How a motile cytoskeleton drives bacterial cell division

Posted on 25 May, 2017 by Kevin

In a recent issue of Science, the discovery of a key mechanism for bacterial cell division was reported. This work was carried out by Dr Seamus Holden’s lab (ICaMB) in collaboration with Professor Cees Dekker (TU Delft), Professor Yves Brun (Indiana University), Professor Mike VanNieuwenhze (Indiana University) and Professor Ethan Garner (Harvard University). Here, Seamus tells us about this discovery and what its implications could be for antimicrobial research.

Bacterial cell division is a lovely mechanistic problem in biology: how do the simplest living organisms build a crosswall at mid cell, against very high outwards pressure (think of a racing bike tyre), without bursting? A ring of protein filaments forms around the future division site, and enzymes associated with this ring build a new crosswall that cleaves the bacteria in half. But what has remained completely mysterious is how these proteins work together as a single nanoscale machine to cut the bacterial balloon skin (cell wall) in two.

Cytoskeletal proteins FtsZ in live bacteria imaged in vertical nanocages

Working together with collaborators in Delft, Indiana and Harvard, we tracked the organization and motion of key division proteins as they build the dividing crosswall, and the organization of the newly built crosswall itself. We began by examining the motion of FtsZ, a cytoskeletal filament that is required for cell division – cytokinesis – in bacteria and is related to the tubulin cytoskeletal protein found in eukaryotic cells. Using high-resolution microscopy techniques, we found that FtsZ filaments move around the division site, traveling around the division ring. We imaged the motion of individual cell wall synthesis enzymes, and saw that the synthesis enzymes ride on FtsZ filaments, building new cell wall as they travel along the division site. This causes the cell wall to be synthesized in discrete sites that travel around the division site during cytokinesis, a process which we were able to observe directly by using dyes that label the bacterial cell wall. Using a variety of experimental techniques, we were able to speed up or slow down how fast FtsZ rotated around the cell. Strikingly, we found that the speed of FtsZ filament motion determines how fast the cell can divide. When FtsZ moves more rapidly, cell wall is produced more quickly, and cytokinesis happens faster. This shows that the motion of FtsZ is the critical overall controller of cell division.

One challenge that we faced was trying to look at the division proteins in actively dividing cells. At the earliest stages of division, it was possible to image division protein organization because the proteins in the partially assembled ring are sparsely distributed. However, a new strategy was required to measure how the dense protein network of actively dividing cells was organized. Normally, bacteria are immobilized flat on a microscope slide, and imaged from underneath, but unfortunately this places the division ring side-on, obscuring the motion and organization of division proteins. To solve this problem, we used nanofabrication technology, originally developed to manufacture computer chips, to create tiny gel nanocages to trap bacteria in an upright position.

Bacteria trapped in vertical nanocages

By trapping individual bacteria upright, we were able to rotate the cell division ring so that it was fully visible on our high resolution microscope. This revealed the dynamic motion of FtsZ filaments as they travel around the entire division site:

Together, these results revealed the basic mechanistic principles of bacterial cell division: that the building of the division crosswall is orchestrated by moving cytoskeletal filaments. Previously, the cytoskeleton was thought to serve as a static scaffold, recruiting other molecules and perhaps exerting some force to divide the cell. This new work demonstrates that all the components of cell division are in constant, controlled motion around the division site, driven by the fundamental dynamics of the cytoskeleton.

In the longer term, this study could open up novel antibiotic targets. Based on the discovery that the treadmilling motion of the bacterial cytoskeleton is critical for division, it may be possible to develop new drugs that specifically inhibit this motion, similar to how the chemotherapy drug taxol suppresses the motion of the cytoskeleton in cancer cells.

==================================

Links

Explanatory animation: https://youtu.be/6dq2_gqKPfU (Animation credit TU Delft / Scixel)

Nanocage Video: nanocage-movie-2.

Science report: http://science.sciencemag.org/content/355/6326/739

Press release: http://bit.ly/2kwsnJf

Any excuse to drink red wine

Posted on 22 March, 2017 by Elisabeth

By Elisabeth Lowe

I’m writing this blog from an interesting perspective – though a member of the lab I’m not involved in the project, except through the (exhaustive!) discussions at coffee time with Harry Gilbert and the rest of the authors on the paper. This work, published today in Nature, describes the way in which one particular species of human gut bacteria is able to degrade a complex plant polysaccharide. Why is this new, you might well ask? Well, after being talked to death on the project, so much so I’d be glad never to hear a word about it again, I’ve actually come to appreciate what a great piece of work it is. The Bolam and Gilbert labs have published quite a bit on plant polysaccharide degradation over the years, but this one is a little different, because the polysaccharide involved – Rhamnogalacturonan-II (RGII) – is thought to be the most complex glycan found in nature. It contains ~13 different sugars linked by 21 different linkages, and all but one can be broken by a single bacterial species, Bacteroides thetaiotaomicron (Bt).

Complex structure of RG-II from red wine

Wade Abbott, a post-doc in Harry’s lab when he was at the Complex Carbohydrate Research Centre, in Athens, Georgia, which is incidentally where the structure of RG-II was determined in the first place by Alan Darvil and Malcolm O’Neill in the 1980s, initiated this project. We knew which loci were upregulated during growth on RG-II from earlier work by myself, Dave Bolam and Eric Martens, so Wade set about cloning and expressing the 30 genes in these loci, and looking for activity with a PhD student, Jeff Xhang. It was a tough job, and little progress was made. When Harry came back to the UK he decided he absolutely had to crack it, and put two post-docs onto the project, Art Rogowski and Didier Ndeh. Though as the project has progressed, more and more people from the lab were sucked in, including the post-docs Alan Cartmell and Aurore Labourel, and Harry’s last PhD student, Ana Sofia Luis. All worked incredibly hard, assisted by Arnaud Baslé from SBL, and between them discovered the activities of 26 enzymes, including seven entirely new glycoside hydrolase families, three new activities, and seven crystal structures, with all the active site mutations, and ligand soaks that go along with a new crystal structure.

