Bacterial cell wall architecture and dynamics

Lead Research Organisation: University of Sheffield
Department Name: Krebs Institute for Biomolecular Researc


Bacteria are able to assume a myriad of different shapes, a property governed by their cell wall. The cell wall is like an external skeleton not only required for shape determination but also for keeping the cell alive as it is able to withstand the considerable internal forces which would otherwise rupture the cell. The major structural element of the cell wall for most bacteria is a polymer called peptidoglycan (PG). PG is unique to the bacteria and is essential for keeping the bacteria alive. This importance of PG is illustrated by the incredibly wide use of cell wall antibiotics such as penicillin and vancomycin, which prevent PG production. PG is a single large, bag-like molecule that surrounds the cell and whilst very strong is also dynamic to allow the cells to grow and divide. Even though PG is chemically only made of relatively simple building blocks how these are assembled to produce an architecture able to fulfil the many functions of PG has remained largely elusive. We have used a high-resolution microscopy technique that has provided exciting new and unexpected information as to the architecture of the PG. Initially in the rod shaped bacterium Bacillus subtilis we have found cables of material running round the cylinder of the cell providing strength. Also where the cell divides, a complex plate of material is laid down in an apparent spiral. Our further work in the round bacterium Staphylococcus aureus has shown a very different architecture of whorls and small knobbles. It is these features, which allow the cells to solve their engineering problems of maintaining cell integrity whilst allowing growth. The proposed project will take an integrated approach across the biological, chemical and physical disciplines to determine the architecture of PG from the molecular to the cellular level. We will address fundamental questions in microbiology that lie at the heart of the ability of bacteria to grow and proliferate.

Technical Summary

Bacterial cell wall peptidoglycan (PG) is essential for the maintenance of cellular viability and shape determination for most bacteria. PG is a polymer of glycan strands cross-linked via peptide sidechains. It is dynamic being synthesised, modified and hydrolysed to allow for cell growth, division and other important roles. The classical models of PG architecture are of a woven fabric surrounding the cell. Using atomic force microscopy with Bacillus subtilis we have revealed a new architecture of cables along the cylinder and apparent spiral septa. The cabling makes sense in terms of bioengineering as it gives strength in maintaining integrity in the face of the internal osmotic pressure. This has led to a new model of PG architecture. Our initial results with Staphylococcus aureus reveal a very different, but equally exciting architecture of ribs interspersed with a knobbly surface. S. aureus is extraordinary in that as a coccus it divides sequentially in 3 planes. We hypothesise that the ribs form an internal pattern able to carry information to set the division plane orientation across generations. S. aureus is also is peculiar in that it grows in between septation events by expanding its wall without new PG synthesis. This may occur by hydrolysis of nascent septal PG resulting in architectural maturation. The project will take an integrated and interdisciplinary approach to understanding PG architecture and its relationship to growth and division. It will involve a synergy between a biological and physical scientist, associated with a range of complementary collaborators. Using state-of-the-art biophysical and microscopic analysis, PG will be studied from the molecular to the cellular level. PG architecture is all about finding engineering solutions to physical problems encountered by the bacteria. Our wide-ranging studies are at the forefront in this area and will address problems at the very heart of fundamental microbiology.

