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PROGRAMME AND ABSTRACTS

Welcome

The biannual Molecular Microbiology Mee�ng seeks to unite scien�sts who study the molecular of microbes with a focus on prokaryotes and fungi.

The Molecular Microbiology Mee�ng has previously been held five �mes at various loca�ons around Europe. In 2019 the mee�ng will be hosted by the Centre for Bacterial Cell Biology at Newcastle , UK.

The mee�ng will span two days and will be composed of both lectures and a poster session, allowing for PhD students, Postdoctoral Research Associates and Inves�gators to present their work. Short talks will be selected from submited abstracts. Early career scien�sts are encouraged to apply.

Please use the Hashtag #micronewcastle for posts and updates on Social Media.

The Organising Commitee,

Tracy Palmer, Jeff Errington, Henrik Strahl, Yulia Yuzenkova, Wendy Grunson, Stephanie Zuger-Legler and Heath Murray

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Sponsors

We would like to acknowledge the generous support of our sponsors:

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Contents

Welcome ...... 2 Sponsors ...... 3 Keynote Speakers ...... 5 General Information ...... 6 Conference venues ...... 6 Registration ...... 6 Talks ...... 6 AV facilities ...... 6 Poster presentations ...... 6 Internet ...... 6 Catering ...... 6 Sponsor Exhibitions ...... 6 Conference dinner ...... 6 Maps ...... 7 Useful Contacts ...... 10 Programme ...... 11 Monday 17th of June ...... 11 Programme ...... 13 Tuesday 18th of June ...... 13 Abstracts Oral Presentations ...... 15 Abstracts Posters ...... 34 List of participants ...... 39

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Keynote Speakers

Susan Gotesman, Na�onal Ins�tutes of Health (USA)

Tracy Palmer, (UK)

Jeanna Salje, University of (UK) / Rutgers (USA)

Tung Le, John Innes Centre (UK)

Waldemar Vollmer, Newcastle University, UK

Mar�n Thanbichler, Max Planck Ins�tute for Terrestrial Microbiology (DE)

Esther Angert, Cornell University (USA)

Morgan Beeby, Imperial Collage (UK)

Tanneke den Blaauwen, (NL)

Manjula Reddy, Centre for Cellular & Molecular Biology (CCMB) (IN)

Bert van den Berg, Newcastle University (UK)

David Grainger, (UK)

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General Information

Conference venues The conference will be taking place in the Catherine Cookson Building and the Baddiley-Clark building. The venues will be sign-posted. Registration The registration desk will be located next to the David Shaw Lecture Theatre in the Catherine Cook Building. Talks All talks will be held in the David Shaw Lecture Theatre in the Catherine Cookson building. A detailed schedule and titles are included in this booklet. AV facilities

• Presenter PC with a standard set of software /HDMI cable to connect your personal device • Speakers to amplify any audio from the PC or your connected personal device • Widescreen (16:9) Digital display (this may either be a data projector or a large screen) Poster presentations The posters will be set- in the Forum and the Seminar Room in the Baddiley-Clark Building. Internet

Eduroam Visiting academics from participating institutions can use eduroam over wireless to login at Newcastle using their local Login Name and Password.

Wireless Guest Service who are not able to connect to eduroam can use the free cloud WiFi network WiFi Guest to access the Internet using their own computer. Catering During the breaks there will be a tea/coffee buffet just outside the David Shaw Lecture Theatre. The lunch buffets will be in the Baddiley-Clark Building. Sponsor Exhibitions Our sponsors will set up stalls next to the David Shaw Lecture Theatre in the Catherine Cookson Building. Conference dinner The evening event will be held at the Wylam Brewery in Exhibition Park – a 10 minute walk from the main venues.

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Maps

FMS / CBCB FMS

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Useful Contacts

Newcastle University Webpage: www.ncl.ac.uk

Institute for Cell and Molecular Biosciences Reception: 0191 20 83492

Organising Committee Email: [email protected] or [email protected]

Online mapping: www.ncl.ac.uk/about/visit/maps

Campus Security General: 0191 208 6817

Emergency: 0191 208 6666

City Information www.newcastlegateshead.co.uk

National Rail Enquiries 08457 484950 / www.nationalrail.co.uk

Newcastle International Airport 0871 882 1121 / www.newcastleairport.com

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Programme

Monday 17th of June 08:00–09:00 Registration 09:00–09:10 Welcome – Jeff Errington

Session 1 Chair: Elisabeth Lowe 09:10–09:40 Tracy Palmer (Newcastle, UK) Genetic and biochemical analysis of the bacterial Tat system 09:40–09:55 Sam Hickman (Oxford, UK) Capturing protein transport in bacteria using ultra-long single-molecule tracking 09:55–10:10 Francesca Cianfanelli (Basel, CH) The PhoPQ two-component system enables Salmonella to adapt to tissue heterogeneity 10:10–10:40 Tanneke den Blaauwen (Amsterdam, NL) Periplasmic party 10:40–11:20 Coffee

Session 2 Chair: David Bolam 11:20–11:50 Bert van den Berg (Newcastle, UK) Outer membrane nutrient processosomes mediate glycan uptake by the human gut microbiota 11:50–12:05 Paula Salgado (Newcastle, UK) S-layer structure and function in C. difficile 12:05–12:20 Marvin Gohrbandt (Osnabrück, DE) Partitioning of proteins into liquid-disordered domains in phase-separated in vivo plasma membranes of Escherichia coli and Bacillus subtilis 12:20–12:35 Rosalyn Leaman (, UK) Illuminating the spatio-temporal dynamics of lipopolysaccharide in the Gram-negative bacterial outer membrane 12:35–12:50 Damon Huber (Birmingham, UK) Tale of a tail: how the C-terminal tail of SecA regulates its activity 12:50–14:00 Lunch

