Chairs
Professor Judith Armitage FRS, University of Oxford, UK
Professor Judith Armitage FRS, University of Oxford, UK
Judy Armitage gained her BSc and PhD in Microbiology from University College London. After a period as a Lister Institute Research Fellow she was appointed a University Lecturer in Biochemistry at the University of Oxford. She became a Full Professor in 1996 and is a Professorial Fellow in Biochemistry at Merton College Oxford. She is a Member of EMBO, the American Academy of Microbiology and the Royal Society. She has spent her research career investigating bacterial behaviour, in particular how bacteria swim and how they control that swimming to reach an optimum environment for growth (chemotaxis). She uses interdisciplinary approaches to address these questions, including quantitative imaging of fluorescent proteins in living cells, combined with biophysics, molecular genetics, biochemistry and mathematical modelling.
09:00-09:30
Structural insights into ribosome-dependent activation of stringent control
Venki Ramakrishnan, President, The Royal Society
Abstract
In order to survive, bacteria continually sense, and respond to, environmental fluctuations. Stringent control represents a key bacterial stress response to nutrient starvation that leads to a rapid and comprehensive reprogramming of metabolic and transcriptional patterns. In general, transcription of genes for growth and proliferation are down-regulated, while those important for survival and virulence are favored. Starvation is sensed as depletion of one, or more, of the aminoacyl-tRNA pools results in accumulation of ribosomes stalled with non-aminoacylated (uncharged) tRNA in the ribosomal A site. RelA is recruited to stalled ribosomes, and activated to synthesize a hyperphosphorylated guanosine analog, (p)ppGpp, which acts as a pleiotropic second messenger. However, structural information for how RelA recognizes stalled ribosomes, its mechanism of activation, and how aminoacylated tRNAs are discriminated against, is missing. Here, we present the electron cryo-microscopy (cryo-EM) structure of RelA bound to the bacterial ribosome stalled with uncharged tRNA at 3 Å resolution. The structure reveals that RelA utilizes a distinct binding site compared to the translational factors, with a multi-domain architecture that wraps around a highly distorted A-site tRNA. The TGS domain of RelA binds the CCA tail to orient the free 3’ hydroxyl group of the terminal adenosine towards a beta-strand, such that an aminoacylated tRNA at this position would be sterically precluded. The structure supports a model where association of RelA with the ribosome suppresses auto-inhibition to activate synthesis of (p)ppGpp and initiate the stringent response. Since stringent control is responsible for the survival of pathogenic bacteria under stress conditions, and contributes to chronic infections and antibiotic tolerance, RelA represents a good target for the development of novel antibacterial therapeutics.
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Venki Ramakrishnan, President, The Royal Society
Venki Ramakrishnan, President, The Royal Society
Venki Ramakrishnan has a long-standing interest in ribosome structure and function. In 2000, his laboratory determined the atomic structure of the 30S ribosomal subunit and its complexes with ligands and antibiotics. This work has led to insights into how the ribosome “reads” the genetic code, as well as into various aspects of antibiotic function. In the last few years, Ramakrishan’s lab has determined the high-resolution structures of functional complexes of the entire ribosome at various stages along the translational pathway, which has led to insights into its role in protein synthesis during decoding, peptidyl transfer, translocation and termination. More recently his laboratory has been applying cryoelectron microscopy to study eukaryotic and mitochondrial translation. Since 1999, he has been on the scientific staff of the MRC Laboratory of Molecular Biology in Cambridge and he is currently President of the Royal Society.
09:30-10:00
In vivo remodelling of the bacterial flagellar motor and related protein complexes
Professor Judith Armitage FRS, University of Oxford, UK
Abstract
It is now clear that bacteria are not bags of diffusing chemicals, dividing in the middle to produce 2 daughter cells, but highly organised organisms. Using live cell imaging, molecular genetics and biophysics we have been following the exchange of proteins in response to the local environment in functioning nanomachines.. We have shown that both the bacterial flagellar motor and injectisome undergo remodelling in response to changes in their environment. Our recent data will be discussed, along with new methods for following in vivo protein dynamics over extended periods.