Planned Impact

The proposed project will build on our exciting recent findings and provide novel insight into how bacteria are able to maintain their viability, grow and divide. There will be a variety of impacts over a range of timescales and in different arenas. The Scientific Community The project will address fundamental questions about the architecture of bacteria. Part of this will be an integral development and utilization of biophysical techniques giving an interdisciplinary approach. The long-term outcome will be a holistic view of how bacteria have evolved engineering solutions underpinning the problems of shape, growth and division. - Development and extension of biophysical approaches. Following on from our initial success the project will utilize many state-of-the-art techniques to address fundamental biological questions. - Establishment of Sheffield as a hub for interdisciplinary biophysical research. The project will enhance our standing in the area and lead to further inward investment. - BBSRC priorities. The project addresses several of the research priorities of the BBSRC (Nanoscience through engineering to application: bionanotechnology; Systems approach to biological research; Technology development for bioscience) and will have potential impacts in them all. - Interdisciplinary research. The project will lead to physical models to explain fundamental biological processes. This will inform future experiments and approaches, leading to potential collaborations with engineers and architects. - Publications. We will produce high quality data that will be published in leading international journals to provide maximum access to user communities. - International collaborations in the field. Wider interactions will develop the area of cell wall research in the UK to maintain and enhance our international standing. - Oral communications. We will participate in national and international meetings and conferences to publicise the work to a diverse audience. Industry, Policy, the Public and UK-PLC The global antibiotics market is in excess of $25billion per annum. The growing spectre of antibiotic resistance has led to renewed emphasis on the development of novel drugs. The project is fundamental, underpinning science in this extremely important area of immense public concern. - Intellectual property. Where appropriate IP will be secured to facilitate income generation in the long-term. - New antibiotic targets. The project will provide fundamental data to inform the development of new antibiotic targets. - Public Engagement. I (SJF) have participated in several radio and newspaper interviews over the years to inform the public concerning our research and other pertinent issues of concern. I have also hosted several school parties in order to inform the younger generation. Training Our interdisciplinary project should not be seen as a stand-alone enterprise, otherwise we will have missed a great opportunity. - Training of project staff in interdisciplinary approaches to biology. Specifically the RA and technician will become experts in a diverse range of skills. - The next generation of scientists. Both the RA and the technician will be actively involved in transfer of their skills and knowledge to PhD students, MSc students and undergraduates in the laboratories of both investigators. - Dissemination of skills and expertise. Visitors from other laboratories in the UK and internationally will be trained in the new technologies. - Teaching. The project will be central to the development of new models to explain the fundamental principles of bacterial shape, growth and division. Already our initial work in B. subtilis has overturned the textbook images of cell walls for this organism. The research will have implications at the level of teaching of microbiology to undergraduates.


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Description 1. Elucidation of the molecular basis of peptidoglycan architecture in B. subtilis
Our previous studies had revealed a totally novel and unexpected architecture for the PG of B. subtilis (Hayhurst, E. et al. (2008) Proc Natl Acad Sci U S A 105, 14603). This comprises of apparent "cables" of PG running around the short axis of the cells. During division the septum appears as an elegant spiral of material. Our current studies have taken a number of approaches to further analyse the architecture of PG in B. subtilis.
This objective has 2 sections. Firstly to further analyse the PG by the use of protoplasts, mutants and other approaches. Secondly to apply a range of biophysical approaches to gain higher resolution AFM.
Biological approaches:
Protoplast regeneration: Protoplasts have had their cell wall removed enzymatically . During regeneration the cell wall regrows and eventually the cell assumes its original rod shape. By following morphology via fluorescence and electron microscopy we have developed a new model for how this process occurs. Initially a web of PG is produced that intercalates to form a cuff of PG around the protoplast. This leads to a tube of PG visible by EM and a dog bone shape to the regenerating protoplast. A division septum is then made giving a single open ended cylinder and finally a new division septum leads to a new rod shaped vegetative cell. Purification and AFM analysis of PG during regeneration reveals a lattice of ropes and cables of material (see Case for Support Fig. 5). This corroborates our cabling model for PG architecture. We are currently using fluorescent amino acid derivatives to begin to determine how the web of PG material is synthesised and develops into the mature wall architecture.
We have also spent some time developing and applying tools to allow PG architecture to be interrogated in a number of ways.
The "Rack": We have developed an apparatus ("The Rack") to allow PG sacculi to be stretched, whilst being viewed by AFM. The main method development stage has been to define a surface to which sacculi will remain attached during the experiment. The final method involves casting a silicon rubber support and coating this with "Cell Tack" to immobilize the sacculi. We have proven, and published, the efficacy of this method with E. coli (Turner et al., 2013).
In situ PG digestion: A method has been developed to allow sacculus imaging in liquid and in situ digestion with a range of PG hydrolases to reveal architectural features. This requires imaging over many hours and correlation of sequential images. We have used this method to successfully reveal PG architecture (Turner et al., 2013) and are currently expanding this to look at a range of species.
Use of B. subtilis mutants: We are currently examining a range of B. subtilis mutants to determine effects on PG architecture. In particular using the tagO (lacking wall teichoic acid (WTA)) coupled with chemical regimes to remove WTA has revealed this secondary polymer to form a carpet like structure across the surface of the PG. This is extremely important as it stresses the need to remove WTA if one is to make conclusions as to the architecture of PG.
Biophysical approaches:
This is the subject of our current main area of research. We are beginning to develop several ultra-resolution microscopy approaches for analysis of PG as described in the Case for Support. This includes Exit Wave Reconstruction Electron Microscopy, which has only just been invented. The techniques are beginning to take us to the next level of resolution and understanding of PG architecture.