Working out the enzyme activities on a polysaccharide as complex as RG-II poses some problems, in that you can’t really buy it, so you have to make it. RGII is concentrated in red wine, so Harry put 150 litres of Asda’s not so finest wine on Dave Bolam’s credit card (don’t ask) and with Art and Didier, took it to Prozomix in Haltwhistle, to use their industrial concentrators.

Starting to concentrate 150 L of wine

As you can see, this did not end well.

It’s all gone horribly wrong

Art then turned to apple concentrate and managed to purify some through an intensive multiple two-week process. Making or buying oligosaccharides is also extremely difficult, so Didier devised a way of killing two birds with one stone – identifying which unknown open reading frames (ORFs) were enzymes, and making oligos at the same time. By deleting the gene of interest, RG-II degradation (which is mainly exo-acting) was halted at the point at which that enzyme acted. Taking the supernatant from these strains grown on RG-II and purifying the sugars released by the bacterium yielded partial breakdown products which could be identified by mass spectrometry with tremendous help from Joe Gray, and then used as a substrate for the enzyme in question.

Getting the data in a format suitable for the tight space restrictions imposed by Nature was almost as challenging as doing the work. Given that Harry is not very artistic, and certainly has no idea how to use Adobe Illustrator, he was fortunate to have three talented artists Ana, Art and Aurore, who ended up doing all the figures. They are now referred to as the art department. This project was definitely a labour of love (for Harry, if no-one else!) and by carefully characterising the breakdown of this polysaccharide, one linkage at a time, has actually revealed new features of the structure of this critical plant polysaccharide. Finding and characterising the vast repertoire of enzymes produced by gut microbes is not only interesting in its own right, but can provide tools to understand complex glycan structure. It’s a fantastic achievement, and I pity the poor lab member who has to try and bake the RG-II cake for the celebrations this week!

Link to paper

A novel copper storage protein discovered by ICaMB scientists

Posted on 17 September, 2015 by Kevin

In a recent issue of Nature the discovery of a new family of proteins that store copper ions inside bacterial cells was reported. This work was carried out by Prof. Chris Dennison’s lab (ICaMB) in collaboration with Dr. Kevin Waldron (ICaMB), Dr. Arnaud Baslé (ICaMB), Dr. Neil Paterson (Diamond Light Source) and Prof. Colin Murrell (UEA). Here, Kevin tells us about this discovery and what its implications could be for the biotechnological exploitation of methane.

Methane, the main component of natural gas, is produced by both geological and anthropogenic processes on Earth, and can be released into the atmosphere. The biosphere’s primary mechanism for limiting the amount of methane (a powerful ‘greenhouse gas’) that escapes into the atmosphere is through its consumption by methane-oxidising bacteria, or methanotrophs (literally ‘methane-eating’), who utilise methane as both a carbon and energy source.

Transmission electron micrograph of a M. trichosporium OB3b cell showing the intracytoplasmic membranes that house the copper-dependent enzyme pMMO.

The key enzyme that methanotrophs use to oxidise methane to produce methanol is called methane monooxygenase (MMO). Almost all methanotrophs possess a membrane-bound form of MMO that requires copper (Cu) for activity, called pMMO (some strains also possess an iron-requiring alternative soluble enzyme, called sMMO). Therefore, these bacteria have an unusually high demand for copper, a metal that is essential for most organisms but is used rather sparingly, mainly because it can also be extremely toxic.

Previous studies of methanotrophs have revealed that they utilise unusual mechanisms for acquiring the copper they need for pMMO. For example, they produce and secrete a small, modified peptide called methanobactin that is involved in high affinity copper uptake, analogous to the more familiar siderophore iron uptake systems.

The model methanotroph, Methylosinus trichosporium OB3b, was analysed in the hope of uncovering novel copper proteins. Soluble extracts were resolved by two-dimensional liquid chromatography, with the copper content of resulting fractions monitored. The most abundant copper pool was found to contain a small, cysteine-rich protein that had not been studied previously and Chris Dennison (PI) and I (Co-I) obtained a BBSRC grant to investigate this and related proteins.

The presence of 13 cysteine residues in a mature protein (after cleavage of its signal peptide) with only 122 amino acids was immediately significant, as its thiol sidechain is often found in copper-binding sites. The recombinant protein was extensively studied using a suite of biochemical and biophysical methods to determine its in vitro properties This novel tetrameric protein is capable of binding up to 52 Cu(I) ions with high affinity, consistent with a potential role in the storage of this essential metal and was therefore named Csp1 (copper storage protein 1). A mutant strain of M. trichosporium OB3b that lacks this Csp1 protein (as well as a closely-related protein, Csp2) shows a phenotype consistent with a copper storage role of these proteins in vivo.

The crystal structure shows that the apo-monomer adopts a well-characterised four-helix bundle motif with all 13 cysteine sidechains pointing into the core of the bundle. The protein structure is essentially unchanged in the presence of copper, except that the core of each monomer has filled with 13 Cu(I) ions, bound by the cysteine sidechains. This is an unprecedented structure for metal storage.

The structure of the Cu(I)-Csp1 tetramer, with the 13 Cu(I) ions bound within the core of each four-helix bundle shown as orange spheres.

The implications of this discovery could be of wide impact. There is a great deal of interest in using methanotrophs and MMOs in industrial biotechnology. Available methane is on the increase due to ‘fracking’, but importantly methane can also be produced from sustainable sources such as through the degradation of biomatter (biogas). Methanotrophs and the MMOs could in future be exploited using synthetic biology approaches for gas-to-liquid conversion, for the production of liquid fuels as well as bulk and fine chemicals from renewable methane.

Any such biotechnological applications require an understanding of how the production of pMMO is regulated and how this complex enzyme is assembled, including the cellular delivery and insertion of the essential copper cofactor. The work published in Nature shows that Csps are an important piece in that assembly jigsaw.

Furthermore, bioinformatics has shown that cytosolic members of the Csp family are also encoded in other bacterial genomes. Most bacteria are thought not to require copper in their cytoplasm, so the presence of cytosolic Csp homologues in their genomes could re-write our basic understanding of their use and homeostasis of copper.

