Structural insights into ribosome-dependent activation of stringent control
Venki Ramakrishnan, President, The Royal Society
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.
In vivo remodelling of the bacterial flagellar motor and related protein complexes
Professor Judith Armitage FRS, University of Oxford, UK
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.
c-di-AMP targets both arms of osmoprotection – potassium and osmolyte uptake systems
Professor Angelika Grundling, Imperial College London, UK
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.
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
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.
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).