EVALUATING ELONGASOME TUG-OF-WAR AS A KEY REGULATOR OF BACTERIAL CELL WALL SYNTHESIS
Lead Research Organisation:
Newcastle University
Department Name: Biosciences Institute
Abstract
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Technical Summary
How bacteria regulate their shape and material properties of their cell wall is a major outstanding problem with substantial relevance to biomedicine and biotechnology. The elongasome is responsible for processive peptidoglycan (PG) synthesis around the cell circumference driving morphogenesis and envelope expansion in a wide range of Gram-positive and Gram-negative rod-shaped bacteria. Despite the major role of the elongasome in determining cell wall properties, how cells regulate elongasome processivity (the length of elongasome synthesis events) and thereby set the length of new elongasome-synthesized glycan strands remains unknown.
In preliminary experiments, we applied single molecule tracking to determine the processivity of the elongasome. We found that elongasome processivity in B. subtilis is likely determined by molecular tug-of-war caused by multiple PG synthases pulling in opposite directions on a single MreB filament. We further found evidence that elongasome processivity modulates B. subtilis cell size, and initial evidence that E. coli elongasome dynamics are regulated by synthase tug-of-war as well.
Based on these results, we hypothesize that elongasome tug-of-war is a conserved key regulator of elongasome processivity, cell wall material properties and cell fitness. We propose a research programme to determine:
- The molecular principles of elongasome bidirectional motility and processivity in B. subtilis
- How elongasome processivity affects B. subtilis cell shape and cell wall properties
- Whether elongasome tug-of-war is a broadly conserved cell wall regulatory process by analysing E. coli elongasome processivity, cell shape and cell envelope properties
- The potential biomedical and biotechnological applications by analysing the consequences of elongasome tug-of-war-linked cell wall properties on susceptibility towards bacteriolytic antibiotics, and lysis during industrially relevant fermentation conditions
In preliminary experiments, we applied single molecule tracking to determine the processivity of the elongasome. We found that elongasome processivity in B. subtilis is likely determined by molecular tug-of-war caused by multiple PG synthases pulling in opposite directions on a single MreB filament. We further found evidence that elongasome processivity modulates B. subtilis cell size, and initial evidence that E. coli elongasome dynamics are regulated by synthase tug-of-war as well.
Based on these results, we hypothesize that elongasome tug-of-war is a conserved key regulator of elongasome processivity, cell wall material properties and cell fitness. We propose a research programme to determine:
- The molecular principles of elongasome bidirectional motility and processivity in B. subtilis
- How elongasome processivity affects B. subtilis cell shape and cell wall properties
- Whether elongasome tug-of-war is a broadly conserved cell wall regulatory process by analysing E. coli elongasome processivity, cell shape and cell envelope properties
- The potential biomedical and biotechnological applications by analysing the consequences of elongasome tug-of-war-linked cell wall properties on susceptibility towards bacteriolytic antibiotics, and lysis during industrially relevant fermentation conditions
Organisations
Publications
Middlemiss S
(2024)
Molecular motor tug-of-war regulates elongasome cell wall synthesis dynamics in Bacillus subtilis
in Nature Communications
| Description | Imbalance between cell biomass production and envelope biosynthesis underpins the bactericidal activity of cell wall -targeting antibiotics |
| Amount | £112,000 (GBP) |
| Organisation | Medical Research Council (MRC) |
| Sector | Public |
| Country | United Kingdom |
| Start | 08/2023 |
| End | 08/2027 |
| Description | Interplay between bacterial multidrug efflux and outer membrane permeability creates an unexpected vulnerability towards antibiotics |
| Amount | £104,000 (GBP) |
| Funding ID | DTP studenthip, Project Reference 2928093 |
| Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
| Sector | Public |
| Country | United Kingdom |
| Start | 08/2024 |
| End | 08/2028 |
| Description | Understanding the functional dynamics of antimicrobial tolerance in the bacterial pathogen Enterococcus faealis |
| Amount | $76,000 (NZD) |
| Funding ID | 23-UOO-015-CSG |
| Organisation | Royal Society of New Zealand |
| Sector | Charity/Non Profit |
| Country | New Zealand |
| Start | 09/2023 |
| End | 09/2025 |
| Description | Bioenergetics of Enterococcus faecalis and its implication to antibiotic mode of action and AMR. |
| Organisation | University of Otago |
| Department | Department of Microbiology & Immunology |
| Country | New Zealand |
| Sector | Academic/University |
| PI Contribution | The collaborative award has funded a 6 month research stay of a PhD students from the group of Professor Gregory Cook (University of Otago) to carry out collaborative research in my laboratory in Newcastle. |
| Collaborator Contribution | The collaborative award funds a 2 month sabbatical stay of myself at the University of Otago, which will take place mid-March to mid-May 2025. |
| Impact | No output other than knowledge exchange yet. |
| Start Year | 2023 |
| Description | Cellular processes underpinning bactericidal and bacteriolytic activities of envelope-targeting antibiotics |
| Organisation | Durham University |
| Department | Department of Biosciences |
| Country | United Kingdom |
| Sector | Academic/University |
| PI Contribution | Lead-supervision of two shared PhD students funded by BBSRC and MRC DTP PhD studentships, respectively. The supervisor team includes Dr Gary Sharples (Durham), Dr Rebecca Corrigan (Sheffield) and Dr Kevin Waldron (Polish Academy of Sciences). |
