Final Report Summary - OPAL (Origins, proliferation and pathogenesis of L-form (cell wall deficient) bacteria)
The bacterial cell wall is an ancient structure that was probably present in their last common ancestor. It is the target of our best antibiotics and fragments of the wall trigger powerful innate immune responses. L-forms are curious bacterial variants that lack a cell wall. They have been reported in the literature since the 1930’s but very little was known of their molecular biology until recently. The aim of the project was to elucidate basic features of L-form molecular biology, including their strange “blebbing” mode of proliferation. The project was extremely successful in that virtually all of the key aims and objectives were achieved, with most of the findings being published in prominent journals.
We isolated a B. subtilis strain (PDC134) that could be efficiently switched between rod (walled) and L-form (wall deficient) states and used this strain to characterize in detail the “natural history” of L-forms. In particular, we discovered that the switch from rods to L-forms is normally actively prevented by homeostatic mechanisms involved in ensuring the fidelity of cell division and controlling the activity of certain wall autolytic enzymes (Domínguez-Cuevas et al.,2012). The PDC134 strain was then used to carry out a genetic screen for mutations that specifically prevent L-form growth. This in turn allowed us to define a novel step in L-form growth, which we call scission, and which is dependent on a certain level of membrane fluidity. We also showed that various mutations affecting other aspects of cell function, particularly various cytoskeletal elements and chromosome segregation factors, had no effect on L-form viability, ruling out earlier hypothetical mechanisms for L-form proliferation (Mercier et al., 2012).
We found that an L-form promoting mutation, accDA*, works by upregulating membrane synthesis. These results led to a simple model for L-form growth in which excess growth of cell surface area (membrane) generates spontaneous shape changes leading to scission and hence cell proliferation (Mercier et al., 2013). The wider significance of the work described above was reviewed by Errington (2013), including implications for thinking about the early evolution of cellular life, and the possible importance of invention of the cell wall.
We showed that L-forms from which essential genes for cell wall synthesis had been completely deleted, could regenerate a normal ell shape after reintroduction of the synthetic genes, which refuted a long standing hypothesis that cell wall morphogenesis requires a template (Kawai et al., 2014).
We showed that the methods and understanding we had obtained from our work on L-forms of B. subtilis could be applied to other organisms by successfully generating L-forms from 3 widely divergent phyla: Staphylococcus aureus, Corynebacterium glutamicum and Escherichia coli. We established that ability to tolerate deletion of essential cell division genes, such as ftsZ, is an excellent operational definition for true L-form cells, removing an historical impediment to work in this field (Mercier at al., 2014).
By isolating “class 2” mutations that fell outside the ispA gene we discovered that they probably work by relieving oxidative stress. This unexpected finding has important implications for our general understanding of cell wall and antibiotic stress (Kawai et al., 2015).
During the last months of the grant we made the serendipitous discovery that when E. coli L-forms deleted for ftsZ were maintained on plates that supported cell wall synthesis (i.e. reversion plates) colonies appeared at low frequency. These turned out to be genuine walled cells that were growing and dividing in the complete absence of the normally essential cell division machinery. We characterised these mutants in detail, including their strange mode of pinching division, and identified the genes in which mutations were needed to enable proliferation. The results have important implications for how certain groups of bacteria that naturally lack ftsZ may have emerged during evolution (Mercier et al., 2016, Nature Microbiology; under revision following favourable review).
We recognised a commercial opportunity in terms of using L-forms for production of hydrophobic compounds and secreted proteins and filed a patent (Errington et al., 2013). We are presently investigating the possibility of licencing this technology or setting up a start up company. Unfortunately, two attempts to obtain funding through the ERC proof of concept scheme were unsuccessful.
We isolated a B. subtilis strain (PDC134) that could be efficiently switched between rod (walled) and L-form (wall deficient) states and used this strain to characterize in detail the “natural history” of L-forms. In particular, we discovered that the switch from rods to L-forms is normally actively prevented by homeostatic mechanisms involved in ensuring the fidelity of cell division and controlling the activity of certain wall autolytic enzymes (Domínguez-Cuevas et al.,2012). The PDC134 strain was then used to carry out a genetic screen for mutations that specifically prevent L-form growth. This in turn allowed us to define a novel step in L-form growth, which we call scission, and which is dependent on a certain level of membrane fluidity. We also showed that various mutations affecting other aspects of cell function, particularly various cytoskeletal elements and chromosome segregation factors, had no effect on L-form viability, ruling out earlier hypothetical mechanisms for L-form proliferation (Mercier et al., 2012).
We found that an L-form promoting mutation, accDA*, works by upregulating membrane synthesis. These results led to a simple model for L-form growth in which excess growth of cell surface area (membrane) generates spontaneous shape changes leading to scission and hence cell proliferation (Mercier et al., 2013). The wider significance of the work described above was reviewed by Errington (2013), including implications for thinking about the early evolution of cellular life, and the possible importance of invention of the cell wall.
We showed that L-forms from which essential genes for cell wall synthesis had been completely deleted, could regenerate a normal ell shape after reintroduction of the synthetic genes, which refuted a long standing hypothesis that cell wall morphogenesis requires a template (Kawai et al., 2014).
We showed that the methods and understanding we had obtained from our work on L-forms of B. subtilis could be applied to other organisms by successfully generating L-forms from 3 widely divergent phyla: Staphylococcus aureus, Corynebacterium glutamicum and Escherichia coli. We established that ability to tolerate deletion of essential cell division genes, such as ftsZ, is an excellent operational definition for true L-form cells, removing an historical impediment to work in this field (Mercier at al., 2014).
By isolating “class 2” mutations that fell outside the ispA gene we discovered that they probably work by relieving oxidative stress. This unexpected finding has important implications for our general understanding of cell wall and antibiotic stress (Kawai et al., 2015).
During the last months of the grant we made the serendipitous discovery that when E. coli L-forms deleted for ftsZ were maintained on plates that supported cell wall synthesis (i.e. reversion plates) colonies appeared at low frequency. These turned out to be genuine walled cells that were growing and dividing in the complete absence of the normally essential cell division machinery. We characterised these mutants in detail, including their strange mode of pinching division, and identified the genes in which mutations were needed to enable proliferation. The results have important implications for how certain groups of bacteria that naturally lack ftsZ may have emerged during evolution (Mercier et al., 2016, Nature Microbiology; under revision following favourable review).
We recognised a commercial opportunity in terms of using L-forms for production of hydrophobic compounds and secreted proteins and filed a patent (Errington et al., 2013). We are presently investigating the possibility of licencing this technology or setting up a start up company. Unfortunately, two attempts to obtain funding through the ERC proof of concept scheme were unsuccessful.