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Session 3 Chair: Paula Salgado 14:00–14:30 Jeanne Salje (Oxford, UK/Rutgers USA) Peptidoglycan in obligate intracellular bacteria 14:30–14:45 Helene Botella (Imperial , UK) Antibiotic tolerance in Mycobacteria 14:45–15:00 Emeline Lawaree (Oxford, UK) Impact of DNA ADP-ribosylation on DNA replication in enteropathogenic Escherichia coli 15:00–15:15 Jani Reddy Bolla (Oxford, UK) MmpL3 is a lipid transporter that binds trehalose monomycolate and phosphatidylethanolamine 15:15–15:30 David Adams (Lausanne, CH) DNA-uptake pili of Vibrio cholerae – one pilus, three fundamental functions 15:30–16:00 Coffee

Session 4 Chair: Henrik Strahl 16:00–16:30 Morgan Beeby (Imperial College, UK) How and why diverse bacterial flagellar motors evolved 16:30–17:00 Martin Thanbichler (Marburg, DE) TBD 17:00–19:00 Poster Session 19:00–22:00 Banquet

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Programme

Tuesday 18th of June Session 5 Chair: Yulia Yuzenkova 09:10–09:40 Susan Gottesman (NIH, USA) Silencing the silencers: new layers of sRNA regulation 09:40–09:55 Josh McQuail (Imperial College, UK) An unusual behaviour of Hfq in long-term nitrogen starved Escherichia coli 09:55–10:10 Kathrin Fröhlich (LMU Munich, DE) Post-transcriptional regulation by a cell-cycle controlled small RNA in Caulobacter crescentus 10:10–10:25 Karrera Djoko (Durham, UK) The interplay between bacterial copper homeostasis and carbon metabolism 10:25–10:40 Dennis Claessen (, NL) Exploitation of a shape-shifting Kitasatospora viridifaciens strain identifies a MurG-like protein required for peptidoglycan synthesis 10:40–11:20 Coffee

Session 6 Chair: Heath Murray 11:20–11:50 Tung Le (Norwich, UK) Determinants of specificity at a protein-DNA interface crucial for chromosome maintenance 11:50–12:05 Azhar Kabli (York, UK) Chromosome segregation in the thermophilic archaeon Sulfolobus solfataricus 12:05–12:20 Agnes Noy (York, UK) The structure and dynamics of DNA under supercoiling stress 12:20–12:35 Jean-Luc Ferat (Paris-Saclay, FR) Two strategies to load bacterial replicative helicases 12:35–14:00 Lunch

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Session 7 Chair: Fiona Cuskin 14:00–14:30 David Grainger (Birmingham, UK) A regulatory mechanism linking population density to natural competence in Vibrio cholerae 14:30–14:45 Jeanine Rismondo (Imperial College, UK) Balanced activity of multiple FtsW and RodA proteins is crucial for maintaining cell shape and antibiotic resistance in Listeria monocytogenes 14:45–15:15 Esther Angert (Cornell, USA) Thinking big: challenges faced and the simple solutions found by giant bacteria 15:15–15:45 Coffee

Session 8 Chair: Seamus Holden 15:45–16:15 Manjula Reddy (Hyderabad, IN) Regulation of lipopolysaccharide biosynthesis in response to fatty acid availability 16:15–16:30 Nathalie Reichmann (Oxford, UK) SEDS-bPBP pairs direct lateral and septal peptidoglycan synthesis in 16:30–17:00 Waldemar Vollmer (Newcastle, UK) Robust peptidoglycan synthesis and maintenance in E. coli 17:00–17:10 Closing – Tracy Palmer 17:10 Departure

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Abstracts Oral Presentations

Sam Hickman (Oxford, UK) Speaker Number 2

Capturing protein transport in bacteria using ultra-long single-molecule tracking

The twin-arginine translocation (Tat) machinery transports folded proteins across the cytoplasmic membrane of bacteria.

In E. coli Tat comprises from three membrane proteins TatA, TatB, and TatC. TatBC constitute a substrate- binding complex that recruits and oligomerises TatA to form the active translocon.

We have developed an ultra-long time scale single-molecule tracking method in live E. coli, which allows the kinetics of substrate transport to be determined under physiological conditions.

Distinguishing whether a substrate molecule has moved into the periplasm from the cytoplasm is technically challenging. To overcome this issue, we employ an amidase deficient strain in which chain of cells have segmented cytoplasms but the periplasmic space is continuous. This allows substrate transport across the cytoplasmic membrane to be clearly identified.

Now that we have the capability to capture transport, a range of Tat mutants can be used gain detailed mechanistic information about each step of the transport cycle.

Samuel J Hickman, Achillefs Kapanidis & Ben Berks

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Francesca Romana Cianfanelli (Basel, CH) Speaker Number 3

The PhoPQ two-component system enables Salmonella to adapt to tissue heterogeneity

Host tissues are characterised by complex anatomy imposing different stresses on invading pathogens. Such complexity results in pathogen subsets residing in distinct microenvironments while being able to respond to specific local conditions. These individual host-pathogen encounters have disparate outcomes and influence disease progression. In Salmonella, the PhoPQ two component system plays a crucial role in sensing such stresses and regulating many genes in response. However, the role of PhoPQ in adaptation to diverse microenvironments is still unclear. Flow cytometry of PhoP activity in single Salmonella cells isolated from infected mice revealed extensive heterogeneity in PhoPQ activation. Proteomics and microscopy analysis suggested that host cell type, within the same tissue, determines the level of PhoPQ activation with differential pH as a potential major driving force of heterogeneity. Together, our data show that intracellular pathogens can use a variety of strategies to adapt to different host cells with divergent stress conditions.

Francesca R. Cianfanelli and Dirk Bumann

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Paula Salgado (Newcastle, UK) Speaker Number 6

S-layer structure and function in C. difficile

Many bacteria are coated with a proteinaceous paracrystalline surface layer (S‑layer) implicated in growth and survival. Despite its key role in many bacteria, structural and functional details of complete S-layer proteins are still lacking.

In C. difficile, the most common cause of antibiotic-associated intestinal infections in humans and a significant cause of morbidity and mortality worldwide, the principal S-layer protein, SlpA displays sequence diversity between strains and has been linked to interactions with host.

Using a combination of X-ray crystallography and 2D electron diffraction, we determined the first complete crystal structure of an S-layer protein in a pathogenic bacterium and the overall assembly of the S-layer. Surprisingly, deleting exposed domains of SlpA resulted in a strain that has attenuated virulence in the mouse model, despite similar colonisation, sporulation and toxin levels, providing the first direct evidence that SlpA plays a role in disease severity.