Links:

Nature article: http://www.nature.com/nature/journal/v525/n7567/full/nature14854.html

News & Views: http://www.nature.com/nchembio/journal/v11/n10/full/nchembio.1918.html

BBSRC website: http://www.bbsrc.ac.uk/news/fundamental-bioscience/2015/150827-pr-bacteria-that-could-protect-our-environment/

ACS Chemical & Engineering news: http://cen.acs.org/articles/93/web/2015/08/Protein-Stores-Copper-Methane-Digesting.html

Press release: http://www.ncl.ac.uk/press.office/press.release/item/could-bacteria-help-protect-our-environment

What did the Black Death ever do for us? The curious route to an Industrial Biotechnology Catalyst Grant

Posted on 12 March, 2015 by Neil

UK Government Minister Vince Cable recently announced the results of the first round in the BBSRC-supported Industrial Biotechnology Catalyst scheme where £20 M was distributed across 23 projects. Here, ICaMB’s Jeremy Lakey describes the curious scientific route that led from researching Yersinia pestis, the bacteria responsible for the bubonic plague, to a potential biotechnology breakthrough.

By Professor Jeremy Lakey

The project that I put together with Neil Perkins (ICaMB), Dave Fulton (Chemistry), Matt German (Dentistry) and Nick Reynolds (Dermatology), called (rather snappily I think) Manufacture of complex protein polymers for industry and medicine, is one of the recently announced Industrial Biotechnology Catalyst awards. It’s a £2.4 million research programme with a BBSRC 80% contribution of £1.8 million and quite honestly a year ago I’d never imagined having this amazing five years funding to realise this project that has nagged at me for at least the last five years.

The early years

My interest in the Caf1 protein first arose from a project with the Defence Science and Technology Laboratory (DSTL) on vaccines against possible bioweapons such as anthrax and plague. Other labs had shown Caf1 to be a chaperone usher (CU) family protein, secreted through the outer membrane of the plague bacterium Y. pestis as non-covalently linked polymers. However, most members of this family were visible under the electron microscope (EM) and had a defined structure. We tried in vain to see it under the EM for a couple of years and had given up but, as luck would have it, the world authority on EM observation of proteins using negative stain, Robin Harris, had retired from his job in Germany and moved to Hexham a town in the Tyne Valley near Newcastle. He agreed to have a look with my student at the time, Andrei Soliakov. By adding very low concentrations of protein and using his magic staining recipe, they got amazing images on the first day (see figure). I was on the other end of a country modem at my brother’s farm and so for me the images unfolded slowly down the screen. Robin, Andrei and I (much later in the day), were thus the first folk ever to see one of the key proteins of the Black Death which, between 1346 and 1353, killed up to half of the population of Europe. It was one of those sobering moments in life.

The Caf1 polymer as seen using an electron microscope (top). The structure (middle) resembles a line of circus elephants (bottom).

We also imaged these amazing molecules on the vaccine adjuvant, they looked like Christmas paper chains hanging off the sides of the adjuvant particles. No one had seen Caf1 or proteins on adjuvants before, even though most of us have been vaccinated using this material. I was swept away by the sheer coolness of the data we had and submitted a paper to Nature, then Science, then somewhere else and somewhere else after that but it soon appeared no one was quite as amazed as I was. This remains true to this day with the eventual paper in Vaccine (Ref 1 below) still only having 2 non self- citations; something I still fail to understand.

It’s love actually

However I was seriously smitten by this molecule and hardly cared. This infatuation was made worse one day when, in a seminar, somebody showed the structure of the extracellular matrix protein fibronectin or at least the domains around the integrin binding sites where the well-known RGDS sequence motif is situated. These protein domains looked just like Caf1 because they were both immunoglobulin-like domains.

Different immunoglobulin-like protein topologies (from Pyburn et al, PLoS Pathog. 2011)

Of course, I should have seen this before as I go on and on and on about protein families in my second year undergraduate lectures. About 70% of cell surface and extracellular matrix proteins are built at least partly from this simple domain structure. Like other superfolds such as the TIM barrel and globin these are protein structures with no sequence homology that are found in large numbers across biology and partly explain why there seems to be a limit to the number of different protein folds (Ref 2). But that is a story for another day.

The immunoglobulin-like fold is found in immunoglobulins (surprise, surprise), MHC, fibronectin, many surface receptors such as EGF receptors etc. etc. It’s also found in the largest protein known, Titin, which stops our muscles over extending when they are stretched. Now it turns up in this Caf1, which is beginning to nag me constantly with the thought that surely I could make things from this amazing polymer. So, off I go to the BBSRC with Mark Birch as co-investigator. With Liz Mitchell we had just shown that we could induce bone formation by growing osteoblasts on surfaces coated with engineered small proteins containing sequences from Bone morphogenic protein 2 and osteopontin (Ref 3). I said I could modify Caf1 to do the same and we could form it into hydrogels and grow 3D bone amongst other tissues – what could possibly go wrong. Funnily, the BBSRC did not agree and forbade any similar grant from darkening their doors for seven generations or something that sounded like that.

Try, try and try again

The project, such as it was, retreated to lick its wounds. At least my lack of citations meant that no one else in the world was likely to be working on it but neither it seemed was anyone interested in funding it. Salvation came in two forms. One was the MRC Industrial Collaboration studentship scheme, which enabled us to apply for money with Orla Protein Technologies Ltd, a spinout company I had co founded with Dale Athey in 2002. Orla sells engineered protein surface coatings such as those we used in the BMP/Osteopontin paper and very good they are too, try them yourselves at www.orlaproteins.com!

The second bit of luck was that Ana Roque applied for the studentship. She is an amazingly tenacious and hardworking scientist who single handedly made the Caf1 project work. First she tackled the ridiculously large Caf1 plasmid and won. We then had a manageable system that could produce mutant Caf1 in good amounts.

