CFILP · Characterization of factors involved in proliferation of Bacillus subtilis L-forms

Characterisation of factors involved in proliferation of Bacillus subtilis L-forms

The cell wall is a crucial protective outside surface layer present in all major branches of the bacterial subkingdom. Interestingly, most bacteria are capable of mutating into a cell-wall-deficient 'L-form' state. Moreover, most classically described L-forms were identified as antibiotic resistant or persistent organisms isolated in association with a wide range of infectious diseases. We recently developed a tractable system for studying the cell biology and genetics of L-forms in the bacterial model Bacillus subtilis. The following main discovery was the identification of an unknown mode of cell division by an unusual membrane cell shape deformation and scission process, independent of the normally essential cell division protein machinery. The proposed project was aimed at unravelling the mechanisms underlying the remarkable processes by which L-forms proliferate. To identify factors involved, we first isolated and studied genetic mutations capable of promoting cell division and proliferation in absence of a cell wall. We discovered that the key mutations for L-form proliferation induce the production of excess membrane by over activation of the fatty acid synthesis system. Furthermore, we showed that artificially increasing the cell surface area / volume ratio in wild type cells lacking the cell wall was sufficient to induce L-form-like membrane deformation and scission followed by the formation of progeny cells. Secondly, we developed, with a strain carrying the prerequisite L-form mutations, an unbiased gene inactivation approach to search for mutants affected in L-form proliferation. We isolated a mutant implicated in the synthesis of a specific component of phospholipid membranes, the branched chain fatty acids. The main phenotype observed in this mutant was a reduction in membrane fluidity. We found that the reduced membrane fluidity blocked L-form proliferation at a membrane scission step, preventing the release of progeny cells. Taken together, our results suggest the following model of L-form division. Unbalanced growth generates an increase in cell surface area relative to cytoplasmic volume, which leads to spontaneous shape deformation. Subsequently, provided the membrane is sufficiently flexible, the disequilibrium between surface area and volume can be corrected by progeny formation (division), because the total surface area of several small cells is more than that of a single cell of equal total volume and similar shape. Interestingly, our results accord with previous theoretical and in vitro studies of membrane vesicle reproduction aimed at understanding the possible replicative mechanisms of primitive cells. In conclusion, our results provide direct support for the notion that purely biophysical effects could have supported an efficient mode of proliferation in primitive cells, before the invention of the cell wall, and provide an extant model for exploration of the possible properties of early forms of cellular life.

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