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Professor Judith Armitage FRS, University of Oxford, UK
Professor Judith Armitage FRS, University of Oxford, UK
Judy Armitage gained her BSc and PhD in Microbiology from University College London. After a period as a Lister Institute Research Fellow she was appointed a University Lecturer in Biochemistry at the University of Oxford. She became a Full Professor in 1996 and is a Professorial Fellow in Biochemistry at Merton College Oxford. She is a Member of EMBO, the American Academy of Microbiology and the Royal Society. She has spent her research career investigating bacterial behaviour, in particular how bacteria swim and how they control that swimming to reach an optimum environment for growth (chemotaxis). She uses interdisciplinary approaches to address these questions, including quantitative imaging of fluorescent proteins in living cells, combined with biophysics, molecular genetics, biochemistry and mathematical modelling.
10:55-11:25
c-di-AMP targets both arms of osmoprotection – potassium and osmolyte uptake systems
Professor Angelika Grundling, Imperial College London, UK
Abstract
Cyclic diadenosine monophosphate (c-di-AMP) is an essential second messenger in Staphylococcus aureus but it physiological function remains enigmatic. In a previous high throughput screen, four c-di-AMP binding partners were identified: a PII like protein of unknown function, a putative cation/proton antiporter, a gating component of a potassium uptake system, and a protein involved in the regulation of a second potassium transport system. The current study revealed an additional c-di-AMP binding protein, named OpuCA, a substrate-binding component of an osmoprotectant ABC uptake system. Biochemical assays showed that c-di-AMP is able to bind with high affinity and specificity to OpuCA. No other nucleotide tested could compete with c-di-AMP for binding even when added in 100-fold excess. Physiological tests indicate that the OpuC system plays a role in osmoprotection through the uptake of the compatible solute carnitine. Experiments are currently under way to determine mechanistically how c-di-AMP regulates the function of the S. aureus OpuC uptake system. The two main mechanisms, which bacteria utilize to respond to osmotic stress, are the rapid uptake of potassium and osmolytes. With the identification of OpuCA as a novel c-di-AMP binding protein, we now linked this signaling molecule to both arms of osmoprotection. This points towards c-di-AMP being a general regulator of the osmotic stress response in S. aureus.
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Professor Angelika Grundling, Imperial College London, UK
Professor Angelika Grundling, Imperial College London, UK
Angelika Gründling is a Reader in Molecular Microbiology at Imperial College London. The research in her lab focuses on the investigation of fundamental processes that are essential for the growth of Gram-positive bacterial pathogens. She combines genetic, biochemical and in collaborative work structural approaches to provide mechanistic insight into cell wall synthesis and nucleotide signaling pathways in Staphylococcus aureus and Listeria monocytogenes. Angelika obtained her Ph.D. in Microbiology from the University of Vienna in 2000. She performed her postdoctoral training at the Harvard Medical School, where she investigated flagallar motility in the bacterial pathogen L. monocytogenes and at the University of Chicago, where she initiated her studies on the cell wall polymer lipoteichoic acid in S. aureus. Angelika relocated to Imperial College London in 2007 and started her independent research career. There, she continues her work on the bacterial cell wall and more recently on the essential signaling nucleotide c-di-AMP.
11:25-11:55
Remarkable functional convergence: Type I and Type II toxin-antitoxins induce persistence by a ‘magic spot’ dependent mechanism
Professor Kenn Gerdes, University of Copenhagen, Denmark
Abstract
Using single-cell technology, we showed previously that, in E. coli, the ubiquitous bacterial stress alarmone (p)ppGpp (Magic Spot) is a central regulator of both spontaneous and environmentally induced persistence1. The (p)ppGpp level varied stochastically in a population of exponentially growing cells and the high (p)ppGpp level in the rare cells induced persistence. Persister cell formation depended on 10 type II toxin – antitoxin (TA) modules encoding RNases that inhibit translation by cleavage of mRNA or rRNA2, 3. A similar mechanism underlies persister formation by Salmonella4.
