Characterization of factors involved in

proliferation of Bacillus subtilis L-forms

Informe

Información del proyecto

CFILP Financiado con arreglo a

FP7-PEOPLE
Identificador del acuerdo de subvención:
255096 Presupuesto
general
€ 172 240,80
Proyecto cerrado
Aportación de la
Fecha de inicio Fecha de UE
finalización € 172 240,80
1 Enero 2011
31 Diciembre 2012

Coordinado por
UNIVERSITY OF NEWCASTLE
UPON TYNE
  Reino Unido

Final Report Summary - CFILP (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

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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.

Última actualización: 5 Julio 2013

Número de registro: 56672

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