The new bacteriology

09:05-09:30 Bacteria:the first 2 billion years

Professor Andrew Knoll, Harvard University, USA

Abstract

The conventional fossil record of bones, shells, tracks and trails traces an evolutionary history of animals some 600 million years in duration.  Phylogenies, however, indicate that animals are evolutionary late-comers, implying a deeper, largely microbial history of life.  Geochronology, in turn, demonstrates the conventional fossil record documents only the last 15 percent of recorded earth history.  Microfossils, stromatolites, preserved lipids, and biologically informative isotopic ratios quadruple the known history of life, providing a substantial record of bacterial diversity and biogeochemical cycles in Proterozoic (2500-541 Ma) oceans that can be interpreted, at least broadly, in terms present day organisms and processes.  Archean (>2500 Ma) sedimentary rocks add an additional billion years to this account, but phylogenetic and functional details are limited.  Geochemistry provides a major constraint on early evolution, indicating that early bacteria were shaped by anoxic environments, with distinct patterns of major and micro-nutrient availability different.  The Archean biosphere may document Earth’s first iron age, as reduced Fe was the principal electron donor for photosynthesis, oxidized Fe the most abundant terminal electron acceptor for respiration and Fe a key cofactor in proteins.  With the permanent oxygenation of the atmosphere and surface ocean ca. 2400 Ma, photic zone O2 limited the access of photosynthetic bacteria to electron donors other than water, while expanding the inventory of oxidants available for respiration and chemoautotrophy.  Thus, nearly halfway through Earth history, the microbial underpinnings of functioning ecosystems began to resemble their present form.  

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09:30-10:00 Cell wall deficient (L-form) bacteria: from cell biology to the origins of life

Professor Jeff Errington FMedSci FRS, Director of the Centre for Bacterial Cell Biology, Newcastle University

Abstract

The peptidoglycan cell wall is a defining structure of the bacteria. It is the target for our best antibiotics and fragments of the wall trigger powerful innate immune responses against infection. Surprisingly, many bacteria can switch almost effortlessly into a cell wall deficient “L-form” state. These cells become completely resistant to many antibiotics and may be able to pass under the radar screen of our immune systems. Studies of L-forms have provided surprising insights into various aspects of bacterial cell physiology and biochemistry, as well as providing a model illuminating how the earliest true cells on the planet might have proliferated. Recent studies have revealed that rapid growth of L-forms, as well as accurate chromosome segregation and assembly of components of the division machinery, can occur in the absence of a cell wall, provided that the L-forms are artificially constrained into a cylindrical shape of appropriate dimensions.

Key references

Leaver et al., 2009, Nature 457, 849-853. Mercier et al., 2013, Cell 152, 997-1007. Errington, 2013, Open Biology 3, 120143. Mercier et al., 2014, eLife 04629. Kawai et al., 2015, Current Biology 25, 1613-1618.

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10:55-11:25 What sequence gazing tells us about bacteria

Professor Julian Parkhill FRS, Sanger Institute, UK

Abstract

Genomes represent not only the set of instructions for an organism, but also a record of how that set of instructions has evolved and adapted over short and long timescales, a record that is enhanced by the ability to compare related genomes. New technologies have allowed us to sequence very large numbers of closely-related bacterial genomes, enabling the construction of phylogenetic relationships within recently-evolved clones of human and animal pathogens. We can now see bacteria as measurably-evolving populations, amenable to analysis with Bayesian tools developed for the analysis of fast-evolving viruses. The resulting data can allow us to answer specific hypotheses, but perhaps more interestingly, these vast data sets allow us to explore without preconceptions, answering questions we didn’t know that we should be asking. Sequence gazing is hypothesis generating, and I will discuss some examples of novelty that has come from such analyses.

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11:25-11:55 The transformative CRISPR-Cas9 genome engineering technology: lessons learned from bacteria

Professor Emmanuelle Charpentier, Max Planck Institute for Infection Biology, Germany

Abstract

The RNA-programmable CRISPR-Cas9 system has recently emerged as a transformative technology in biological sciences, allowing rapid and efficient targeted genome engineering, chromosomal marking and gene regulation in a large variety of cells and organisms. In this system, the endonuclease Cas9 or catalytically inactive Cas9 variants are programmed with single guide RNAs (sgRNAs) to target site-specifically any DNA sequence of interest given the presence of a short sequence (Protospacer Adjacent Motif, PAM) juxtaposed to the complementary region between the sgRNA and target DNA.

