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Figure S1 Repression of ispA Expression Supports L-Form Growth, Related to Figure 1 Show full caption (A) Growth of strains Bs115 (P xyl -murE-B, left), LR2 (ispA P xyl -murE-B, right) and RM82 (ispA P xyl -murE-B, amyE:: xseB-ispA+, bottom) cultured on NB/MSM plates to support L-form growth. Provision of an ectopic copy of ispA+ prevents growth in the L-form state. (B) Growth of strain YK1410 (P xyl -murE-B P spac -ispA) streaked on NA/xylose plates (left) to monitor walled cell growth and on NB/MSM plates with (middle) or without (right) 1mM IPTG, to monitor L-form growth.

Figure 1 Effects of ispA and murE-B Mutations on L-Form Growth Show full caption (A–C) Strains LR2 (ispA P xyl -murE-B; A), 168CA (wild-type; B), and Bs115 (P xyl -murE-B; C) were grown in the walled state then converted to protoplasts, incubated in L-form-supporting medium (NB/MSM, no xylose) with benzamide (FtsZ inhibitor), and observed by time-lapse phase contrast microscopy. (C) Deformed cells are labeled with arrows, the remains of lysed cells with hashes, and a star points to a successful division event. Elapsed time (min) is shown in each panel. Scale bars, 3 μm. See also Figure S1 and Movie S1

We previously showed that repression of the PG precursor pathway using a repressible P-murE-B construct, together with a single point mutation of the ispA gene encoding a polyisoprenoid synthase, results in L-form growth of B. subtilis 168 (). Consistent with this, complementation of the ispA mutation prevented L-form growth in a similar strain ( Figure S1 A available online). We also built a strain with P-murE-B and a repressible allele of ispA and showed that this strain also grew well in the L-form state when both promoters were repressed ( Figure S1 B). Investigations of L-form phenotypes are complicated by several factors: (1) the heterogeneity of the population in terms of cell shape and size; (2) strong selection for compensating mutations that enhance the growth rate or cell stability; and (3) requirement for “escape” mutations that facilitate the emergence of L-forms from a population of rods (). To help assess the effects of the different mutations (e.g., repressible ispA and P-murE-B) on L-form growth, we developed a protocol in which cells of a defined genotype were grown in the rod state, then converted to protoplasts by stripping the cell wall with lysozyme and cultured in our standard L-form medium. The medium contains an osmoprotectant (sucrose) and an inhibitor of cell division (benzamide;) that efficiently kills rods, but not L-forms. For reasons that are not understood, reversion of protoplasts or L-forms to the walled state (regeneration) occurs at a very low frequency, even if they are capable of synthesizing wall material. Benzamide is added to prevent the rare regenerated rods, which grow much more rapidly than L-forms, from overrunning the cultures. Figure 1 A (and Movie S1 A) shows the transition from protoplasts to proliferating L-forms for strain LR2 (ispA P-murE-B). In contrast, wild-type cells or ispA mutant cells showed only a slow increase in size over many hours ( Figure 1 B; Movie S1 B). Remarkably, even though they showed very little growth and no detectable division, the cells remained intact for many days under these conditions (data not shown). In contrast, after a limited amount of growth, the P-murE-B protoplasts frequently initiated L-form-like pulsating shape changes but the cells then lysed. Figure 1 C shows a detailed time lapse (from Movie S1 C) of a small group of cells over a period of 395 min in L-form medium. Hashes point to the remains of cells that had undergone lysis at some point after the preceding frame. Arrowheads point to these cells in previous frames during which they exhibited L-form-like shape changes. An asterisk highlights one cell that successfully produced at least one smaller progeny cell ( Figure 1 C, 395 min). It was evident from these and similar time lapses that these cells are capable of initiating L-form-like shape perturbations and occasionally producing progeny cells, but they do not undergo prolonged proliferative increase because the shape changes are almost always a prelude to cell lysis.