Bacterial cell wall architecture and dynamics

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.

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.

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.