Lead Research Organisation: University of Warwick
Department Name: School of Life Sciences


Almost all bacteria are surrounded by a mesh-like peptidoglycan cell wall that is essential for their survival. Due to the cell's high internal osmotic pressure (turgor), defects in cell wall structure cause bacteria to die by cell lysis. How bacteria regulate cell wall synthesis to build a strong, precisely shaped and structured cell wall remains a major puzzle bridging physics and bacteriology. Many rod-shaped bacteria, including major antibiotic resistant pathogens, grow by adding new material to the cylindrical portion of their cell wall, thus leading to cell elongation. This is achieved by an essential multi-protein synthesis machinery called the elongasome, which inserts glycan strands around the circumference of the cell thereby elongating and reinforcing the cell wall and giving cells their rod shape. It is likely that the length of new elongasome-synthesized circumferential glycan strands has substantial effect on key cell wall properties including vulnerability to lysis upon antibiotic treatment or changing environmental conditions. However, how cells regulate the length of circumferential glycan strands is unknown.

This proposal focusses on two current knowledge gaps: how do rod-shaped bacteria regulate the length of circumferential glycan strands, and how does this affect bacterial cell wall properties and cell fitness?

We will address these questions by testing two central hypotheses:
(1) Molecular motor tug-of-war, where multiple synthesis complexes pull individual elongasomes in opposite directions, is a key regulator of elongasome processivity, ie the length of elongasome synthesis events, which, in turn, determines the length of new circumferential glycan strands.
(2) Elongasome processivity and associated glycan strand length are key determinants of cell wall material properties and cell fitness in rod-shaped bacteria

We will test these hypotheses in key Gram-positive and -negative model organisms Bacillus subtilis and Escherichia coli.

-During a successful BBSRC DTP PhD studentship supervised by the co-PIs, we developed a single molecule tracking method that allows us to accurately determine elongasome processivity for the first time.
-We found strong evidence that B. subtilis elongasome processivity is regulated by synthase tug-of-war, and that elongasome processivity has substantial effect on cell shape.
-This makes us uniquely placed to determine (i) the molecular mechanism of elongasome tug-of-war in B. subtilis, (ii) whether tug-of-war is a conserved cell wall regulatory mechanism active in Gram-positive and -negative rod-shaped model bacteria B. subtilis and E. coli, and (iii) how it affects the cell wall properties and cell fitness of both organisms.

This project leverages established specialist instrumentation: a unique Holden lab custom single molecule microscope, a BBSRC 19ALERT funded microscope with integrated microfluidics, a small scale bacterial fermentation setup, and extensive apparatus for cell wall biochemical analyses. The project thus requires no investment in equipment except ongoing maintenance. Furthermore we have a large number of relevant and readily constructed bacterial strains from ongoing BBSRC DTP PhD and responsive mode grants and highly relevant interdisciplinary expertise from all PIs. As one PI has an ongoing Wellcome fellowship, first 11 months of his PI time are of no cost to UKRI.

-Fundamental knowledge gain: The proposal will substantially advance our understanding of bacterial envelope biophysics, bacterial molecular machines, and bacterial cell biology.
-Potential applications in biomedicine and biotechnology: The pilot studies carried out in WP4 will evaluate the utility of our fundamental research in biomedicine (to identify novel synergistic antibiotic combinations) and in biotechnology (to reduce cell lysis during fermentation condition used in heterologous protein production).

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


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