Recently, Jan Michiels’ group showed that a type I TA module (hokB/sokB) can induce persistence by a mechanism that also depends on (p)ppGpp and, and surprisingly, the highly conserved GTPase Obg5. Type I TAs encode small proteins that depolarize the cell membrane and confer membrane damage and rapid cell killing when overexpressed whereas moderate expression depletes the ATP pool5, 6, 7. Expression of these highly toxic proteins is repressed by cis-acting antisense RNAs. A complex mRNA folding pathway allows the mRNA to escaping irreversible inactivation by the antisense and expression of the toxin in the absence of transcription8.
Together, these results reveal Magic Spot as the central regulator and toxin - antitoxins as the central effectors of persistence in E. coli and other enterics.
References
1. Maisonneuve, E., Castro-Camargo, M. & Gerdes, K. (p)ppGpp controls bacterial persistence by stochastic induction of toxin-antitoxin activity. Cell 154, 1140-1150 (2013).
2. Germain, E., Roghanian, M., Gerdes, K. & Maisonneuve, E. Stochastic induction of persister cells by HipA through (p)ppGpp-mediated activation of mRNA endonucleases. Proc Natl Acad Sci U S A 112, 5171-6 (2015).
3. Maisonneuve, E., Shakespeare, L.J., Jørgensen, M.G. & Gerdes, K. Bacterial persistence by RNA endonucleases. Proc.Natl.Acad.Sci.U.S.A 108, 13206-13211 (2011).
4. Helaine, S. et al. Internalization of Salmonella by macrophages induces formation of nonreplicating persisters. Science 343, 204-8 (2014).
5. Verstraeten, N. et al. Obg and Membrane Depolarization Are Part of a Microbial Bet-Hedging Strategy that Leads to Antibiotic Tolerance. Mol Cell 59, 9-21 (2015).
6. Gerdes, K. et al. Mechanism of Postsegregational Killing by the Hok Gene-Product of the parB System of Plasmid R1 and Its Homology with the RelF Gene-Product of the Escherichia coli relB Operon. EMBO Journal 5, 2023-2029 (1986).
7. Gerdes, K., Rasmussen, P.B. & Molin, S. Unique Type of Plasmid Maintenance Function - Postsegregational Killing of Plasmid-Free Cells. Proceedings of the National Academy of Sciences of the United States of America 83, 3116-3120 (1986).
8. Moller-Jensen, J., Franch, T. & Gerdes, K. Temporal translational control by a metastable RNA structure. J Biol Chem 276, 35707-13 (2001).
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Professor Kenn Gerdes, University of Copenhagen, Denmark
Professor Kenn Gerdes, University of Copenhagen, Denmark
Kenn Gerdes is a Professor and centre leader at the Department of Biology at the University of Copenhagen. He has profound experience within the field of molecular microbiology. At present, his work is focussed on bacterial stress responses. Early on, Kenn discovered type I toxin – antitoxin (TA) modules and unravelled a complicated antisense RNA-regulated control system (hok/sok). Turning to the almost ubiquitous type II TA modules, he discovered several toxin targets and revealed how TA modules are regulated. In parallel, Kenn uncovered the first bacterial DNA segregation machinery (ParM). More recently, Kenn showed that type II TA modules are effectors of persistence (multidrug tolerance) in bacteria and that the “alarmone” (p)ppGpp induces persistence by activating TA modules. Based on the latter discoveries Kenn and his colleagues established the Centre for Bacterial Stress Response and Persistence (BASP) funded by the Danish National Research Foundation, the Novo Nordisk Foundation and the ERC.