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

Abstract

SummaryL-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 FtsZdependent 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.

An Acyl-Linked Dimer of Daptomycin Is Strongly Inhibited by the Bacterial Cell Wall

“…These were generated from the Bacillus subtilis strain PDC134, in which expression of the murE operon, which is required for murein synthesis, has been made dependent on xylose, so that xylose withdrawal triggers conversion to L-forms (22). On these L-forms, the MIC of the monomer at 5 mM Ca ++ decreased from 0.5 to 0.075 µg/mL; this increase in activity agrees with previous findings (23).…”

“…With the dimer, however, activity increased dramatically, resulting in an MIC of 0.2 µg/mL which was only slightly (but reproducibly) above that of the monomer. Also note that the MIC for vegetative cells of PDC134 is similar to that observed with B. subtilis strain 1046, indicating that the mutations that characterize PDC134 (22) have no major effect on the susceptibility of vegetative cells.…”

“…The Bacillus subtilis strain PDC 134 has been engineered to make expression of several enzymes in murein synthesis dependent on xylose, so that L-forms can be obtained simply by withdrawing xylose (22). Xylose-free LB medium supplemented with MSM (MgCl 2 , 20 mM; maleic acid, 20 mM; sucrose, 500 mM) at pH 7 was inoculated with a liquid culture of PDC 134 grown beforehand in the presence of xylose (0.5% w/v) and chloramphenicol (20 mg/ml).…”

The Rcs Stress Response and Accessory Envelope Proteins Are Required for De Novo Generation of Cell Shape in Escherichia coli

“…The lysozyme treatment is simple and transient and leaves most cells viable, and the resulting spheroplasts are similar to wild-type cells in size. Other model systems (e.g., ␤-lactam treatment) produce cells that are 4 to 30 times larger (61)(62)(63) and are therefore less useful for examining morphological recovery.…”

A Highly Unstable Transcript Makes CwlO D,L-Endopeptidase Expression Responsive to Growth Conditions in Bacillus subtilis

“…Autolysins have the potential to disrupt the integrity of the cell wall, with potentially lethal consequences. For example, unregulated cwlO transcription, generated by a constitutively active WalR response regulator, compromises the integrity of the cell wall to the extent that protoplasts emerge from the sacculus in isotonic medium (39,40). The degree of control required to allow normal autolysin function without compromising cell viability is strikingly illustrated by the multiplicity of controls on CwlO expression and enzyme activity.…”