Immunoelectron microscopy of Y. pestis showing macrophage resistant hydrogel capsule From Du Y et al. Infect. Immun. 2002;70:1453-1460

She then showed that cells were not keen on growing on Caf1 surfaces. Not good. What turned it around was that by simply adding an RGDS motif we could make Caf1 act like fibronectin with cells sticking to it like last night’s pigs trotters. Caf1 makes a macrophage resistant hydrogel capsule around the Y. pestis cell that prevents interaction with cells (see figure). Ana had thus shown that it keeps its non-adhesive properties in vitro… whoo-hoo!. Thus, Caf1 is a non-stick, tough, flexible, polymer – all things very difficult to build into a protein by design. It can be genetically modified to imitate at least one Extra Cellular Matrix (ECM) protein and physically resembles many more. Ana then showed that we could cross-link it into hydrogels and her pièce de résistance was to show that we could make mixed polymers by expressing differently modified domains in the same cell and letting them mix randomly in the emerging polymer. This was published in Advanced Materials (Ref 4), which will do nicely as one of my REF papers although I am not waiting for the citations to roll in any time soon.

Ana returned to Portugal and the Caf1 project was halted again. We got some short term funding to pay Helen Waller’s salary to make more protein and more mutants.

In November 2013 the Technology Strategy Board (TSB – now Innovate UK) announced the Industrial Biotechnology Catalyst with £20M in the first round. Although I have been involved with the bioprocessing industry for many years I did not think the Catalyst was for me, as it seemed to be either biopharmaceuticals or bioenergy. However, I read the outline for the feasibility awards and it seemed to fit Caf1. I was thinking of a post doc for three years etc but the award size started at £2M, so some imagination was required. This led to the current project. Protein engineering to design and produce new polymers will be done in my laboratory. This will be done by Helen Waller and a new post-doc. David Fulton will employ another post-doc to make our cross-linked hydrogels smart and responsive to a range of stimuli like temperature, light and pH. Matt German will use impressive kit like his Atomic Force Microscope (AFM) to measure the material properties of the gels whilst Nick Reynolds will continue our work on wound care. Last, but not least, ICaMB blog’s very own Neil Perkins will work with another new post doc to understand how cells respond to and remodel the different materials. Neil’s suggestion that his qualification was that he was the first person I met that day is unfounded, the truth is that his title of Prof Gene Expression and Signalling fitted the number of words left in that section of the form (I maintain that the previous 2 people Jeremy had bumped into that day had turned him down – Neil).

To boldly go…

So, we have five years to turn Caf1 into a range of 3D cell culture products. By exploiting bacterial production we hope to reduce costs and provide bulk materials at affordable prices for a series of applications. These will include research tools for cell culture, better culture conditions for industrial processes, tissue and regenerative engineering materials and many more. I have produced products in the past and it is very different to our normal hypothesis driven discovery research. In discovering things about nature you generally have observed something and then set out to find out why it’s like that or how it works. In product invention you never know if it will ever work because, by definition, no one knows (and that is particularly true of protein engineering!). The Industrial Biotechnology Catalyst money is aimed at expanding the UK biotechnology industry and, in taking the money, we must be aware that quite justifiably the tax payer wants to see a return on the investment. Ultimately we hope we can create jobs in a new area based upon this project. What’s more, collaborating across disciplines to invent amazing new materials and use them to cure disease is a great way to spend ones working life.

[1] Soliakov A, Harris JR, Watkinson A, Lakey JH. The structure of Yersinia pestis Caf1 polymer in free and adjuvant bound states. Vaccine. 2010;28:5746-54.

[2] Orengo CA, Jones DT, Thornton JM. Protein superfamilies and domain superfolds. Nature. 1994;372:631-4.

[3] Mitchell EA, Chaffey BT, McCaskie AW, Lakey JH, Birch MA. Controlled spatial and conformational display of immobilised bone morphogenetic protein-2 and osteopontin signalling motifs regulates osteoblast adhesion and differentiation in vitro. BMC Biology. 2010;8:57.

[4] Roque AI, Soliakov A, Birch MA, Philips SR, Shah DS, Lakey JH. Reversible Non-Stick Behaviour of a Bacterial Protein Polymer Provides a Tuneable Molecular Mimic for Cell and Tissue Engineering. Advanced Materials. 2014;26:2704-9.

Donating hope – a success for parents in danger of transmitting mitochondrial disease

Posted on 5 February, 2015 by Paula

by Prof Bob Lightowlers

It was 2pm on a Tuesday afternoon and there I was watching television in my office. An unusual experience (honestly) but it was to be a truly momentous occasion. For the next 90 minutes, members of parliament would be debating whether to sanction the procedure of mitochondrial donation. What on earth does that mean ? Well, mitochondria are essential compartments (or organelles) that provide numerous key functions for the cell. They also have their own genome, called mitochondrial DNA – mtDNA – that contains the information to make up just 13 proteins, all of which are important in their function of producing main energy source of the cell, ATP. This is why mitochondria are often referred to as the cell’s batteries or the powerhouse of the cell. It is important to note that mtDNA is very small when compared to the nuclear DNA component: 16 thousand mitochondrial nucleotides (that is, the “letters” in the genetic code) vs more than 3 billion nucleotides in the nuclear DNA.

OK, but why would you want to donate mitochondria?

Mitochondrial DNA is strictly passed down via our mothers, unlike nuclear DNA which is comes from our father and mother. Almost 2,500 women in the UK have mutations in some or all of their mtDNA that can cause disease. Defects of this mitochondrial genome, can be responsible for a wide spectrum of mainly muscle and neurodegenerative disorders for which there is no treatment. By identifying those women with defective mtDNA, we could potentially prevent transmission of their unhealthy DNA by substituting their mitochondria for organelles from a donor. Simple on paper!

What’s the problem?

There are two major barriers. First, how safe would any technique be for mitochondrial donation ?

The pronuclear transfer method: Embryos are shown with mitochondria carrying normal (green) or mutant (red) mtDNA.
As the embryos begin to develop, pronuclei become visible. Pronuclei from the normal donor embryo are removed (blue, top panel ‘enucleation’) and are replaced with the nuclear DNA from the patients (red, karyoplast). The resultant embryo carries nuclear DNA from the patients and mtDNA from the donor (mitochondrial donor zygote).

