The rod to L-form transition of Bacillus subtilis is limited by a requirement for the protoplast to escape from the cell wall sacculus.

L-forms are variants of common bacteria that can grow and proliferate without a cell wall. Little is known about their molecular cell biology but they undergo a remarkable mode of proliferation that is independent of the normally essential FtsZ-dependent division machinery. We have isolated a strain of Bacillus subtilis that can quickly and quantitatively convert from the walled to the L-form state. Analysis of the transition process identified an unexpected 'escape' step needed for L-form emergence from the rod. Mutations in two different genes, walR and sepF, contribute to the high frequency of escape: walR, a transcriptional regulator involved in cell wall homeostasis; and sepF, required for accurate and efficient cell division. Time-lapse imaging shows that the mutations act by facilitating the release of the L-form from its walled parent cell but that they act in different ways. The walR mutation renders the activity of the protein partially constitutive, inappropriately upregulating the activity of autolytic enzymes that weaken the cell wall. The sepF mutation probably works by perturbing the formation of a properly constructed division septum, generating a mechanical breach in the wall. The new strain provides a powerful experimental system for studying the genetics and cell biology of L-forms.