Paola Lanzoni-Mangutchi, Anna Barwinska-Sendra, BugSLayers Consortium, Paula S. Salgado

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Marvin Gohrbandt (Osnabrück, DE) Speaker Number 7

Partitioning of proteins into liquid-disordered domains in phase-separated in vivo plasma membranes of Escherichia coli and Bacillus subtilis

Vital chemico-physical properties of cell membranes are determined by their lipid composition. Depletion of fluidity-promoting unsaturated fatty acids in a thermo-sensitive Escherichia coli strain or of branched-chain fatty acids in an auxotrophic Bacillus subtilis strain allowed gradual changes of the membrane fatty acid composition and the analysis of key membrane properties such as fluidity, miscibility, diffusion, and permeability in vivo. Here we show that the lower limit of membrane fluidity capable for supporting growth was associated with large-scale phase separation into liquid-ordered and liquid-disordered domains in both organisms, accompanied by exclusive partitioning of WALP23 and FOF1-ATP-synthase into the liquid- disordered phase. In vivo single-molecule tracking of FOF1 revealed a corresponding major reduction in lateral mobility. Thus, by demonstrating a liquid-ordered and disordered phase-separation and an associated protein partitioning for the first time in protein-crowded in vivo membranes, our results provide strong support for the corresponding in vitro and in silico models.

Marvin Gohrbandt, André Lipski, Zunera Baig, Rainer Kurre, Henrik Strahl & Gabriele Deckers-Hebestreit

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Rosalyn Leaman (York, UK) Speaker Number 8

Illuminating the spatio-temporal dynamics of lipopolysaccharide in the Gram-negative bacterial outer membrane

The Gram-negative bacterial outer membrane (OM) is an asymmetric bilayer with an inner leaflet composed of phospholipid and an outer leaflet of lipopolysaccharide (LPS). This LPS leaflet creates an impermeable barrier to environmental challenges including many commonly used antibiotics. It is known that OM proteins do not diffuse freely within the OM and are confined to `islands’. Previous results on LPS diffusion within the OM are contradictory with both mobile and immobile behaviour observed.

We have visualised LPS on the surface of bacteria using a metabolic labeling technique to incorporate a sugar analogue into LPS. This analogue can be fluorescently labeled with a specific bio-orthogonal click-it reaction. Using both fluorescence recovery after photobleaching and single-molecule tracking experiments we have demonstrated that LPS is unable to diffuse laterally in the OM. This immobility is of LPS polysaccharide structural complexity and dependent on metal ion mediated LPS-LPS interactions.

Rosalyn Leaman, Sam Lenton, Leonore Mantion, Martin A. Fascione, Dmitri O. Pushkin, Mark C. Coles, Christoph G. Baumann

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Damon Huber (Birmingham, UK) Speaker Number 9

Tale of a tail: how the C-terminal tail of SecA regulates its activity

In bacteria, the translocation of proteins across the cytoplasmic membrane by the Sec machinery requires SecA. SecA can bind ribosomes and recognise nascent substrate proteins, but the mechanism of recognition is unknown. We investigated the role of the C-terminal tail (CTT) of SecA in nascent substrate protein recognition. The CTT consists of a flexible linker (FLD) and a small metal-binding domain (MBD). Phylogenetic analysis and ribosome binding experiments indicated that the MBD interacts with 70S ribosomes. Disruption of the MBD alone and the entire CTT had opposing effects on ribosome binding, substrate-protein binding, ATPase activity and in vivo function, suggesting that the CTT influences the conformation of SecA. In addition, site-specific crosslinking experiments indicated that binding to nascent chains affects the interaction of SecA with the ribosome. Structural studies provided into the CTT-mediated conformational changes in SecA. Our results suggest a mechanism for nascent substrate protein recognition.

Mohammed Jamshad, Timothy J. Knowles, Scott A. White, Douglas G. Ward, Fiyaz Mohammed, Kazi Rahman, Gareth W. Hughes, Günter Kramer, Bernd Bukau, and Damon Huber

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Helene Botella (London, UK) Speaker Number 11

Antibiotic tolerance in Mycobacteria

Mycobacterium tuberculosis (Mtb)-the agent of tuberculosis (TB)-is one of several bacterial pathogens for which some clinical isolates are now resistant to most or all antibiotics approved for treatment of the infections they cause. Heritable antimicrobial resistance in Mtb emerges with interruption of treatment, and the long duration of TB treatment provides many opportunities for interruption. Prolonged treatment is necessary because of phenotypic tolerance, or persistence, a phenomenon by which genetically-sensitive bacteria transiently survive exposure to antibiotics. Although entrance by an individual cell into a persistent state is regulated by phenotypic rather than genotypic changes, paradoxically, mutations have been identified that increase the proportion of individual cells that become persisters within a given population. We have developed a novel method that uses a recombination strategy to enable the isolation of such mutants in Mtb that form during infection upon exposure to stresses imposed by host immunity and chemotherapy.

Helene Botella, Sabine Ehrt, Julien Vaubourgeix

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Emeline Lawaree (Oxford, UK) Speaker Number 12

Impact of DNA ADP-ribosylation on DNA replication in enteropathogenic Escherichia coli

Protein ADP-ribosylation is involved in fundamental cellular processes and has been extensively studied. However, the importance of DNA ADP-ribosylation is unknown.

In enteropathogenic Escherichia coli (EPEC), we have found that a toxin:antitoxin system catalyzes reversible DNA ADP-ribosylation. The toxin DarT ADP-ribosylates single-stranded DNA (ssDNA), while DarG, the antitoxin, is a glycohydrolase that removes ADP-ribose from DNA. Expression of DarT reduces bacterial viability by stalling DNA replication. Furthermore, we show that the SOS response is induced following expression of the toxin, indicating DNA ADP-ribosylation is perceived as a DNA damage by bacteria. In EPEC, we demonstrate that growth inhibition following DNA-ADP ribosylation is reversed by RecF-mediated homologous recombination, that is predicted to convert ssDNA ADP-ribosylation to modified double stranded DNA (dsDNA). This DNA modification is then eliminated by the nucleotide excision repair pathway that removes the dsDNA lesion.

