Evolutionary rescue of spherical mreB deletion mutants of the rod-shape bacterium Pseudomonas fluorescens SBW25
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Abstract
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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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 …
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.
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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 …
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!
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