The technique being championed in Newcastle is that of pronuclear transfer. The idea is to take the nuclear DNA from a fertilised embryo and transfer the DNA to a donor with no nuclear DNA, ie, where the nucleus was removed. The newly made embryo then has nuclear DNA from the mother and father but has only mtDNA from the donor.
As you can tell, there is quite a lot of tricky manipulation here. Further, although the mtDNA that has been replaced carries only a very small number of genes, could these somehow be incompatible with the nuclear DNA?
After many years of very careful analysis, there is no evidence to suggest that this procedure is unsafe. The question of incompatibility would also seem to be highly unlikely. After all, nature has been performing the experiment of mixing and matching nuclear and mtDNA since the evolution of Homo sapiens. The idea that, for example, there would be something wrong with the child of an aboriginal woman and a inuit man due to the substantial differences in their mitochondrial DNA would seem laughable.

In addition, does the replacement of DNA, albeit the complete mitochondrial genome, constitute genetic manipulation? This is a contentious issue and a strict definition of what constitutes genetic manipulation, particularly when concerning mtDNA, is difficult to agree on. It must be remembered that mtDNA is completely separate from nuclear DNA and needs to be considered as such.

A separate issue is that many people are ethically uncomfortable with this process. Can embryo manipulation ever be acceptable? Certainly, many religious people have a deeply felt objection to this.
Even when we accept that these methods could offer such an immeasurable benefit for many couples, it is clear that there were many questions to be tackled before we could consider the prospect of mitochondrial donation. For this reason, experimentation and public consultations had to be initiated.

The Parliamentary Under-Secretary of State for Health, Jane Ellison, when presenting the vote on Tuesday highlighted the measures taken to date to assess the safety and ethical concerns surrounding mitochondrial donation.

To cut a very long story short, both have been carried out for many years, including supportive public consultations and independent review by the Human Fertilisation and Embryology Authority (HFEA) reporting that the procedure was not dangerous, as we’ve covered before (here and here). Following these procedures, Professors Turnbull, Herbert and numerous members of the Wellcome Trust Centre for Mitochondrial Research were instrumental in persuading the government to finally hold a debate in the House of Commons.

The vote scheduled for Tuesday afternoon would decide whether the HFEA would have the right to offer a licence to perform mitochondrial donation, a first for the UK and the world. The week leading up to the debate was a white-knuckle ride. Letters of support were published in leading newspapers from Nobel Laureates and other eminent scientists. Just when it appeared that the tide was turning, the Church of England announced it could not support mitochondrial donation. This was a great disappointment and rather a shock, as they had been involved throughout the lengthy consultation processes and had not indicated their level of concern.

Now a back-benh MP, former Minister for Science, David Willets, made a clear case in support of mitochondrial donation.

Now a back-bench MP, former Minister for Science, David Willetts, made a clear case in support of mitochondrial donation. You can read the whole debate here.

Back to me watching the television. At 3:45pm, the members had cast their votes and the count was in – 382 for the motion, 128 against! This is a fantastic result. It gives couples who may be at risk of having a baby with mitochondrial disease the chance to choose whether they want to try mitochondrial donation, just like couples have been able to choose in vitro fertilisation since the 1970’s.

Obviously, this result has gathered lots of media interest and even I was rolled out to perform a couple of interviews.

There is still more to be done, however. Further important research to support the safety of the procedure is currently in review but it must be recognised that every clinical procedure carries a risk. What this vote does is to empower the HFEA to licence this procedure in the UK, but this is still an important barrier and many issues are still to be addressed. And it also needs to be discussed by the House of Lords, of course.

Regardless, today’s vote was a wonderful day for anyone who has been touched by mitochondrial disease in any form. Twenty years ago, Doug Turnbull and I used to discuss this idea. He and his colleagues have done a remarkable job to make this pipedream a reality.

Beer, Bread and Bacteria

Posted on 8 January, 2015 by Kevin

Prof. Harry Gilbert

The diverse organisms that live in our gut, collectively termed the gut microbiota, can have a major impact on our health. A new paper from Prof. Harry Gilbert’s lab in ICaMB, published today in Nature, shows that one member of our gut microflora uses a ’selfish’ mechanism to degrade a component of the cell wall of the yeast in our diet.

By Drs. Elisabeth Lowe and Fiona Cuskin.

Have you over-indulged this Christmas? One too many ales, or too many turkey sandwiches?

Beer: full of yeast.

The good news is that, while your waist line may be expanding and you may be feeling a little the worse for wear, the organisms of your gut microbiota are thriving!

Some of the bacteria in the human gut, called Bacteroides, can break down the complex carbohydrates in our diet into simple sugars, which they use for energy.

: Bread: full of yeast

Our work, published in Nature today, shows that one of these bacteria can also break down some of the carbohydrate cell wall of yeasts, called mannan, a polymer of mannose. This includes mannan from Saccharomyces cerevisiae (Baker’s yeast), and the pathogenic gut fungus, Candida albicans. The current understanding of polysaccharide digestion by gut bacteria suggests a cooperative environment, in which break-down products are shared between different members of the microbiota. However our data shows that this species (Bacteroides thetaiotaomicron) is ‘selfish’ when it degrades mannan, and doesn’t share any of the digestion products with other bacteria.

Turkey: er… full of yeast?

To achieve this selfish degradation, the large and multiply-branched mannan structure undergoes only very limited degradation in the extracellular space (see figure). The resulting processed mannan fragments are then imported into the Bacteroides periplasmic space, where they are further degraded by a suite of glycoside hydrolase enzymes to yield the sugar mannose. Achieving complete breakdown within the periplasm prevents the mannose from being shared with competing bacteria.

This selfish approach to mannan utilisation could offer the opportunity to design bespoke prebiotics targeted to a specific bacterial population within the gut. Food products containing yeast mannan would be expected to promote growth of B. thetaiotamicron, whereas other complex polysaccharides may specifically promote other members of the microbiota.

Complex polysaccharide mannan is sequentially degraded by a ‘selfish’ mechanism to yield periplasmic mannose.

A number of inflammatory bowel diseases such as Crohn’s disease are associated with intolerance to yeast, and particularly cell wall polysaccharides, and have also been linked to low levels of Bacteroides species in the gut. Our work provides a potential mechanism for how Bacteroides might be able to influence the effect of yeast on patients with Crohn’s disease, and in fact a drug named Thetanix (which is a live formulation of Bacteroides thetaiotaomicron) has been licensed for treatment of paediatric Crohn’s disease in the USA.