Further experiments are underway to assess the role of DarTG in persistence.

Lawaree E., Jankevicius G., Cooper C., Ahel I., Uphoff S. & Tang C. M.

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Jani Reddy Bolla (Oxford, UK) Speaker Number 13

MmpL3 is a lipid transporter that binds trehalose monomycolate and phosphatidylethanolamine

The cell envelope of Mycobacterium tuberculosis is notable for the abundance of mycolic acids (MAs) essential to mycobacterial viability. Yet, exactly how lipidic elements are transported across the cell envelope for cell wall biosynthesis is unclear. Mycobacterial membrane protein Large 3 (MmpL3) is essential and required for transport of trehalose monomycolates (TMMs), precursors of MA-containing trehalose dimycolates (TDM) and mycolyl arabinogalactan peptidoglycan, but the exact function of MmpL3 remains elusive. Here, we report a crystal structure of M. smegmatis MmpL3 at a resolution of 2.59 Å. A previously unknown MmpL3 ligand, phosphatidylethanolamine, was discovered inside this transporter. We also show, via native mass spectrometry, that MmpL3 specifically binds both TMM and PE, but not TDM, in the micromolar range. These observations provide insight into the function of MmpL3 and suggest a possible role for this protein in shuttling a variety of lipids to strengthen the mycobacterial cell wall.

Chih-Chia Su, Philip Klenotic, Jani Reddy Bolla, Georgiana Purdy, Carol V Robinson, Edward W Yu

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David Adams (Lausanne, CH) Speaker Number 14

DNA-uptake pili of Vibrio cholerae – one pilus, three fundamental functions

How bacteria colonise surfaces and how they sense the individuals around them are fundamental biological questions. Here we directly visualise the DNA-uptake pilus of Vibrio cholerae, which is produced specifically during growth upon its natural habitat - chitinous surfaces. As predicted, these pili are highly dynamic and retract prior to DNA-uptake during competence for natural transformation. Unexpectedly, however, we discovered that these pili bind to chitinous surfaces and are required for chitin colonisation under flow. Moreover, we go on to show that DNA-uptake pili can self-interact, but that extensive strain-to-strain variability in the major pilin subunit PilA creates a set of highly specific interactions, enabling cells producing pili composed of different PilA subunits to distinguish between one another. Our results suggest a model whereby DNA-uptake pili function to promote inter-bacterial interactions during surface colonisation and highlight a simple and potentially widespread mechanism for bacterial kin recognition.

David. W. Adams, Sandrine Stutzmann, Candice Stoudmann and Melanie Blokesch

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Josh McQuail (London, UK) Speaker Number 18

An unusual behaviour of Hfq in long-term nitrogen starved Escherichia coli

RNA binding proteins play diverse roles in the regulation of RNA metabolism. In many bacteria, the RNA binding protein Hfq is a pleiotropic regulator of RNA metabolism and contributes to post-transcriptional regulation of gene expression by stabilising small non-coding RNAs and promoting their interactions with cognate mRNAs, ribosome biogenesis and indirectly on nucleoid conformation. Using single molecule photoactivated localisation microscopy analysis of individual Hfq molecules in live bacterial cells we now unravel an unusual feature of Hfq. We demonstrate that Hfq forms distinct foci in long-term nutrient starved Escherichia coli. We describe the discovery and properties of the Hfq foci and propose that they represent subcellular structures in bacteria which are analogous to processing bodies (P-bodies) found in stressed eukaryotic cells.

Josh McQuail & Ramesh Wigneshweraraj

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Kathrin Fröhlich (Munich, DE) Speaker Number 19

Post-transcriptional regulation by a cell-cycle controlled small RNA in Caulobacter crescentus

Small RNAs (sRNAs) are a prominent class of regulators of bacterial gene expression. Most sRNAs characterized act at the post-transcriptional level, and engage in direct base-pairing interactions to modulate translation and/or stability of target mRNAs. In many cases, the ubiquitous RNA chaperone, Hfq, contributes to the establishment of functional interactions.

The water-dwelling alpha-proteobacterium C. crescentus is an important model organism for the bacterial cell cycle, however its fundamental principles of RNA-mediated regulation have not been studied in detail. Here we report on the identification of sRNAs associated with Hfq in C. crescentus, and for the first time the characterization of Hfq-dependent post-transcriptional regulation in this organism. Using transcriptomics we have uncovered a large set of targets controlled by one Hfq-bound sRNA which is differentially expressed over the C. crescentus cell cycle, and have validated its activity both in vivo and in vitro.

Laura Vogt; Anna Schäpers; Nikolai Peschek; Kai Papenfort; Kathrin Fröhlich

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Karrera Djoko (Durham, UK) Speaker Number 20

The interplay between bacterial copper homeostasis and carbon metabolism.

Metals are nutrients, required by half of bacterial enzymes, but are poisons if present in excess or bound to the wrong proteins. Most bacterial organisms possess metal homeostasis systems, which, in the case of copper (Cu), relies on an efflux pump (CopA) that removes surplus Cu from the cytoplasm. In the Gram- positive pathogen Streptococcus pyogenes, deletion of copA leads to growth inhibition by added Cu salts and accumulation of Cu in the cytoplasm. However, a Cu stress phenotype is observed only in the late exponential phase – growth ceases abruptly, viability decreases sharply, and carbon and nitrogen metabolism stops suddenly. To determine the mechanism of Cu stress, a transposon mutant is screened for genes that confer Cu tolerance. Only four genes meet our criteria, including copA and SPy_1616, which encodes a putative pyridoxal phosphate salvage enzyme. The newly identified role of SPy_1616 in Cu tolerance will be described.