Originally, CRISPR-Cas is an RNA-mediated adaptive immune system that protects bacteria and archaea from invading mobile genetic elements. Short crRNA (CRISPR RNA) molecules containing unique genome-targeting spacers guide Cas protein(s) to invading cognate nucleic acids to affect their maintenance. CRISPR-Cas has been classified into three main types and further subtypes. CRISPR-Cas9 originates from the type II system that has evolved unique molecular mechanisms for maturation of crRNAs and targeting of invading DNA, which my laboratory has identified in the human pathogen Streptococcus pyogenes. During the step of crRNA biogenesis, a unique CRISPR-associated RNA, tracrRNA, base pairs with the repeats of precursor-crRNA to form anti-repeat-repeat dual-RNAs that are cleaved by RNase III in the presence of Cas9, generating mature tracrRNA and intermediate forms of crRNAs. Following a second maturation event, the mature dual-tracrRNA-crRNAs guide Cas9 to cleave cognate target DNA and thereby affect the maintenance of invading genomes. We have shown that Cas9 can be programmed with sgRNAs mimicking the natural dual-tracrRNA-crRNAs to target site-specifically any DNA sequence of interest. I will discuss the biological roles of CRISPR-Cas9, the mechanisms involved, the evolution of type II CRISPR-Cas components in bacteria and the applications of CRISPR-Cas9 as a novel genome engineering technology.

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13:20-13:50 Mechanisms of local signalling by the second messenger c-di- GMP in Escherichia coli

Professor Regine Hengge, Humboldt Universitat, Germany

Abstract

Bacterial biofilms are architecturally and morphologically highly structured multicellular communities with essentially tissue-like properties. Structure and functions of a biofilm depend on an elaborate spatiotemporal control of the synthesis of an extracellular matrix that usually contains adhesins, amyloid fibres and exopolysaccharides (e.g. cellulose) (1). In E. coli the production of amyloid curli fibres and cellulose is activated by the biofilm regulator CsgD, whose expression requires the stationary phase sigma factor RpoS and the nucleotide second messenger c-di-GMP. The latter is synthesized by 12 diguanylate cyclases (DGC, with GGDEF domains) and degraded by 13 phosphodiesterases (PDE, with EAL domains). Some of these DGCs and PDEs show specialized functions in matrix control that are based on highly specific macromolecular interactions, i.e. 'local c-di-GMP signaling'.
The key player in the switch that turns on CsgD expression in cells that begin to starve, is the PDE PdeR (formerly YciR). It acts as a 'trigger PDE'  that – in response to the rising c-di-GMP level generated by RpoS-driven induction of the DGC DgcE – allows the equally RpoS-controlled DGC DgcM and the transcription factor MlrA to co-activate csgD transcription by an intriguing mechanism: PdeR initially inhibits both DgcM and MlrA by direct specific interactions, which are relieved when c-di-GMP levels get high enough for PdeR to efficiently bind and degrade c-di-GMP. As the prototype of a 'trigger PDE', PdeR thus combines three activities, i.e. it is (i) a direct and therefore locally acting antagonist for DgcM and MlrA, (ii) a sensor and effector for the cellular c-di-GMP level (driven up by DgcE), and (iii) a PDE (2). 
A second mode of local c-di-GMP signaling in E. coli is exemplified by DgcC and PdeK that directly interact with each other and with the cellulose synthase complex BcsA/BcsB. DgcC and PdeK thus dynamically determine the local c-di-GMP concentration in close vicinity to the regulatory c-di-GMP-binding PilZ domain of BcsA. However, PdeK does not act as a 'trigger PDE', i.e. its direct interaction with cellulose synthase does not have a direct regulatory impact, but rather allows it to act as a local sink for c-di-GMP and thus to specifically counteract local c-di-GMP accumulation driven by DgcC.

(1) Serra and Hengge (2014) Environ. Microbiol. 16, 1455-1471.
(2) Lindenberg et al. (2013) EMBO J. 32, 2001-2014.