2. Analysis of architecture and dynamics during growth and division of S. aureus
S. aureus is a true coccus in which division occurs equatorially and on specific sequentially orthogonal planes in three dimensions resulting, after incomplete cell separation, in the "bunch of grapes" cluster organization that defines the genus. S. aureus also has a remarkable growth mode in that PG is only laid down during septation. The organism divides to form a pseudohemisphere and then the septal plate expands to allow the organisms to become round. This expansion occurs without any new PG synthesis and occurs via maturation of existing material. The PG architecture, and its dynamics, in S. aureus were examined and correlated with the cell cycle. AFM of purified sacculi demonstrated that the material can assume 2 different morphologies within a single organism. Initially the septum is produced as a likely spiral of thin ropes of material. After division this is remodelled by the action of PG hydrolases to take on a knobbly structure. Fluorescence microscopy with vancomycin labeling was used to determine the dynamics of peptidoglycan synthesis and remodeling throughout the cell cycle and intergenerationally. This revealed that at each division, each daughter cell has sectors of PG from generations before, leading to a characteristic pattern of PG of different ages in its wall. This means that within a population, cells have a heterogeneous cell wall age. The sectoring has also led to the first hypothesis as to how S. aureus is able to divide with fidelity in 3 planes. At the presumptive septum, cells were found to form a large belt of peptidoglycan in the division plane prior to the centripetal formation of the septal disc. This "piecrust" forms the interface between the new septal plate and the existing cell wall. During division the piecrust is split in two, with each daughter inheriting half (called ribs). Over the generations the ribs are inherited forming the boundaries of the sectors of PG of different ages. The ribs form a set pattern and can carry the structural information necessary for correct septal placement over the generations. We propose that this epigenetic information, a previously unknown form of structural inheritance, is used to enable S. aureus to divide in sequentially orthogonal planes. This would explain how a spherical organism can maintain division plane localization with fidelity over many generations, which leads to the characteristic "bunch of grapes" cellular distribution. This work has been now published (Turner et al., 2010).
We have gone on to further characterise the growth mode of S. aureus as to how it is able to round up after division without any new cell wall synthesis. Key characteristics of S. aureus PG are that it has short glycan strands (mostly <10 disaccharides) and there is evidence of substantial glucosaminidase (PG hydrolase) activity. We hypothesised that the glucosaminidases may be required for growth after division. There are 4 putative glucosaminidases in the genome and any combination of 3 genes can be mutated. A triple mutant with the fourth gene under the control of an inducible promoter showed that together the enzymes are required for growth. When the inducer is removed the cells are able to divide but not show subsequent growth and cells get stuck at the post-septation pseudohemispherical stage. Glucosaminidase activity is also required for the short glycan strands, as a strain missing one of the enzymes (SagB) has much longer strands (>100 disaccharides). This is an important finding as it shows that S. aureus (like other Gram positives) synthesises long glycans but it is a requirement of the growth mode that leads to short chains. This also means that the glucosaminidases of S. aureus are potential targets for novel therapeutics.