| Collaborator Contribution | Co-supervision of a shared DTP PhD students including hosting the students during research stays. |
| Impact | No outputs yet beyond funding for a BBSRC and MRC DTP PhD studentship, respectively. |
| Start Year | 2021 |
| Description | Cellular processes underpinning bactericidal and bacteriolytic activities of envelope-targeting antibiotics |
| Organisation | Polish Academy of Sciences |
| Country | Poland |
| Sector | Public |
| PI Contribution | Lead-supervision of two shared PhD students funded by BBSRC and MRC DTP PhD studentships, respectively. The supervisor team includes Dr Gary Sharples (Durham), Dr Rebecca Corrigan (Sheffield) and Dr Kevin Waldron (Polish Academy of Sciences). |
| Collaborator Contribution | Co-supervision of a shared DTP PhD students including hosting the students during research stays. |
| Impact | No outputs yet beyond funding for a BBSRC and MRC DTP PhD studentship, respectively. |
| Start Year | 2021 |
| Description | Cellular processes underpinning bactericidal and bacteriolytic activities of envelope-targeting antibiotics |
| Organisation | University of Sheffield |
| Country | United Kingdom |
| Sector | Academic/University |
| PI Contribution | Lead-supervision of two shared PhD students funded by BBSRC and MRC DTP PhD studentships, respectively. The supervisor team includes Dr Gary Sharples (Durham), Dr Rebecca Corrigan (Sheffield) and Dr Kevin Waldron (Polish Academy of Sciences). |
| Collaborator Contribution | Co-supervision of a shared DTP PhD students including hosting the students during research stays. |
| Impact | No outputs yet beyond funding for a BBSRC and MRC DTP PhD studentship, respectively. |
| Start Year | 2021 |
| Description | How do membrane potential and efflux pumps interact to affect bacterial physiology, antibiotic susceptibility and solvent tolerance? |
| Organisation | University of Birmingham |
| Country | United Kingdom |
| Sector | Academic/University |
| PI Contribution | This is a new interdisciplinary collaboration with Prof Jessica Blair (University of Birmingham, Institute of Microbiology and Infection) and Dr Tim Overton (University of Birmingham, School of Chemical Engineering). At this early stage, we were involved in collecting key preliminary data for a collaborative BBSRC grant proposal that is currently under evaluation. |
| Collaborator Contribution | The research partners provided further preliminary data for the grant proposal. |
| Impact | The collaboration has resulted in a multi-disciplinary grant proposal that us currently under evaluation. |
| Start Year | 2023 |
| Description | Hydrogen and carbon dioxide biochemistry in the bacterial energy-transducing membrane |
| Organisation | Max Planck Society |
| Department | Max Planck Institute of Biophysics |
| Country | Germany |
| Sector | Charity/Non Profit |
| PI Contribution | This is a new collaboration with Prof Frank Sargent (Newcastle University) and Dr Bonnie Murphy (Max Planck Institute of Biophysics, Germany) funded by a BBSRC responsive mode grant BB/Y004302/1 due to start in 2024. At this early stage, we were involved in collecting key preliminary data for the grant proposal, which was building upon knowledge and techniques established in the framework of grant BB/S00257X/1, and using equipment funded by grant BB/T017570/1. |
| Collaborator Contribution | The research partners Prof Frank Sargent (Newcastle University) and Dr Bonnie Murphy (Max Planck Institute of Biophysics, Germany) provided further preliminary data for the successful grant proposal. |
| Impact | Funded BBSRC grant BB/S00257X/1 |
| Start Year | 2023 |
| Description | Hydrogen and carbon dioxide biochemistry in the bacterial energy-transducing membrane |
| Organisation | Newcastle University |
| Country | United Kingdom |
| PI Contribution | This is a new collaboration with Prof Frank Sargent (Newcastle University) and Dr Bonnie Murphy (Max Planck Institute of Biophysics, Germany) funded by a BBSRC responsive mode grant BB/Y004302/1 due to start in 2024. At this early stage, we were involved in collecting key preliminary data for the grant proposal, which was building upon knowledge and techniques established in the framework of grant BB/S00257X/1, and using equipment funded by grant BB/T017570/1. |
| Collaborator Contribution | The research partners Prof Frank Sargent (Newcastle University) and Dr Bonnie Murphy (Max Planck Institute of Biophysics, Germany) provided further preliminary data for the successful grant proposal. |
| Impact | Funded BBSRC grant BB/S00257X/1 |
| Start Year | 2023 |
| Description | Transmembrane potential regulates lipid packing and protein binding |
| Organisation | Durham University |
| Department | Department of Physics |
| Country | United Kingdom |
| Sector | Academic/University |
| PI Contribution | This is a new cross-disciplinary collaboration with Profs Kislon Voichovsky and Margarita Staykova (Durham University, Department of Physics). At this early stage, we were involved in collecting key preliminary data for a BBSRC collaborative grant that is currently under evaluation. |
| Collaborator Contribution | The research partners provided further preliminary data for the successful grant proposal. |
| Impact | This is a multidisciplinary research project bringing in vivo membrane biophysics and soft matter physics. |
| Start Year | 2023 |
| Description | School class visit |
| Form Of Engagement Activity | Participation in an open day or visit at my research institution |
| Part Of Official Scheme? | No |
| Geographic Reach | Local |
| Primary Audience | Schools |
| Results and Impact | 10 members of Emmanuel College (Gateshead) Genetics Club visited the Newcastle University Centre for Bacterial Cell Biology. The visit included a tour around our research facilities , presentations of our research activities by several academics including me, and a vibrant discussion with the students about current challenges linked to microbiology with focus on antibiotics and AMR. |
| Year(s) Of Engagement Activity | 2024 |