Researchers Max Temple, Lis Lowe and Fiona Cuskin of the Gilbert lab.

Yeast is commonly in our diet in the form of some of our favourite things: bread, beer, wine and fermented food products. So it may be dry January but if your willpower fails and you succumb to an alcoholic beverage, then make sure it’s an unfiltered one with plenty of yeast!

This research in the Gilbert lab was funded by the Wellcome Trust and the European Research Council.

Links:

Nature article: http://www.nature.com/nature/journal/v517/n7533/full/nature13995.html

Press release: http://www.ncl.ac.uk/press.office/press.release/item/beer-and-bread-yeast-eating-bacteria-aid-human-health

Gilbert Lab: http://www.ncl.ac.uk/camb/staff/profile/harry.gilbert

More ICaMB winners! Doctoral Thesis Prize Success.

Posted on 15 July, 2014 by Suzanne

‘The Faculty of Medical Sciences Doctoral Thesis Prize is a mark of recognition of an outstanding level of achievement by the end of a research doctorate. Prizes are awarded biannually on a very limited basis following nomination by thesis examiners.’ Dr Tim Cheek, Post Graduate Tutor

Doctoral Bling!

Prizes were first awarded in 2009 and included two ICaMB students, Holly Anderson and Monika Olahova. This was followed in 2011 by David Adams and in 2012 by Graham Scholefield. However, 2013, was an absolute triumph, with three out of only five potential Faculty Prizes being bestowed on theses submitted by ICaMB students. Dr Andrew Foster from Professor Nigel Robinson’s Lab (currently a post-doc in the Robinson lab in Durham), Dr Fiona Cuskin from Professor Harry Gilbert’s lab (currently a post-doc in the Gilbert lab) and Dr Kristoffer Winther from Professor Kenn Gerdes lab (currently a post-doc in Gerdes lab). With their new roles keeping them busy, our 3 winners only just managed to get together recently to be presented with their medals by the Dean of Post Graduate studies. Andrew and Fiona tell us about their past and present research.

Dr Andrew Foster

Abstract by Dr Andrew Foster. Achieving metal selectivity is often more difficult than one might first imagine as the inherent chemical properties of metals often mean that a metalloprotein will preferentially select an incorrect metal over a correct one.

My PhD studies involved understanding metal selectivity among a group of proteins called metal sensors. These metal sensing, transcriptional regulators control the expression of genes of metal homeostasis and therefore influence the metallation of other proteins within the cell. I characterised a novel nickel sensor InrS and showed for the first time how metal selectivity could correlate with relative metal affinity across a class of proteins. The nickel sensor InrS has a tighter nickel affinity than the other sensors within the cell, thus InrS responds to nickel activating

Nickel

a nickel efflux gene so that the buffered nickel concentration within the cell does not rise high enough to mis-populate the sensors of other metals.

During my PhD studies our lab moved from Newcastle to Durham University but I remained registered at Newcastle. This move was obviously very disruptive but at the same time made me more focussed and determined to make a success of the work in spite of the disruption.

Busy Andrew

I am currently working with Professor Nigel Robinson at Durham University. My current work seeks to understand how the affinity of a metal sensor relates to the available concentration of the sensed element within the cell. Our model system involves the nickel sensor I discovered, InrS, and nickel supply to hydrogenase, a nickel enzyme capable of hydrogen production. Metal supply to enzymes will be a key biotechnological challenge as we seek to utilise microbial factories for the production of fuel and other useful products.

Dr Fiona Cuskin.

Abstract by Dr Fiona Cuskin. The use of complex carbohydrates in the food industry is wide and varied; a few examples include the use of polysaccharides and oligosaccharides as gelling agents, emulsifiers and fat replacements. Small oligosaccharides are being increasingly used as prebiotics for the vast array of “friendly” bacteria in the gut of both humans and animals. The addition of small fructose oligosaccharides by the food industry into yoghurts, amongst other foods, has been shown to promote a healthy gut flora, which in turn has a positive effect on the host gut health and immune system.

Having been in the lab for just a month my supervisor abandoned me and moved to America. Not to worry I tracked him down and moved there too for a few months. The subject of my PhD was to investigate how bacteria use enzymes called glycoside hydrolases to breakdown complex carbohydrates for utilisation. Part of this was to characterise a glycoside hydrolase that degraded the fructose containing polysaccharide, levan.This glycoside hydrolase contained two

Happy gut?

modules, the catalytic module and non-catalytic carbohydrate-binding module (CBM). CBMs are usually attached to enzymes that catalyse the breakdown of recalcitrant insoluble substrates to help target the catalytic module to the right carbohydrate. However, the CBM characterised in my PhD bound soluble fructan polysaccharides and potentiated the activity of the catalytic module ~100 fold. This work adds valuable knowledge to how bacteria breakdown complex polysaccharides. This knowledge can be exploited to better inform the use of prebiotics and to also choose enzymes that are efficient for the production of small oligosaccharides from polysaccharides.

We are very proud of our current winners. Who will be in the next batch of Doctoral Thesis Prize winners, adding to a growing list of ICaMB winners?

Deep Impact

Posted on 24 April, 2014 by Suzanne

Alginate bread on a pedestal under show-biz lights.

Another excellent post by ICaMB’s Dr Matthew Wilcox as the fame of alginate spreads and seaweed bread goes on tour!

Well, I was kindly invited along to the BBSRC Fostering Impact awards ceremony in London a couple of weeks ago and although I wasn’t up for anything, Newcastle University were.

Fostering impact is a scheme run by the BBSRC to capture the economic and social impact of research funded by them. There are three competitions that fit the fostering impact scheme; ‘innovator of the year’, ‘activating impact’ and ‘excellence with impact’. The first is for an individual researcher, the second is for the knowledge exchange teams and the final award is for research organisations (runs from 2013 – 2015).

The knowledge exchange and commercialisation team at Newcastle University has changed substantially over the past couple of years. What was once a centralised Business Development Directorate has now become the Research Enterprise Service, comprised of three teams, each embedded in one of the Faculties. Each Institute or School now has their own dedicated business development manager (BDM), with ICAMB’s BDM being Laura Rush (who is very nice). They are now much easier to contact, whether it’s just a quick question or the drafting of patents.