3. Determination of the conservation of peptidoglycan architectural solutions across the bacteria
This objective has been highly successful and has resulted in significant findings for a number of organisms. During the project our AFM resolution capability has increased leading us to be able to tackle a series of Gram negative bacteria in particular.
Escherichia coli, Caulobacter crescentus, Pseudomonas aeruginosa and Campylobacter jejuni:
PG architecture was determined for a range of Gram negative bacteria and found to be largely conserved. PG is made up of circumferentially oriented bands of material interspersed with a more porous "filigree" network. The observed architecture was not an artefact of sacculus shrinkage during purification as our PG stretching device ("The Rack") revealed that features are still present under tension. In situ digestion of material using PG hydrolases revealed the glycans to likely run parallel to the cell membrane and that peptides hold glycan together within a structure of heterogeneous thickness. This architecture was unlike any previously hypothesised and led to the question of how these bacteria are able to grow? In order to address this we developed vancomycin labelling of cells and sacculi for super-resolution fluorescence microscopy. This necessitated building our own Stochastic Optical Reconstruction Microscope (STORM) to give the required resolution. STORM revealed an unexpected discontinuous, patchy, synthesis pattern. We have developed a consolidated model of growth via architecture-regulated insertion, where we propose only the more porous regions of the peptidoglycan network are permissive for synthesis. This work has been now published (Turner et al., 2013).
The ovococci (Streptococcus pneumoniae, Enterococcus faecalis and Lactococcus lactis) have an ovoid cell shape. AFM analysis showed preferential orientation of the peptidoglycan network parallel to the short axis of the cell, with distinct architectural features associated with septal and peripheral wall synthesis. Super-resolution three-dimensional structured illumination fluorescence microscopy was applied for the first time in bacteria to unravel the dynamics of peptidoglycan assembly in ovococci. Our observations reveal the ovococci to have a unique peptidoglycan architecture and growth mode not observed in other organisms. This work has been now published (Wheeler et al., 2011).
Other organisms:
We are currently examining the PG architecture of a range of other organisms to identify underlying rules and conservation of structure across the bacteria. These are all being carried out in collaboration (Mycobacterium and Corynebacterium (Prof. Besra, University of Birmingham); Bdellovibrio (Prof. Sockett, University of Nottingham; Haemophilus and Helicobacter (Prof. Ivo Boneca, Inst. Pasteur, Paris, France; Myxococcus (Dr Higgs, Max Planck, Marburg, Germany).

- Turner, R.D., Hurd, A.F., Cadby, A., Hobbs, J.K. & Foster, S.J. (2013) Cell wall elongation mode in Gram-negative bacteria is determined by peptidoglycan architecture. Nature Communications 4, 1496-1504.
- Wheeler, R., Mesnage, S., Boneca, I.G., Hobbs, J.K. & Foster, S.J. (2011) Super-resolution microscopy reveals cell wall dynamics and peptidoglycan architecture in ovococcal bacteria. Molecular Microbiology, 82, 1096-1109.
- Turner, R.D., Ratcliffe, E.C., Wheeler, R., Golestanian, R., Hobbs, J.K. & Foster, S.J. (2010) Peptidoglycan architecture can specify division planes in Staphylococcus aureus. Nature Communications 1(3), 1-9.
Exploitation Route Drug companies
Sectors Pharmaceuticals and Medical Biotechnology

Description The findings have been used to inform our understanding of the architecture of the bacterial cell wall
First Year Of Impact 2010
Description Reserach Grant
Amount £676,775 (GBP)
Funding ID BBL006162/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 03/2014 
End 03/2018
Description Public seminar during Science Week 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Public talk on infectious disease, control and the spread of resistance.
Year(s) Of Engagement Activity 2016
Description School visits 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Introduction to microbiology and the interaction of microbes with the human host. A mixture of presentation and practical

No subsequent impact
Year(s) Of Engagement Activity 2009,2010,2011,2012,2013
Description Shambala Festival August 2016 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Krebsfest went on tour to the Shambala Festival with series of talks and workshops.
Year(s) Of Engagement Activity 2016
Description Virtual reality E. coli 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact We developed a virtual reality E. coli experience that was used at Festival of the Mind in September 2016. Hugely successful.
Year(s) Of Engagement Activity 2016