Evolutionary rescue of spherical mreB deletion mutants of the rod-shape bacterium Pseudomonas fluorescens SBW25

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    This valuable study combines evolution experiments with molecular and genetic techniques to study how a genetic lesion in MreB that causes rod-shape cells to become spherical, with concomitant deleterious fitness effects, can be rescued by natural selection. The results are convincing, although the statistical analyses and figure presentation could be improved, and the concrete contribution of the paper and how it relates to previous literature clarified.

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Maintenance of rod-shape in bacterial cells depends on the actin-like protein MreB. Deletion of mreB from Pseudomonas fluorescens SBW25 results in viable spherical cells of variable volume and reduced fitness. Using a combination of time-resolved microscopy and biochemical assay of peptidoglycan synthesis we show that reduced fitness is a consequence of perturbed cell size homeostasis that arises primarily from differential growth of daughter cells. A 1,000-generation selection experiment resulted in rapid restoration of fitness with derived cells retaining spherical shape. Mutations in the peptidoglycan synthesis protein Pbp1A were identified as the main route for fitness restoration with genetic reconstructions demonstrating causality. The pbp1A mutations targeting transpeptidase activity enhance homogeneity in cell wall synthesis on lateral surfaces, thus restoring cell size homeostasis in the population. Together our experimental approach emphasizes the new knowledge to be gained from strategies that exploit the power of natural selection to rescue fitness-compromised mutants.

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  1. eLife assessment

    This valuable study combines evolution experiments with molecular and genetic techniques to study how a genetic lesion in MreB that causes rod-shape cells to become spherical, with concomitant deleterious fitness effects, can be rescued by natural selection. The results are convincing, although the statistical analyses and figure presentation could be improved, and the concrete contribution of the paper and how it relates to previous literature clarified.

  2. Reviewer #1 (Public Review):


    The authors performed experimental evolution of MreB mutants that have a slow-growing round phenotype and studied the subsequent evolutionary trajectory using analysis tools from molecular biology. It was remarkable and interesting that they found that the original phenotype was not restored (most common in these studies) but that the round phenotype was maintained.


    The finding that the round phenotype was maintained during evolution rather than that the original phenotype, rod-shaped cells, was recovered is interesting. The paper extensively investigates what happens during adaptation with various different techniques. Also, the extensive discussion of the findings at the end of the paper is well thought through and insightful.

    I find there are three general weaknesses:

    (1) Although the paper states in the abstract that it emphasizes "new knowledge to be gained" it remains unclear what this concretely is. On page 4 they state 3 three research questions, these could be more extensively discussed in the abstract. Also, these questions read more like genetics questions while the paper is a lot about cell biological findings.

    (2) it is not clear to me from the text what we already know about the restoration of MreB loss from suppressors studies (in the literature). Are there suppressor screens in the literature and which part of the findings is consistent with suppressor screens and which parts are new knowledge?

    (3) The clarity of the figures, captions, and data quantification need to be improved.

  3. Reviewer #2 (Public Review):

    Yulo et al. show that deletion of MreB causes reduced fitness in P. fluorescens SBW25 and that this reduction in fitness may be primarily caused by alterations in cell volume. To understand the effect of cell volume on proliferation, they performed an evolution experiment through which they predominantly obtained mutations in pbp1A that decreased cell volume and increased viability. Furthermore, they provide evidence to propose that the pbp1A mutants may have decreased PG cross-linking which might have helped in restoring the fitness by rectifying the disorganised PG synthesis caused by the absence of MreB. Overall this is an interesting study.


    Do the small cells of mreB null background indeed have have no DNA? It is not apparent from the DAPI images presented in Supplementary Figure 17. A more detailed analysis will help to support this claim.

    What happens to viability and cell morphology when pbp1A is removed in the mreB null background? If it is actually a decrease in pbp1A activity that leads to the rescue, then pbp1A- mreB- cells should have better viability, reduced cell volume and organised PG synthesis. Especially as the PG cross-linking is almost at the same level as the T362 or D484 mutant.

    What is the status of PG cross-linking in ΔmreB Δpflu4921-4925 (Line 7)?

    What is the morphology of the cells in Line 2 and Line 5? It may be interesting to see if PG cross-linking and cell wall synthesis is also altered in the cells from these lines.

    The data presented in 4B should be quantified with appropriate input controls.

    What are the statistical analyses used in 4A and what is the significance value?

    A more rigorous statistical analysis indicating the number of replicates should be done throughout.

  4. Reviewer #3 (Public Review):

    This paper addresses an understudied problem in microbiology: the evolution of bacterial cell shape. Bacterial cells can take a range of forms, among the most common being rods and spheres. The consensus view is that rods are the ancestral form and spheres the derived form. The molecular machinery governing these different shapes is fairly well understood but the evolutionary drivers responsible for the transition between rods and spheres are not. Enter Yulo et al.'s work. The authors start by noting that deletion of a highly conserved gene called MreB in the Gram-negative bacterium Pseudomonas fluorescens reduces fitness but does not kill the cell (as happens in other species like E. coli and B. subtilis) and causes cells to become spherical rather than their normal rod shape. They then ask whether evolution for 1000 generations restores the rod shape of these cells when propagated in a rich, benign medium.

    The answer is no. The evolved lineages recovered fitness by the end of the experiment, growing just as well as the unevolved rod-shaped ancestor, but remained spherical. The authors provide an impressively detailed investigation of the genetic and molecular changes that evolved. Their leading results are:

    (1) The loss of fitness associated with MreB deletion causes high variation in cell volume among sibling cells after cell division.