Andrew Turner, Yoann LeBreton, Kevin McIver, Mark Walker, Alastair McEwan, Louisa Stewart, Elisabeth Lowe, Kevin Waldron, Karrera Djoko

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Dennis Claessen (Leiden, NL) Speaker Number 21

Exploitation of a shape-shifting Kitasatospora viridifaciens strain identifies a MurG-like protein required for peptidoglycan synthesis

Bacteria are enveloped by a cell wall structure that is generally considered to provide cellular protection. Despite this critical role, some bacteria are able to shed their wall when exposed to environmental insults, thereby adopting a transient cell wall-deficient state. Here, we use a derivative of the filamentous bacterium Kitasatospora viridifaciens with the unique ability to switch between a wall-deficient and filamentous state to discover an enzyme that is critically important for restoring cell wall synthesis. This newly identified enzyme, called MglA, shares less than 30% sequence identity to MurG but is able to functionally replace its activity. Contrary to each single mutant, the absence of both murG and mglA abolishes switching from the wall- deficient to the walled state. These findings highlight MglA as an important cell wall biosynthetic enzyme with the ability to make lipidII in the absence of MurG.

Karina Ramijan, Le Zhang, Lizah van der Aart, Joost Willemse, Gilles van Wezel, Dennis Claessen

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Azhar Kabli (York, UK) Speaker Number 23

Chromosome segregation in the thermophilic archaeon Sulfolobus solfataricus

Chromosome segregation is a vital process for all organisms. The mechanisms underpinning chromosomal partitioning in the archaeal domain remain elusive. Our group has identified the first chromosome segregation system in thermophilic archaea. Sulfolobus solfataricus partition system consists of SegA, an orthologue of bacterial Walker-type ParA proteins; SegB, an archaea-specific DNA binding protein and a cis- acting DNA region.

Molecular biology and microscopy approaches were used to study the involvement of the SegAB complex in chromosome segregation. Immunofluorescence microscopy showed the subcellular localisation of SegA as diffused fluorescent signals suggesting non-specific binding to the nucleoid. In contrast, SegB was detected as multiple bright and discrete foci indicating binding of SegB to different chromosomal sites. In agreement with the microscopy data, ChIP-seq experiments disclosed multiple SegB binding sites scattered over the chromosome and revealed a novel DNA binding motif. Atomic Force Microscopy showed the dynamics of the DNA-binding features of the proteins.

Azhar Kabli & Daniela Barilla

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Agnes Noy (York, UK) Speaker Number 24

The structure and dynamics of DNA under supercoiling stress

In vivo, DNA is supercoiled due to cellular processes, such as transcription and replication. Here I investigate how DNA supercoiling influences DNA recognition properties, and how these deformations can be transmitted through the DNA itself or across DNA loops. In the former, I detect that the probability for a DNA side to melt depends of the whole topological domain [2]. In the latter, I found plectonemes promote non- specific DNA-protein bridges due to close approximation between DNA strands [3]. I also study the effect of supercoiling on the DNA-bending protein IHF. To this end, I apply molecular dynamics simulation techniques to DNA minicircles, as they can be analyzed experimentally and theoretically at the same time, becoming ideal systems to explore the topology of the DNA.

1. Sutthibutpong et al., Nuc Acids Res, (2016). 44, 9121.

2. Noy et al., Biophys J, (2017), 112, 523.

Agnes Noy

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Jean-Luc Ferat (Paris-Saclay, FR) Speaker Number 25

Two strategies to load bacterial replicative helicases

In bacteria, replicative helicases (DnaB) loading is assisted by designated loaders, among which DnaC and DnaI are well characterized. DciA is a prospective loader, recently discovered and prevailing in the bacterial domain. Its management of the helicase is unknown.

We characterized the function of DciA and established that it stimulates the loading, and more surprisingly, the translocation of helicases on DNA. Comparative analyses of helicase loading revealed major differences between DciA- and DnaC-assisted helicases. To understand the rationale behind these discrepancies, we crystallized a DciA:DnaB complex. We showed that DnaB is organized in a close or semi-close conformation, incompetent for the loading. This contrasts with the loading-competent staircase conformation of the helicase in a DnaC:DnaB complex and suggests that bacterial replicative helicases are loaded according to two distinct mechanisms. Models will be presented in light of the strategy adopted by phage loader to hijack the host helicase.

S Marsin, Y Adam, J Andreani, S Baconnais, P Legrand, I Gallay-Li de la Sierra, C Possoz, A Humbert, M Aumont, C Velours, F Ochsenbein, D Durand, E Le Cam, S Quevillon-Cheruel, JL Ferat

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Jeanine Rismondo (London, UK) Speaker Number 27

Balanced activity of multiple FtsW and RodA proteins is crucial for maintaining cell shape and antibiotic resistance in Listeria monocytogenes

The cell division- and cell elongation-specific enzymes FtsW and RodA have recently been identified as peptidoglycan glycosyltransferases. Listeria monocytogenes encodes up to six FtsW/RodA homologs, however their functions have not yet been investigated. Analysis of de(p)letion strains led to the identification of the essential FtsW protein, FtsW1. Interestingly, L. monocytogenes encodes a second FtsW protein, FtsW2, which can compensate for the lack of FtsW1, when overexpressed. L. monocytogenes also possesses three RodA homologs, RodA1, RodA2 and RodA3 and their combined absence is lethal. Cells of a rodA1/rodA3 double mutant are shorter and have increased antibiotic and lysozyme sensitivity. Results from promoter activity assays revealed that expression of rodA3 and ftsW2 is induced in the presence of antibiotics targeting binding proteins. Taken together, L. monocytogenes encodes a multitude of functional FtsW and RodA enzymes and their expression needs to be tightly regulated to maintain growth, cell division and antibiotic resistance

Jeanine Rismondo, Sven Halbedel and Angelika Gründling

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Nathalie Reichmann (Oxford, UK) Speaker Number 30

SEDS-bPBP pairs direct Lateral and Septal Peptidoglycan Synthesis in Staphylococcus aureus

Bacteria maintain cell shape by directing the incorporation of peptidoglycan (PGN), the major component of the bacterial cell wall, to distinct regions of the cell. This is largely achieved through the localisation of late stage PGN synthesis proteins including two key protein families, SEDS transglycosylases and bPBP transpeptidases, thought to function in cognate pairs. Although rod-shaped bacteria have two SEDS-bPBP pairs involved in elongation and division, we questioned why the coccoid Staphylococcus aureus would possess two SEDS-PbPBP pairs. We demonstrate that RodA-PBP3 and FtsW-PBP1 likely constitute the cognate pairs of S. aureus. We found that the lack of RodA-PBP3 led to more spherical cells indicating deficient sidewall PGN synthesis, while depletion of FtsW-PBP1 prevented normal septal PGN synthesis and led to divisome proteins appearing as multiple rings/arcs that drive lateral PGN incorporation. We propose that together these cognate pairs balance their activity to maintain the S. aureus coccoid morphology.

