The cell wall is an essential structure for virtually all bacteria, forming a tough outer shell that protects the cell from damage and osmotic lysis. It is the target of our best antibiotics. L-form strains are wall-deficient derivatives of common bacteria that have been studied for decades. However, they are difficult to generate and typically require growth for many generations on osmotically protective media with antibiotics or enzymes that kill walled forms. Despite their potential importance for understanding antibiotic resistance and pathogenesis, little is known about their basic cell biology or their means of propagation. We have developed a controllable system for generating L-forms in the highly tractable model bacterium Bacillus subtilis. Here, using genome sequencing, we identify a single point mutation that predisposes cells to grow without a wall. We show that propagation of L-forms does not require the normal FtsZ-dependent division machine but occurs by a remarkable extrusion-resolution mechanism. This novel form of propagation provides insights into how early forms of cellular life may have proliferated.
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We thank D. Swan for assistance with bioinformatics; A. Danchin for providing unpublished sequence data; E. J. Allan for providing the L-form strain that sparked our interest; T. Davey and V. Thompson of the Electron Microscopy Research Service of Newcastle University for technical assistance with electron microscopy; S. Gruber for help with quantitative real time PCR; J.-W. Veening and H. Murray for supplying strains and advice; and D. Claessen, K. Gerdes, H. Murray and W. Vollmer for critical reading of the manuscript. M.L. was supported by the UK Biotechnology and Biological Sciences Research Council. P.D.-C. was supported by an EMBO Long-Term Fellowship.
Author Contributions M.L. and J.E. designed the research and wrote the manuscript. M.L. performed the experiments. P.D.-C. constructed plasmids and strains and contributed to discussions. J.M.C. performed the genome sequencing. R.A.D. constructed strains, and contributed to discussions and the design of the research. All authors commented on the manuscript.
This file shoes time-lapse microscopy of a field of growing L-forms. (See file s1 for full legend).
This file shows time-lapse microscopy of an L-form producing an extrusion that resolves to form multiple progeny. (See file s1 for full legend).
This file shows time-lapse microscopy of an L-form dividing to produce pairs of progeny. (See file s1 for full legend).
This file shows time-lapse microscopy showing many small bodies erupting form the surface of a large L-form. (See file s1 for full legend).
About this article
Nature Microbiology (2016)