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

Reviewer #1 (Public review):

Summary:

The authors performed experimental evolution of MreB mutants that have a slow growing round phenotype and studied the subsequent evolutionary trajectory using analysis tool 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.

Strengths:

The finding that the round phenotype was maintained during evolution rather than that the original phenotype, rod shape 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.

Reviewer #3 (Public review):

This paper addresses a long-standing 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 is 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.

Author response:

The following is the authors’ response to the previous reviews.

We made a serious effort to address the reviewers comments. If we have come up short, then let this be stated and explained in the eLife review. But we would be grateful if you did not include in the revised eLife review, comments that were corrected / addressed last time – unless of course there is disagreement, or if our response was unsatisfactory. If either of the latter, then please explain and we will respond.

As to the exceptionally minor issue, namely, correction for multiple statistical tests (minor because the data and the error are presented in the text). We have now conducted one-way ANOVA to back the data displayed in Fig 4A., and Supp. Figs 19 and 21. In each case ANOVA revealed a highly significant difference among means: Dunnett’s post hoc test was then used to test each result against SBW25, with the multiple comparisons corrected for in the analysis.

This resulted in changes to the description of the statistical analysis in the following captions:

To Figure 4.

Where we previously referred to paired t-tests we now state: ANOVA revealed a highly significant difference among means [F_7,16 = 8.19, p < 0.001] with Dunnett’s post-hoc test adjusted for multiple comparisons showing that five genotypes (*) differ significantly (p < 0.05) from SBW25.

To Supplementary Figure 19.

Where we previously referred to paired t-tests we now state: ANOVA revealed a highly significant difference among means [F_7,16 = 16.74, p < 0.001] with Dunnett’s post-hoc test adjusted for multiple comparisons showing that three genotypes (*) differ significantly (p < 0.05) from SBW25.

To Supplementary Figure 21.

Where we previously referred to paired t-tests we now state: ANOVA revealed a highly significant difference among means [F_7,89 = 9.97, p < 0.0001] with Dunnett’s post-hoc test adjusted for multiple comparisons showing that SBW25 ∆mreB and SBW25 ∆PFLU4921-4925 are significantly different (*) from SBW25 (p < 0.05).