Nathalie T. Reichmann, Andreia C. Tavares, Bruno M. Saraiva, Ambre Jousselin, Patricia Reed, Ana R. Pereira, João M. Monteiro, Rita G. Sobral, Michael S. VanNieuwenhze, Fábio Fernandes and Mariana G. Pinho

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Abstracts Posters

Mayokun Ajeigbe (/UK) Cloning and expression of a putative microcin gene cluster from Erwinia persicina

Fuad Alatawi (Newcastle University/UK) Secondary metaboilt production of Bacillus spp

Nettie Alevropoulos-Borrill (/UK) The Translocation Mechanism of a Hexameric Helicase

John Allan (Newcastle University/UK) RC/GC: Remote Control of Genetic Circuits

John Armstrong (University of York/UK) Separating DNA rings – investigating plasmid segregation mechanisms in the archaeon Sulfolobus.

Emma Banks (University of /UK) Discovering the role of an L,D-carboxypeptidase in the cell shape determination of Bdellovibrio bacteriovorus

Anna Barwinska-Sendra (Newcastle University/UK) Structural characterisation of the Clostridium difficile S-layer

Lisa Bowman (Newcastle University/UK) Investigations into the Type VII Secretion System of the Staphylococcus aureus strain EMRSA-15 and the Role of Secreted Effectors

Alejandro Carrion Sanabria (John Innes Centre/UK) “The Tn5-seq screen identified a new small protein that plays a role in DNA-damage repair in Caulobacter crescentus”

Jonathan Chapman (Newcastle University/UK) Uncovering the specialised metabolites behind mycetoma

Jingqi Chen (Groningen University/NL) Subcellular localization of a lantibiotic biosynthesis machinery in Bacillus subtilis

Jon Cherry (Newcastle University/UK) Mechanistic analysis of the minimalistic twin-arginine translocation system found in Bacillus subtilis

Laura Clark (University of York/UK) Invesitgating the function of staphylococcal surface proteins SasG and Aap

James Connolly (Newcastle University/UK) Distinct intra-species virulence mechanisms regulated by a conserved transcription factor

Daniel Devlitsarov (Ludwig Maximilian University of Munich (LMU)/DE) Regulation of motility in Vibrio cholerae by RNA-binding protein ProQ

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Alex Finney (Newcastle University/UK) No, it is not lysis - Adaptation of a phage lysis cassette for two-step protein secretion in Serratia marcescens

Francesco Fiorentino (/UK) The Different Effects of Substrates and Nucleotides on the Complex Formation of ABC Transporters

Hannah Gaimster (Newcastle University/UK) Lethal depletion of an essential bacterial cell wall synthesis protein can be rescued by slowing DNA replication

Hannah Gibson (Newcastle University/UK) The biochemical mechanism of bacterial exopolysaccharide utilisation by Bacteroides species

James Grimshaw (Newcastle University/UK) Induced formation of membrane associated RNA degradosome in Bacillus subtilis

Alexandria Holland (/UK) The molecular mechanism linking metabolism to DNA replication in Bacillus subtilis

Adrián Izquierdo Martínez (University of Marburg/DE) Coordination of autolysins during cell division in Caulobacter crescentus

Adam Jalal (John Innes Centre/UK) Structural and molecular insights into ParB spreading on bacterial chromosomes

Calum Jukes (Newcastle University/UK) The Great Divide: How a Motile Cytoskeleton Drives Bacterial Cell Division

Azhar Kabli (University of York/UK) Chromosome segregation in the thermophilic archaeon Sulfolobus solfataricus

Nikol Kadeřábková (/UK) The role of the Disulfide Bond Formation system in antimicrobial resistance

Eleni Karinou (Newcastle University/UK) Elucidating the dynamics of the septal cell wall synthase PBP2B in Bacillus subtilis

Abigail Kelly (Newcastle University/UK) Peptidoglycan remodelling throughout early engulfment in Clostridium difficile sporulation.

Giorgio Lai (Newcastle University/UK) Towards a synthetic bioclock

Ting Lai (University of Nottingham/UK) Testing Acinetobacter susceptibility to predation by Bdellovibrio bacteriovorus

Paola Lanzoni-Mangutchi (Newcastle University/UK) Constructing a crystal shell: the lattice of Clostridium difficile S-layer 35 | Page 6th Molecular Microbiology Meeting Newcastle upon Tyne

Frederic Lauber (University of Oxford/UK) Molecular analysis of the Type IX Secretion System

Alex Laverick (Newcastle University/UK) Iterative, automatic design and optimisation of chemically defined media

Robert Lawrence (/UK) EnvZ Transmembrane Helices - A Cryptographic Conundrum

Erin Lewis (Newcastle University/UK) Comparative genomics of Listeria monocytogenes isolated from fresh produce, meat and clinical casesComparative genomics of Listeria monocytogenes isolated from fresh produce, meat and clinical cases

Joshua Loh (Newcastle University/UK) Identification of hydrocarbon biosynthetic pathways in Ascocoryne sarcoides

Swati Mahapatra (/UK) Highly Conserved Protein SpoVG Binds RNA in vivo and has Pleiotropic Functions in Bacillus subtilis

Chriselle Mendonca (Newcastle University/UK) Characterisation of Staphylococcus aureus Type VII protein secretion system of MRSA252

Ángela Mérida-Floriano (Universidad de Sevilla/ES) Expression patterns of LexA-regulated genes

Stuart Middlemiss (Newcastle University/UK) Investigating the regulation of vegetative growth by the MreB cytoskeleton in rod-shaped bacteria using single molecule imaging.