13:50-14:20 Using images to understand structure, function and dynamics of Type VI secretion systems

Professor Marek Basler, University of Basel Biozentrum, Switzerland

Abstract

The bacterial Type VI secretion system (T6SS) is a large dynamic organelle that is functionally analogous to contractile tails of bacteriophages. T6SS is used by Gram-negative bacteria to inhibit adjacent cells via translocation of toxic effector proteins and thus plays an important role in bacterial pathogenesis and ecology. Time-lapse fluorescence light microscopy revealed that T6SS sheath, which powers the secretion, cycles between assembly, quick contraction, disassembly and re-assembly. Single cell analysis of subcellular localization of T6SS assembly revealed that T6SS organelle encoded by Pseudomonas aeruginosa H1-T6SS cluster is assembled and aimed to specifically retaliate against attack by other bacteria. I will present latest update on the structure, function and dynamics of T6SS as well on mechanisms of effector delivery and function in various T6SS+ organisms. I will also focus on the structure of the T6SS sheath solved by cryo-electron microscopy and the implications for T6SS dynamics and assembly. I will show examples of how various imaging techniques helped us to understand many aspects of T6SS.

15:35-16:05 From homeostasis to pathology: decrypting microbe-host symbiotic signals in the intestinal crypt

Professor Philippe Sansonetti ForMemRS, Institut Pasteur, France

Abstract

Our immune system has likely evolved under the double constraint to tolerate the commensal microbiota, particularly in the gut, where bacteria can reach astronomic numbers, and to quickly recognize and eliminate the few pathogenic bacteria that reach and colonize this same surface. The cross talks that allow the host to discriminate between the « good » microbes and the « bad » microbes are starting to be fully recognized.

We have focused on the intestinal crypt that accounts for epithelial regeneration, due to the presence of stem cells, their environmental niche and the transit amplifying epithelial compartment. We have identified a « Crypt-specific core microbiota (CSCM) » corresponding to a limited group of strictly aerobic bacteria that populate the cecal and colonic crypts of mammals. We can now decipher the molecular crosstalks between CSCM members and the crypt regenerative apparatus. For instance we have shown that, thanks to activation of a Nod-2 dependent signaling process by muramyl dipeptide from the microbiota, stem cells under stress express increased surviving capacities, thus securing better epithelial restitution. This illustrates the depth of our symbiosis with gut commensals and illuminates the function of Nod-2 beyond innate immune regulation. Alternatively, we have identified the intestinal crypt as the site of invasion by Shigella, a bona fide enteric pathogen. We are currently deciphiring the cross-talks occuring in these various situations.

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16:05-16:35 Microbiota-Genetic-Environment interplay in intestinal inflammation

Dr Benoit Chassaing, Institute for Biomedical Sciences, Georgia State University, USA.

Abstract

The intestinal tract is inhabited by a large and diverse community of microbes collectively referred to as the gut microbiota. While the gut microbiota provides important benefits to its host, disturbance of the microbiota–host relationship is associated with numerous chronic inflammatory diseases, including inflammatory bowel disease and metabolic syndrome. A primary means by which the intestine is protected from its microbiota is via multi-layered mucus structures that cover the intestinal surface, allowing the vast majority of gut bacteria to be kept at a safe distance from epithelial cells that line the intestine. Thus, agents that disrupt mucus–bacterial interactions might have the potential to promote diseases associated with gut inflammation. We recently reported that, in mice, relatively low concentrations of two commonly used emulsifiers, detergent-like molecules and ubiquitous component of processed foods, induced low-grade inflammation and obesity/metabolic syndrome in wild-type hosts and promoted robust colitis in mice predisposed to this disorder. Emulsifier-induced metabolic syndrome was associated with microbiota encroachment, altered species composition and increased pro-inflammatory potential. Use of germ-free mice and faecal transplants indicated that such changes in microbiota were necessary and sufficient for both low-grade inflammation and metabolic syndrome. These results support the emerging concept that perturbed host–microbiota interactions resulting in low-grade inflammation can promote adiposity and its associated metabolic effects. Moreover, they suggest that the broad use of emulsifying agents might be contributing to an increased societal incidence of obesity/metabolic syndrome and other chronic inflammatory diseases.

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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. 

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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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.

