X., and Buck, M. $ (2007) Sensor I threonine of the AAA ATPase transcriptional activator PspF is involved in coupling nucleotide triphosphate hydrolysis to the restructuring of sigma 54-RNA polymerase, J Biol Chem 282, 9825-9833.
Schumacher, J$ ., Joly, N., Rappas, M., Zhang, X., and Buck, M. (2006) Structures and organisation of AAA enhancer binding proteins in transcriptional activation, J Struct Biol 156, 190-199.
Rappas, M., Schumacher, J*., Niwa, H., Buck, M., and Zhang, X. (2006) Structural basis of the nucleotide driven conformational changes in the AAA domain of transcription activator PspF, J Mol Biol 357, 481-492.
Joly, N., Schumacher, J., and Buck, M. (2006) Heterogeneous Nucleotide Occupancy Stimulates Functionality of Phage Shock Protein F, an AAA Transcriptional Activator, J Biol Chem 281, 34997-35007.
Buck, M., Bose, D., Burrows, P., Cannon, W., Joly, N., Pape, T., Rappas, M., Schumacher, J., Wigneshweraraj, S., and Zhang, X. (2006) A second paradigm for gene activation in bacteria, Biochem Soc Trans 34, 1067-1071.
Wigneshweraraj, S. R., Burrows, P. C., Bordes, P., Schumacher, J., Rappas, M., Finn, R. D., Cannon, W. V., Zhang, X., and Buck, M. (2005) The second paradigm for activation of transcription, Prog Nucleic Acid Res Mol Biol 79, 339-369.
Rappas, M., Schumacher, J., Beuron, F., Niwa, H., Bordes, P., Wigneshweraraj, S., Keetch, C. A., Robinson, C. V., Buck, M., and Zhang, X. (2005) Structural insights into the activity of enhancer-binding proteins, Science 307, 1972-1975.
Schumacher, J., Zhang, X., Jones, S., Bordes, P., and Buck, M. (2004) ATP-dependent transcriptional activation by bacterial PspF AAA protein, J Mol Biol 338, 863-875.
Cannon, W. V., Schumacher, J., and Buck, M. (2004) Nucleotide-dependent interactions between a fork junction-RNA polymerase complex and an AAA transcriptional activator protein, Nucleic Acids Res 32, 4596-4608.
Bordes, P., Wigneshweraraj, S. R., Schumacher, J., Zhang, X., Chaney, M., and Buck, M. (2003) The ATP hydrolyzing transcription activator phage shock protein F of Escherichia coli: identifying a surface that binds sigma 54, Proc Natl Acad Sci U S A 100, 2278-2283.
Zhang, X., Chaney, M., Wigneshweraraj, S. R., Schumacher, J., Bordes, P., Cannon, W., and Buck, M. (2002) Mechanochemical ATPases and transcriptional activation, Mol Microbiol 45, 895-903.
Elderkin, S., Jones, S., Schumacher, J., Studholme, D., and Buck, M. (2002) Mechanism of action of the Escherichia coli phage shock protein PspA in repression of the AAA family transcription factor PspF, J Mol Biol 320, 23-37.
Ton-Hoang, B., Salhi, M., Schumacher, J., Da Re, S., and Kahn, D. (2001) Promoter-specific involvement of the FixJ receiver domain in transcriptional activation, J Mol Biol 312, 583-589.
Chaney, M., Grande, R., Wigneshweraraj, S. R., Cannon, W., Casaz, P., Gallegos, M. T., Schumacher, J., Jones, S., Elderkin, S., Dago, A. E., Morett, E., and Buck, M. (2001) Binding of transcriptional activators to sigma 54 in the presence of the transition state analog ADP-aluminum fluoride: insights into activator mechanochemical action, Genes Dev 15, 2282-2294.
Da Re, S., Schumacher, J*., Rousseau, P., Fourment, J., Ebel, C., and Kahn, D. (1999) Phosphorylation-induced dimerization of the FixJ receiver domain, Mol Microbiol 34, 504-511.
Birck, C., Mourey, L., Gouet, P., Fabry, B., Schumacher, J., Rousseau, P., Kahn, D., and Samama, J. P. (1999) Conformational changes induced by phosphorylation of the FixJ receiver domain, Structure 7, 1505-1515.
Schumacher J. (1997) Molecular mechanism of the response regulator FixJ. Molecular Microbiology, Vol 3, 334 pages. Springer. Verlag ISBN 3-540-63873-3.