Nancy Mulvenna (Imperial College London/UK) Xenogeneic regulation of the ClpCP protease of Bacillus subtilis by a phage-encoded adaptor-like protein

Manuel Pazos (Newcastle University/UK) Z-ring membrane anchors associate with cell wall synthases to initiate bacterial cell division

Caroline Pearson (University of York/UK) Functional analysis of Salmonella O-antigen acetyltransferases

Katharina Peters (The Centre for Bacterial Cell Biology/UK) Copper inhibits peptidoglycan LD- transpeptidases suppressing β-lactam resistance due to by-pass of Penicillin-binding proteins

Matthaios Pitoulias (University of Nottingham/UK) The link between Replication and Metabolism in B.subtilis

Samuel Prudence (University of East Anglia/UK) Composition & Competition: Streptomyces, Type VII Secretion & the Wheat Root-Microbiome

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Alidivinas Prusokas (Newcastle University/UK) Metagenomic-Enabled Discovery of the Plant Virome

Amber Riaz-Bradley (Newcastle University/UK) The Transcription Machinery of Cyanobacteria

Magali Roger (Newcastle University/UK) Efficient hydrogen-dependent carbon dioxide reduction by Escherichia coli.

Jad Sassine (Newcastle university, ICaMB/UK) Balanced cell wall synthesis and hydrolysis is crucial to maintain rod-shape in B. subtilis

Alexander Scott (University of York/UK) How does the cold killer puncture a bacterium? – Probing the molecular basis of Gram-negative cell envelope damage by low temperature plasma

Augustinas Silale (University of Oxford/UK) Characterisation of the bacterial DNA transporter involved in natural transformation

Nadine Silber (Tübingen University/DE) Degradation of FtsZ by antibiotic-activated ClpP involves unfolding of the FtsZ N-terminal domain

Daniel Stevens (Newcastle University/UK) Interaction of the bacterial initiator protein DnaA with the DnaA-trio replication origin element

Aaron Ming Zhi Tan (Newcastle University/UK) Impact of Long-Term Prophylaxis on Bladder Colonisation by Escherichia coli in CISC Patients

Sarah Tindall (University of York/UK) How do bacteria change their coats? Structural analysis of acyltransferases involved in O-antigen modification

Nicholas Tregidgo (Newcastle University/UK) Things that divide us: Elucidating the coupling mechanisms between the cytoskeleton and synthesis machinery in the Bacillus subtilis division complex

Fatima Ulhuq (Newcastle University/UK) A membrane-depolarizing toxin substrate of the Staphylococcus aureus Type VII protein secretion system

Lizah van der Aart (Newcastle University/UK) Thermoactinomycetaceae are tip-growing, multicellular Firmicutes

Marjan Van der Woude (University of York/UK) Role and action of Salmonella spp. O-antigen modifying enzymes

Víctor Velasco (University of York/UK) Study of DNA Flexibility

Ziyi Wang (Newcastle University/UK) INVESTIGATING THE BIOLOGICAL CONTRIBUTION OF C. difficile TYPE IV PILI PROTEINS

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George Watson (University of York/UK) Atomistic simulations unveil the influence of DNA topology on IHF–DNA interaction

Kevin Whitley (Newcastle University/UK) Imaging dynamics of division proteins in B. subtilis cells oriented vertically in microholes during rapid chemical perturbations

Zoe Wilkinson (Institute for Cell and Molecular Biosciences/UK) Structural and functional studies of Type IV pili

Stephan Wimmi (Max Planck Institute for Terrestrial Microbiology/DE) Functional and molecular adaptation of the Yersinia enterocolitica type III secretion system to local pH

Charles Winterhalter (Newcastle University/UK) DNA replication initiation in B. subtilis: how is it done?

Alexander Wood (Newcastle University/UK) Spatiotemporal Effects of Transcription Factor- Promoter Distance on Gene Regulatory Networks

Jason Woodgate (Newcastle University/UK) Backtracked RNAP is pushed forward along mRNA by the ribosome in vitro

Mengru Yang (Institute of Integrative Biology, /UK) Quantifying the stoichiometry and modularity of bacterial metabolosomes

YONG ZHANG (University of /DK) (p)ppGpp Regulates a Bacterial Nucleosidase by an Allosteric Two-Domain Switch

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List of participants

Title Name Surname Organisation Dr Abdessamad Ababou University East London Horeyah Abdalkrim Newcastle University Javier Abellon-Ruiz Newcastle University Dr David Adams EPFL, Switzerland Mayokun Ajeigbe University of Sunderland Yousef Alanazi Newcastle University Fuad Alatawi Newcastle University Dr Felicity Alcock Newcastle University Dr Phil Aldridge Newcastle University Nettie Alevropoulos-Borrill University of York Alaa Aljohani CBCB Dr John Allan Newcastle University Professor Esther Angert Cornell Univeristy, USA John Armstrong University of York Ben Arnold Thermo Fisher Scientific Dr Adam Azzi Newcastle University Dr Alice Banks Newcastle University Emma Banks University of Nottingham Dr Daniela Barilla University of York Joseph Barry Newcastle University Anna Barwinska-Sendra Newcastle University Dr Arnaud Basle ICaMB Dr Alex Bateman EMBL-EBI Dr Christoph Baumann University of York Dr Morgan Beeby Imperial College, London Dr Satya Prathyusha Bhamidimarri Newcastle University Dr Jacob Biboy Newcastle University Dr Lewis Bingle University of Sunderland Dr Eleanor Boardman Newcastle University Dr Dave Bolam Newcastle University Dr Jani Reddy Bolla University of Oxford Dr Simon Booth Newcastle University Dr Helene Botella Imperial College, London Lisa Bowman Newcastle University Dr Grant Buchanan Newcastle University Dr Jessica Buttress Newcastle University Ekaterina Buzun Newcastle University Alejandro Carrion Sanabria John Innes Centre Guilllermina Casabona Newcastle University Elliot Chan University of York Jon Chapman Newcastle University Dr Jon Cherry Newcastle University