Home baking practice.

Newcastle’s application for the Activating Impact award was submitted back in October 2013 and used the wonderful research done by the beautiful people of ICAMB as its basis. In January the RES team found out that they had successfully made it to the final five (from 18) and through to the grand final in London. Newcastle was up against the knowledge exchange and commercialisation teams from King’s college London, Queen Mary University of London, University College London and University of Aberdeen. One of the requirements of the competition was to bring along someone to the final who had worked with RES, a ‘user’ (according to the BBSRC). They also wanted an iconic object?! Alginate bread it was then. Back in the kitchen I went. How many loaves would I need to feed the people there? Two should do it, right?

Martin and Laura on the train gearing up for competition.

In London Martin Cox presented the case for Newcastle in front of a panel of scientists, business types and other technology transferers, assembled by the BBSRC. Demonstrating what Newcastle does well, how BDM’s have been embedded into each institute and also what they would do with the money if we won (£100k). He also described the additional internal funds that are available to help activate impact. FMS has two funds available; the first is to help with data collection for translational grant applications, the second is to support further claims in patent applications. The two internal grants can both potentially support a post doc salary for three months, plus consumables.

Dengue fever carrying mosquito.

The awards ceremony combined the ‘innovator of the year’ and ‘activating impact’. I got a glitzy stand for my bread and also had the chance to look around the other pretty amazing stuff that was on display, like Dr Luke Alphey’s work. Luke ended up being named both social innovator and overall innovator of the year for his work on the genetic control of pests, including the dengue fever carrying mosquito.

I even got to meet the new (ish) CEO of the BBSRC, Professor Jackie Hunter, who was definitely not snapped stuffing free stuff into her bag!

BBSRC's CEO Jackie Hunter enjoying the exhibition

On the right BBSRC’s CEO Professor Jackie Hunter enjoying the exhibition.

Unfortunately Newcastle did not win, but being down to the last five of the competition is brilliant and should give confidence to ICAMB scientists that when help is required in achieving impact (social or economic), we have a great team to help.

Queen Mary University of London, whose entry was also being supported by a previous BBSRC Enterprise Fellow and King’s College London, ended up being joint winners each scooping £100k.

Perhaps if a few more of the world leading researchers in ICaMB engaged with the Enterprise team, they might not have to only take some eejit and his bread to the competition next year and increase NU’s chance of winning!

Skinny Seaweed

Posted on 13 March, 2014 by Suzanne

Obesity has reached epidemic proportions globally, with at least 2.8 million people dying each year as a result of being overweight or obese (WHO). Dr Matthew Wilcox from Professor Jeff Pearson’s lab tells us about his research into the slimming powers of brown seaweed. A simple and sustainable food additive.

“Seaweed bread” by Dr Matt Wilcox

Seaweed bread

Over 40% of the calories in your diet can come from fat (mainly triacylglycerol) and you digest and absorb between 95 and 100% of all the fat that you eat! There is one enzyme (pancreatic lipase) that accounts for 80% of all fat digestion and if the activity of this enzyme can be reduced then we will absorb less fat from our diet.

The Pearson lab has shown that alginate (a dietary fibre extracted from brown seaweed) can reduce the activity of pancreatic lipase by up to 80%. If you don’t digest fat you can’t absorb it.

Lipase is a recognised target as a treatment for obesity. In 2010 the pharmaceutical drug orlistat (Xenical) which acts on this enzyme accounted for 98% of all UK prescriptions for the treatment of obesity. The remaining 2% was for Sibutramine which has since been withdrawn. We are therefore heavily reliant upon one drug which can have some rather unpleasant side effects (brown trouser syndrome) greatly reducing patient compliance. So even though it’s the only pharmaceutical drug available, not everyone who is prescribed it will actually take it!

Tasteless alginate is already found in a number of foods (E400-405), salad dressings, ice-creams ….. even beer.

Thankfully there is still hope (and clean trousers) since some, if not all of the side effects of orlistat can be eliminated if it is taken with a high fibre product. Luckily, it just so happens that alginate is not only a lipase inhibitor but is also a dietary fibre itself, and to top it all off makes great bread!

Most commercial alginates come from seaweed, hence the seaweed bread. The extraction process is relatively simple and you end up with a dry white (ish) powder, great to use in products with flour. There is no ‘seaweed’ taste (although seaweed bread is actually quite nice) and in blinded tests people actually prefer the alginate bread over standard white bread. Likely to be due to the retention of moisture so it’s like eating fresh bread, even if it’s a day or so old

Seaweed culture and harvesting is big business around the world from the Shetlands to Shanghai and can even be seen from space. The extracted alginates are already used in the food industry, just not necessarily the right ones or in the right concentrations. The majority we test in the lab are from Norwegian or Scottish seaweeds.

The latest technology in seaweed harvesting.

The alginate project in the lab started nearly eight years ago with simple colourimetric assays. These initial experiments showed that certain seaweed alginates can inhibit lipase. From this we developed a model of the upper gastrointestinal tract to test the alginates in a system as close to a human as possible without being human. Our model gut has now been used to show that alginate is released from the bread (amongst other things) and has also featured in a BBC3 program. We have just finished the first proof of concept trial in humans, so there will be more on this story to follow.

The Pearson Lab line-up

The big fat truth is that fat in food is often vital for the enjoyment, try eating cream crackers without butter (3 cracker challenge). It’s hard! So, if we can still get the enjoyment but not all the calories from our food then we can significantly reduce the amount of calories we take in. It’s not just a reduction in the absorption of fat in the food that contains the alginate; it’s the entire meal that you eat. Not that you should eat loads of burgers but if we could put alginate into burger buns then you would reduce the amount of fat absorbed from the burger, the cheese, the fries and the ridiculously oversized milkshake that you have with it.

Low fat lunch?

Perhaps that’s the best way to end a blog with the thought of a ridiculously oversized milkshake and also by thanking the BBSRC for all their funding and support!