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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13:20-13:50 Inhibiting intracellular growth of Mycobacterium tuberculosis

Professor Stewart Cole FRS, Ecole Polytechnique Federale de Lausanne, Switzerland

Abstract

Tuberculosis (TB) was one of the first infectious diseases to be rationally treated and the golden age of TB drug discovery led not only to new antibiotics but also to the development of combination therapy.  Until recently, no new TB drugs had been developed since the 1960s but, in response to the HIV-pandemic and widespread multidrug resistance, intensive efforts have been made to establish a pipeline of candidate TB drugs. Mycobacterium tuberculosis primarily occupies an intracellular niche in humans and this constitutes an additional barrier to drug activity.  Using an innovative intracellular screen we sought new leads for drugs that kill tubercle bacilli ex vivo but not in vitro.  Screening a library of FDA-approved drugs led to the identification of the proton pump inhibitor, prevacid, a widely used over-the-counter heartburn remedy, as a prodrug that is activated by the host cell to kill M. tuberculosis.  In an extension of this screen, we discovered small molecule inhibitors of the ESX-1 protein secretion system with immunomodulatory activity.  ESX-1 is the major virulence determinant used by the tubercle bacillus so its inactivation results in attenuation not death, an unorthodox means of disease control.

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13:50-14:20 Phage-inducible chromosomal islands

Professor José Penades, University of Glasgow, UK

Abstract

Bacteria are successful as commensal organisms or pathogens in part because they adapt rapidly to selective pressures. Mobile genetic elements (MGEs) play a central role in this adaptation process and are a means to transfer genetic information (DNA) among and within bacterial species. During the last years, we and other have characterized a novel family of mobile staphylococcal pathogenicity islands, the SaPIs, which are the only source of several important superantigens, including toxic shock syndrome toxin-1 and enterotoxins B and C, as well as the source of other virulence factors related to host adaptation. In this talk we will report that similar elements occur widely in bacteria, comprising a unique class of mobile genetic elements, the phage-inducible chromosomal islands (PICIs). Remarkably, PICIs have an unprecedented dual role in gene transfer: they not only mediate their own transfer, but they independently direct the transfer of unlinked chromosomal segments containing virulence genes through a novel mechanism of phage-mediated transduction. These findings represent the discovery of a novel agency of horizontal dissemination of virulence and other important accessory genes among bacteria.

15:35-16:05 RNA-seq approaches to unveil noncoding RNA functions in bacterial pathogens

Professor Jörg Vogel, University of Würzburg, Germany

Abstract

Bacteria express many small RNAs for which the regulatory roles in pathogenesis have remained poorly understood due to a paucity of robust phenotypes in standard virulence assays. We have developed a generic ‘Dual RNA-seq’ approach to simultaneously profile RNA expression in pathogen and host during Salmonella infection to reveal the molecular impact of bacterial riboregulators. We identify a PhoP-activated small RNA, PinT, which upon bacterial internalization temporally controls the expression of both invasion-associated effectors and virulence genes required for intracellular survival. This riboregulatory activity causes pervasive changes in coding and noncoding transcripts of the host. Inter-species correlation analysis links PinT to host cell JAK–STAT signalling and we identify infection-specific alterations in multiple long noncoding RNAs. Our study provides a paradigm for a sensitive RNA-based analysis of intracellular bacterial pathogens and their hosts without physical separation, as well as a new discovery route for hidden functions of pathogen genes.

REFERENCES
Westermann AJ, Gorski SA, Vogel J (2012) Dual RNA-seq of pathogen and host. Nature Reviews Microbiology 10(9):618-30 
Westermann AJ, Förstner KU, Amman F, Barquist L, Chao Y, Schulte LN, Müller L, Reinhardt R, Stadler PF, Vogel J (2016) Dual RNA-seq unveils noncoding RNA functions in host-pathogen interactions. Nature, in press

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16:05-16:35 Translating microbial sequencing into diagnostic and public health microbiology: are we nearly there yet?

Professor Sharon Peacock CBE, FMedSci, Bloomsbury Research Institute, UK

Abstract

The latest generation of benchtop DNA sequencing platforms can provide an accurate whole genome sequence for a broad range of bacteria in less than a day. These could be employed in routine practice to more effectively contain and treat the spread of infectious disease, including multidrug resistant pathogens. This talk will focus on the opportunities that rapid microbial WGS presents for the investigation of nosocomial outbreaks caused by multidrug resistant bacteria, and the identification of genetic determinants of antimicrobial resistance associated with a stratified medicine approach to patient care. Barriers to implementation will also be discussed.

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