    (2) Fitness recovery is largely driven by a single, loss-of-function point mutation that evolves within the first ~250 generations that reduces the variability in cell volume among siblings.

    (3) The main route to restoring fitness and reducing variability involves loss of function mutations causing a reduction of TPase and peptidoglycan cross-linking, leading to a disorganized cell wall architecture characteristic of spherical cells.

    The inferences made in this paper are on the whole well supported by the data. The authors provide a uniquely comprehensive account of how a key genetic change leads to gains in fitness and the spectrum of phenotypes that are impacted and provide insight into the molecular mechanisms underlying models of cell shape.

    Suggested improvements and clarifications include:

    (1) A schematic of the molecular interactions governing cell wall formation could be useful in the introduction to help orient readers less familiar with the current state of knowledge and key molecular players.

    (2) More detail on the bioinformatics approaches to assembling genomes and identifying the key compensatory mutations are needed, particularly in the methods section. This whole subject remains something of an art, with many different tools used. Specifying these tools, and the parameter settings used, will improve transparency and reproducibility, should it be needed.

    (3) Corrections for multiple comparisons should be used and reported whenever more than one construct or strain is compared to the common ancestor, as in Supplementary Figure 19A (relative PG density of different constructs versus the SBW25 ancestor).

    (4) The authors refrain from making strong claims about the nature of selection on cell shape, perhaps because their main interest is the molecular mechanisms responsible. However, I think more can be said on the evolutionary side, along two lines. First, they have good evidence that cell volume is a trait under strong stabilizing selection, with cells of intermediate volume having the highest fitness. This is notable because there are rather few examples of stabilizing selection where the underlying mechanisms responsible are so well characterized. Second, this paper succeeds in providing an explanation for how spherical cells can readily evolve from a rod-shaped ancestor but leaves open how rods evolved in the first place. Can the authors speculate as to how the complex, coordinated system leading to rods first evolved? Or why not all cells have lost rod shape and become spherical, if it is so easy to achieve? These are important evolutionary questions that remain unaddressed. The manuscript could be improved by at least flagging these as unanswered questions deserving of further attention.

    The value of this paper stems both from the insight it provides on the underlying molecular model for cell shape and from what it reveals about some key features of the evolutionary process. The paper, as it currently stands, provides more on which to chew for the molecular side than the evolutionary side. It provides valuable insights into the molecular architecture of how cells grow and what governs their shape. The evolutionary phenomena emphasized by the authors - the importance of loss-of-function mutations in driving rapid compensatory fitness gains and that multiple genetic and molecular routes to high fitness are often available, even in the relatively short time frame of a few hundred generations - are well-understood phenomena and so arguably of less broad interest. The more compelling evolutionary questions concern the nature and cause of stabilizing selection (in this case cell volume) and the evolution of complexity. The paper misses an opportunity to highlight the former and, while claiming to shed light on the latter, provides rather little useful insight.

  5. Author response:

    Thank you for handling our paper and our thanks to the reviewers for their engagement, comments and valuable suggestions. We will take the opportunity to provide a full response and submit a revised version in the coming weeks.

  6. dear Atanas, that you for taking the time to comment and I'm very glad to learn of your enthusiasm for the work. As far as your question is concerned, there is nothing in the DNA sequence of pbp1A that would lead one to conclude that it is more mutable than any other housekeeping gene. Moreover, identification of pbp1A mutations at generation 50 (see Supp Table 2), shows 13 different mutations across all replicate lines, and while one insertion occurred twice, there is nothing in these data to suggest that there is any kind of "contingency" behaviour (elevated mutability) associated with the gene. My guess is that the prevalence of mutations in pbp1A has a pretty conventional explanation: a combination of low fitness of ∆mreB SBW25 (it's pretty sick), large population sizes, short generation times, and a genetic target that delivers a fitness benefit through loss-of-function mutations. As to mutation rate in SBW25 more generally, Mike Lynch and team looked a mutation rate in P, fluorescens and specifically in SBW25 reporting (doi.org/10.1093/gbe/evu284) one of the lowest ever mutation rates (4.25 × 10−9 mutations per generation). This said we are well aware that mutation rate is uneven across the genome with certain genes (and even specific codons) mutating at rates as high as 10-5.

  7. Thank for all of your work on this paper! We have learned so much about the bacterial cell wall yet there is so much that remains unknown. I particularly enjoyed your use of Pseudomonas fluorescens in your studies, which is still well-studied but definitely not as much as E. coli or B. subtilis. Even for “closely” related species, there is so much easily-accessible novelty that we could discover. I’m glad scientists are taking advantage of this treasure trove. I was curious about something you mentioned in your discussion: “The prevalence of pbp1A mutations in the work reported here likely reflects the fact that loss-of-function mutations in pbp1A are more readily achieved compared to gain-of-function mutations in ftsZ.” Generally, there are more mutations for any gene that are likely to lead to a loss-of-function phenotype relative to the number of mutations that could lead to a gain-of-function phenotype. Do you know if there is anything intrinsically different about the gene locus of pbp1a? Is there anything that suggests it may be a hotspot for mutations? Is there any knowledge on the wild-type strain regarding the rate of mutation across the genome under normal growth conditions? Thank you so much for your hard work and your time!