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Dr Francesca Romana Cianfanelli University of Basel Dr Dennis Claessen Dr Laura Clark University of York John Clark-Corrigall ICaMB Dr James Connolly Newcastle University Rachael Cooper University of York Federico Corona Newcastle University Peter Craggs The Institute Dr Lucy Crouch Newcastle University Dr Fiona Cuskin Newcastle University Maria Dakes Stavrakakis Newcastle University Dr Richard Daniel Newcastle University Professor Tanneke den Blaauwen University of Amsterdam Daniel Devlitsarov Ludwig Maximilian University of Munich Dr Karrera Djoko Dr Kaveh Emami Newcastle University Professor Jeff Errington Newcastle University Stepan Fenyk Newcastle University Professor Jean-Luc Ferat CNRS-I2BC Dr Alex Finney Newcastle University Francesco Fiorentino University of Oxford Dr Paulina Focht Newcastle University Dr David Forrest University of Birmingham Max Fritsch New BioLabs Dr Kathrin Fröhlich Ludwig Maximilian University of Munich Dr Hannah Gaimster Newcastle University José Gallego-Parrilla Newcastle University Dr Gina Gamble Nikon Instruments Andrea Giachino Newcastle University Dr Hannah Gibson Newcastle University Marvin Gohrbandt University of Osnabrück Dr Grace Goldsmith Newcastle University Bethany Gollan Newcastle University Professor Susan Gottesman National Institutes of Health, USA Professor David Grainger University of Birmingham Dr Declan Gray Newcastle University James Grimshaw Newcastle University Mr Aurelie Guyet Newcastle University Sophie Handson Newcastle University Professor Colin Harwood Newcastle University Dr Victor Hernandez-Rocamora Newcastle University Dr Sam Hickman University of Oxford

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Dr Seamus Holden Newcastle University Dr Alexandria Holland University of Nottingham Dr Adam Hopkins Newcastle University Dr Damon Huber University of Birmingham Geoff Hughes Sarstedt Limited Adrián Izquierdo Martínez University of Marburg Adam Jalal John Innes Centre Mr Alexander Johns Newcastle University Calum Jukes Newcastle University Christina Julius Newcastle University Dr Azhar Kabli University of York Nikol Kaderabkova Imperial College London Maxim Kapralov Newcastle University Eleni Karinou Newcastle University Yoshikazu Kawai Newcastle University Dr Abigail Kelly Newcastle University Dr Ciarán Kelly Newcastle University Berhard Kepplinger Newcastle University Dr Anjam Khan Newcastle University Dr Alan Koh Newcastle University Sandra Laborda Anadon Newcastle University Giorgio Lai Newcastle University Ting Lai University of Nottingham Paola Lanzoni-Mangutchi Newcastle University Dr Frederic Lauber University of Oxford Alex Laverick Newcastle University Dr Emeline Lawaree University of Oxford Dr Tung Le John Innes Centre Rosalyn Leaman University of York Dr Erin Lewis Newcastle University Dr Siobhan Lister Newcastle University Dr Hilary Logan Microbiology Society Joshua Loh Newcastle University Dr Elisabeth Lowe Newcastle University Swati Mahapatra University of Warwick Dr Despoina Mavridou Imperial College London Josh McQuail Imperial College London Dr Chriselle Mendonca Newcastle University Eilidh Menzies Thermo Fisher Scientific Angela Mérida-Floriano University of Seville Katarzyna Mickiewicz Newcastle University Stuart Middlemiss Newcastle University Hamed Mosaei-Sejzi Newcastle University Emily Mulligan Newcastle University Professor Heath Murray Newcastle University Soren Nielsen Newcastle University

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Dr Agnes Noy University of York Professor Tracy Palmer Newcastle University Manuel Pazos Newcastle University Caroline Pearson University of York Simone Pelliciari Newcastle University Sudheer Peneti Newcastle University Katharina Peters Newcastle University Mr Matthaios Pitoulias University of Nottingham Professor Jennifer Potts University of York Dr Alidivinas Prusokas Newcastle University Dr Maria Puiu Newcastle University Dr Nicholas Read University of York Professor Manjula Reddy Centre for Cellular & Molecular Biology, India Dr Nathalie Reichmann University of Oxford Amber Riaz-Bradley Newcastle University Dr Jeanine Rismondo Imperial College London Dr David Roberts Newcastle University Dr Magali Roger Newcastle University Jack Rowan Starlab Michelle Rudden University of York Dr Julian Rutherford Newcastle University Dr Zoe Rutter Newcastle University Dr Paula Salgado Newcastle University Dr Jeanne Salje University of Oxford Dr Jad Sassine Newcastle University Alexander Scott University of York Dr Agnese Serafini The Dr Emmanuele Severi Newcastle University August Silale University of Oxford Nadine Silber University of Tübingen Dr Jack Smallwood Newcastle University Daniel Stevens Newcastle University Dr Christopher Stewart Newcastle University Henrik Strahl Newcastle University Aaron Ming Zhi Tan Newcastle University Professor Martin Thanbichler Max Planck Institute for Terrestrial Microbiology, Gavin Thomas University of York Sarah Tindall University of York Nicholas Tregidgo Newcastle University Alexandra Trigg University of Birmingham Fatima Ulhuq Newcastle University Professor Bert Van Den Berg Newcastle University Lizah van der Aart Newcastle University

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Dr Marjan Van der Woude University of York Dr Julien Vaubourgeix Imperial College, London Victor Manuel Velasco Berrelleza University of York Professor Waldemar Vollmer Newcastle University Lucas Walker University of Birmingham Professor Gilly Ziyi Wang Newcastle University University of York Dr Kevin Whitley Newcastle University Professor Ramesh Wigneshwerataj Imperial College, London Dr Zoe Wilkinson Newcastle University Benjamin Willson University of York Stephan Wimmi Max Planck Institute for Terrestrial Microbiology, Germany Charles Winterhalter Newcastle University Jason Woodgate Newcastle University Dr Ling Wu Newcastle University Mengru Yang University of Liverpool Dr Hamish Yau Newcastle University Dr Yulia Yuzenkova Newcastle University Nikolay Zenkin Newcastle University Dr Yong Zhang

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