Matt’s PhD was a BBSRC CASE studentship sponsored by Technostics

This work has been patented

Full Links

The patent: http://worldwide.espacenet.com/publicationDetails/biblio?DB=worldwide.espacenet.com&II=1&ND=3&adjacent=true&locale=en_EP&FT=D&date=20121129&CC=US&NR=2012302521A1&KC=A1

Technostics: http://www.technostics.com/

Matthew Wilcox: http://www.ncl.ac.uk/camb/staff/profile/matthew.wilcox

Jeff Pearson: http://www.ncl.ac.uk/camb/staff/profile/jeffrey.pearson

Pruning the Tree of Life

Posted on 13 December, 2013 by Neil

Dr Tom WIlliams

Anyone who has studied biology has seen an image of the tree of life in the text books. Most of us think of this as being set in stone, one of the rock solid foundations on which evolutionary biology is built. However, all is not quite as settled as it seems. Recently, a Nature article from the laboratory of ICaMB’s Professor Martin Embley challenges the traditional three domain structure of the root of life. Here, first author on the paper, Dr Tom Williams, tells us the story.

By Dr Tom Williams

Our modern understanding of the tree of life began in 1977 when Carl Woese and his colleagues discovered the Archaea, a group of prokaryotes originally isolated from extremely hot or salty environments. Although Archaea looked indistinguishable from Bacteria under the microscope, their gene sequences were at least as different to those of Bacteria as from the eukaryotes – the group of organisms, including fungi, animals and plants, whose cells contain a mitochondrion and a nucleus. According to these analyses, living cells should be classified into three main groups: Bacteria, Archaea and eukaryotes – rather than the two (prokaryotes and eukaryotes) that had previously been established based on cell structure. In 1990, Woese and his colleagues published another seminal paper in which they argued for this “three domains” classification. This three-domains tree has become an iconic image in biology, and is often found in the popular science literature, as well as many textbooks – you’ve probably seen it before. Here it is from a 1997 review by Norman Pace:

The traditional 3 domain Tree of Life. From: A molecular view of diversity and the biosphere. Pace NR Science (1997) 276: 734-740

Professor Martin Embley

This was certainly the tree of life that I was familiar with, first as an undergrad and later as a Ph.D. student at Trinity College Dublin. So I was surprised and very intrigued when a certain Martin Embley came to talk at an Irish bioinformatics meeting, claiming that support for the three-domains tree was not as strong as you might expect. New work from his lab instead favoured the “eocyte tree”, in which the eukaryotes (or, at least, some of their genes) actually evolved from within the Archaea. If true, this tree would imply that there were originally only two types of cells – Bacteria and Archaea – and that the eukaryotes (i.e., us!) originated later in a partnership between the two primary domains.

The new model of the Tree of Life proposed by the Embley lab

Fast-forward a couple of years, and I was thinking about where I wanted to do my postdoc. I remembered Martin not only from that talk, but also from some interesting work (2nd link) he had done on a group of parasitic fungi called Microsporidia. I joined his group and began working on microsporidians, but I was still very interested in the tree of life and the origin of eukaryotes. In the meantime, DNA sequencing technology had been improving, and microbial ecologists were beginning to publish genomes from new groups of Archaea that could not be grown in the lab, and so had never been studied before. One of the really exciting findings from these studies was that some Archaea contained genes that looked very similar to fundamental components of our own cells, such as actin and tubulin – two proteins that help to define the microscopic “skeleton” of eukaryotic cells. When we added these new genomes to our analyses, we found even stronger support for the eocyte tree; those findings were reported last year in Proceedings B. At about the same time, a number of other researchers were reporting something similar: as our view of archaeal biodiversity increased, support for the three-domains tree was on the wane. Given the prominent position of the three-domains tree in the literature, and the importance of this question for understanding early life on Earth, we decided to write a review summarizing these recent developments in the field – it came out in Nature this week, and it’s the reason for this blog post!

As we delved back into the 30 years of literature on the molecular tree of life, one of the most interesting discoveries for me was a seam of eocyte literature that I hadn’t been aware of previously. Although many analyses over the past three decades have recovered the three-domains tree, and it appears in all the textbooks, the literature has actually never been unanimous in its support. Nonetheless, it is only in the last five years or so that support for the eocyte hypothesis has reached critical mass, perhaps due to improvements in our statistical methods and, more recently, sampling of archaeal biodiversity.

The Embley lab: Back row, left-to-right: Kacper Sendra, Martin Embley, Tom Williams, Robert Hirt. Front row: Shaojun Long, Ekaterina Kozhevnikova, Andrew Watson, Paul Dean, Maxine Geggie,Alina Goldberg-Cavalleri, Sirintra Nakjang.

Of course, our latest work is almost certainly not going to be the last word on the relationship between eukaryotes and other cells. Our methods are getting better – in part thanks to the statisticians we are collaborating with here in Newcastle – but there is much room for improvement, and so much about the microbial world that we still have to discover. Still, if the eocyte tree is correct – and it appears to be the best-supported tree on the current evidence – then that has important implications for how we understand early life on Earth and the origin of our own cells. For one thing, it rules out the eukaryotes as a primordial cellular lineage, as old as the Bacteria and Archaea. Instead, it suggests that the Bacteria and Archaea were established and diversifying on Earth before the origin of eukaryotes, resurrecting the concept of an “Age of Prokaryotes” on the early Earth. Of course, when you think about the phenomenal number of Bacteria and Archaea that live in your own body, never mind the wider environment, you might well argue that it never ended…

This work was supported by a Marie Curie postdoctoral fellowship to Tom Williams. Martin Embley acknowledges support from the European Research Council Advanced Investigator Programme and the Wellcome Trust.

Links

The Nature Article: http://www.nature.com/nature/journal/v504/n7479/full/nature12779.html?WT.ec_id=NATURE-20131212

The Proceedings B paper: http://rspb.royalsocietypublishing.org/content/279/1749/4870

The Embley lab website: http://research.ncl.ac.uk/microbial_eukaryotes/

Microsporidia papers: http://www.nature.com/nature/journal/v452/n7187/full/nature06606.html

http://www.nature.com/nature/journal/v453/n7194/full/nature06903.html

ICaMB – Inside Cells and Molecules Blog

Category Archives: Exciting research