Stress-induced formation of cell wall-deficient cells in filamentous actinomycetes

The cell wall is a shape-defining structure that envelopes almost all bacteria and protects them from environmental stresses. Bacteria can be forced to grow without a cell wall under certain conditions that interfere with cell wall synthesis, but the relevance of these wall-less cells (known as L-forms) is unclear. Here, we show that several species of filamentous actinomycetes have a natural ability to generate wall-deficient cells in response to hyperosmotic stress, which we call S-cells. This wall-deficient state is transient, as S-cells are able to switch to the normal mycelial mode of growth. However, prolonged exposure of S-cells to hyperosmotic stress yields variants that are able to proliferate indefinitely without their cell wall, similarly to L-forms. We propose that formation of wall-deficient cells in actinomycetes may serve as an adaptation to osmotic stress.

The work of Ramijan and colleagues describes the intriguing observation that some sporulating actinobacteria grown under high osmotic conditions extrude membrane vesicles of varying sizes and composition (DNA, extent of cell wall, etc.). The authors have termed these vesicles 'S-form cells', and distinguish them from the similar-appearing L-forms in that they originate as a result of exposure to osmotic stress, as opposed to in response to cell wall-targeting enzymes/antibiotics. These S-form cells appear to be able to replicate in a manner reminiscent to that reported previously for L-forms, contain DNA but appears to lack a conventional segregation mechanism as many lose a resident megaplasmid, and may be able to switch back to conventional mycelial growth.
1-While the S-form cells shown here are striking, it isn't clear how these differ from L-forms, which have previously been reported for the K. viridifaciens strain investigated here (previously known as S. viridifaciens -Innes et al 2001). Based on a recent review by Errington and colleagues (2016), L-form cells are loosely defined as: "variants of normally walled bacteria that have adapted to grow in the complete absence of cell wall synthesis". While the S-form cells seem to be generally larger than L-form ones, there is no evidence presented that suggests other obvious differences. Also the consistently large S-form cells seem to be specific to K. viridifaciens, based on the data presented in Extended Data Table 4. 2-Additional membrane synthesis would make sense in order to facilitate the release of these membrane vesicle-like cells. However, the information provided in Extended Data Tables 1 and 2 is challenging to understand. What is meant by 'branching frequency'? What does 'membrane fraction' mean? -would it not be expected that all hyphae should be encased by membranes? I think these are explained in the methods, but it would also be useful to have this info in the Table itself. How is it decided where a hypha starts and ends with respect to defining membrane fraction (and presumably 'length'?)? Collecting data for only 10 hypha seems low. of contaminating spores or small mycelia that may have passed through the filtration steps used to collect S-cells (lines 406-421). It sounds like the lysozyme treatment (what is in the 'lysozyme solution? -please provide a recipe or reference) used to remove residual hyphal fragments may actually help in promoting L-forms, and it is not clear how such a treatment would remove hyphal fragments, nor how it would impact any contaminating spores apart from possibly helping to promote germination. Are there any fluorescent markers -e.g. specific for spores -that could be included as an additional internal check to ensure no spore contamination? I have watched the S4 Video repeatedly, and at least some of the mycelial outgrowth seems to be coming from small spore-like cells (in other cases I can't tell where they originate).
4-The frequent loss of the megaplasmid KVP1 after re-growth of S-cells on MYM medium is interesting. Is a similar thing seen if osmotically stressed cells (not filtered for S-cells) are plated? It would be interesting to know if this was a general response to osmotic stress, or if this was something specific to S-cells. This could be tested using strains that aren't known to form S-cells (e.g. S. coelicolor).
5-The M1/M2 mutants are particularly fascinating, and additional follow up work on these strains would help to further strengthen the manuscript. It would be important to complement the mutations associated with these two strains, as that would provide important insight into the basis for S-form proliferation. If it isn't possible to complement the mutant phenotype, then it may suggest a role for gene regulation or epigenetic factors.
When considering the M1 and M2 strains, were these strains viable when plated without high osmolarity? If yes, what did the colonies look like? Were they able to return to mycelial-type growth, or did the cells all die? 6-The idea put forward in the discussion, suggesting that S-cells may be able to take up DNA from the environment is intriguing but at this point is entirely speculative. In the absence of additional experimental support, it would be worth toning down this section. It seems like an idea that would be very straightforward to test in principle: add DNA with an antibiotic resistance gene on it and see if it gets taken up/incorporated into the chromosome by testing for resistance. 7-Do the authors have any thoughts as to why some strains appeared to generate S-cells whereas others did not? This would seem like an appropriate discussion point. Figure S1D-at 96h, there seems to be little overlap between the membrane and DNA stains, and no obvious S-cells. Can the authors comment? Although cell wall is normally essential structure for the bacterial growth and viability, it has been known that under certain conditions many bacteria are able to switch into a cell wall-deficient (CWD) state, which is completely resistant to many antibiotics working on cell wall synthesis. This is an interesting study on still poorly understood but highly interesting cellular pathways involved in the generation of CWD bacteria. The switch to CWD state from parental walled cells is generally induced by directly inhibiting cell wall synthesis with antibiotics, lysozyme and/or genetic modifications under osmoprotective conditions. In this work the authors convincingly show that, in a wide range of filamentous actinobacteria, the CWD state (S-cells) can be induced in the presence of high levels of sucrose or NaCl, even without artificial direct inhibition of cell wall synthesis. The authors also show that the S-cells can further develop to S-forms, which can proliferate without walls, by prolonged culture in the presence of sucrose or NaCl. It is not clear why sucrose or NaCl should promote the switch to S-forms, but this mechanism is probably beyond the scope of this paper. The manuscript is very well written and the experiments are sound. This study would attract a broad audience interested in basic bacterial physiology responding to environmental effects.

Specific comments
The key initial observation in this work is the inhibition of tip growth and the generation of excess membrane in the presence of higher levels of osmolytes (sucrose or NaCl) in walled cells. The membrane-bound S-cells are released from hyphal tips on the original cell wall probably due to ongoing autolytic activity, suggesting uncoupling of synthesis of the cell envelope layers is induced by the osmolytes. When the S-cells are cultured for prolonged periods in the presence of osmolytes, they are able to switch to S-forms. The authors conclude that the S-form switch is induced by hyperosmotic stress. However, on the other hand, the osmolytes also work to provide an isotonic environment, sufficiently osmoprotective to support viability/growth of these CWD bacteria. In this context, it seems to be not really clear about significance of hyperosmotic stress on generation of CWD bacteria, especially for S-forms. Excess sucrose or NaCl could also bring toxic side effects, other than hyperosmotic stress. 1) Sucrose or its derivatives may be taken and utilised in actinomycetes? Utilization of excess amount of sugar might affect growth and viability. 2) NaCl causes not only osmotic stress but also ionic toxicity. The possible effects of sucrose or NaCl on the generation or selection of CWD bacteria should be more carefully interpreted.