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  1. Author Response

    Reviewer #1 (Public Review):

    This study aimed to identify the genetic foundation favoring common, nearly predictable selection of lasR mutants in laboratory and clinical isolates from persons with CF. They selected these mutants using a predictable and quantitative framework of evolution experiments and then identified their genetic underpinnings by a a suppressor screen. The role of cbrAB as a key intermediate is important and ties together several reports of nutrient-dependent advantages of lasR like phenylalanine, including those reported recently (Scribner et al JBact 2021).

    Thank you for this accurate summary of our work. We included this important reference in the revised version.

    The metabolomic study is interesting and offers a plausible correlation between the evolution of lasR mutants during infections of pwCF and the nutritional conditions that select these mutants. Naturally, these are not causative, which should be clarified.

    We agree that we may have stated the correlation between the higher concentrations of metabolites and the rise of specific mutants in the lung too strongly and thus attenuated this statement in the abstract.

    The summative figure describing a model of metabolic and hence genetic diversity of PA is also elegant. The figures and writing are clear and of high quality.

    Thank you!

    Reviewer #2 (Public Review):

    In this paper, the authors thoroughly explore the selective advantage of LasR- mutants of Pseudomonas aeruginosa. As the authors state, selection of loss of function mutations in quorum sensing regulators, including LasR, is frequently observed during chronic infections and laboratory culture, but the drivers of this selection are poorly understood. Mould et al. utilize mathematical modeling, evolution experiments, and whole genome sequencing to show that metabolic advantages are sufficient for selection of LasR- mutants. Further, the authors use a reverse genetic screen paired with evolution experiments to identify the CbrA/CbrB pathway as necessary for this selection. Subsequently, the authors characterize the roles of genes within this pathway with regard to LasR- phenotypes. The authors also determined the nutrients enriched in bronchoalveolar lavage fluid from people with cystic fibrosis and show that LasR- strains have advantages in this nutrient environment. The authors' conclusions are well supported by their data and thoroughly verified using complementary approaches. In addition, the authors provide extensive supplementary data exploring alternative hypotheses related to their findings.

    We appreciate these supportive comments.

    There are several notable strengths of this work. For instance, the authors performed many experiments using both the PA14 laboratory strain and a cystic fibrosis isolate to illustrate the applicability of their findings to distinct genetic backgrounds. In addition, the authors' use of a mathematical model to test the hypothesis that metabolic advantages of LasR- mutants are sufficient to explain their selection and their application of a reverse genetic screen to evolution experiments are particularly clever approaches.

    We appreciate these supportive comments.

    The authors' finding that lasR mutations arise less frequently on a ∆cbrA or ∆cbrB mutant background is very interesting. Also, among the most compelling findings of this study was the parallel evolution of mutations in the downstream crc gene in ∆cbrB mutant cultures. Together, these findings strongly suggest that increased CbrB expression of lasR mutants plays an important role in their selection, as stated in the paper.

    We agree that the crc mutants are a compelling element of these studies and add additional text describing why they aren’t frequently found in natural isolates as discussed in detail below.

    Reviewer #3 (Public Review):

    The work of Mould et al. focuses on a protein LasR, which is a transcription factor involved in quorum sensing in Pseudomonas aeruginosa, which can frequently cause disease in patients with Cystic Fibrosis (CF). Isolates with loss-of-function mutations are frequently found in both environmental and clinical samples, and are associated with more severe outcomes during infection in people with CF. The authors set out to determine why strains with these loss-of-function have a seeming advantage over wild-type (WT) cells, both based on growth and mechanistically. They use mathematical modeling, experimental evolution, sequencing, and metabolome analysis to come to their well-supported conclusions. They determine that LasR- mutants can quickly take over cell populations when competing with WT cells using serial passage. Using reverse genetics, they then identify a pathway which contributes to this advantage. They ultimately determine that LasR- mutants alter metabolism in a way that they can grow on compounds most commonly found in the lungs of patients with CF via the CbrAB pathway.

    The conclusions in this paper well-supported, and the experiments mainly add to these conclusions. These methodologies and conclusions will add to the evolution field by helping to understand more about why certain genetic changes give an advantage to cells, even when there may also be disadvantages associated with those mutations. However, there are a few passages in the writing which confuse the conclusions a bit, and there a few places in the writing where it is unclear that the comparisons between cultures are done using the same methods. Specifically:

    1. It is not clear whether or why ∆anr or ∆rhlR strains are used to compare rates of LasR- mutations.

    This comment was included in essential comments and is addressed above.

    1. The logic describing why the authors expect higher activity of the CbrA-CbrB-crcZ pathway in LasR- strains, and therefore more loss of function alleles in Crc or Hfq, and then confirm this theory with the data showing that they have mutations in crc or hfq in ∆cbrA and ∆cbrB mutants (but not in WT strains), where there should not be LasR- mutations, is not clear.

    We apologize for this confusion. We have modified the text to clarify this point.

    lasR mutants still responded to succinate; succinate reduced crcZ levels in ∆lasR and enabled ∆lasR growth on medium with FAA due to Crc activity (Fig. 2E, inset). This indicated that ∆lasR retains the Crc-Hfq mediated translational repression when succinate is present.”

    1. It is not clear that all growth curves, which are compared to the mathematical model throughout the paper, are performed the same way (i.e. passaged every 48 hours).

    We have ensured that the appropriate details are in the Figure Legends and Methods sections. Importantly, the growth conditions for the determination of growth parameters (Fig. 1A and Figure 1 – figure supplement 1) and the evolution experiments were performed using the same methods.

    Some differences in methods elsewhere in the paper (e.g. single carbon source growth assays) were due to supply chain disruptions that affected the availability of cuvettes or microtiter plates; other choices were made to accommodate the differences in optical densities for different media.

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  2. Evaluation Summary:

    This study aimed to identify the genetic foundation favoring selection of lasR mutants in laboratory and clinical isolates from persons with CF. They selected these mutants using a predictable and quantitative framework of evolution experiments and then identified their genetic underpinnings by a a suppressor screen. The role of cbrAB as a key intermediate is important and ties together several reports of nutrient-dependent advantages of lasR like phenylalanine.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)

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  3. Reviewer #1 (Public Review):

    This study aimed to identify the genetic foundation favoring common, nearly predictable selection of lasR mutants in laboratory and clinical isolates from persons with CF. They selected these mutants using a predictable and quantitative framework of evolution experiments and then identified their genetic underpinnings by a a suppressor screen. The role of cbrAB as a key intermediate is important and ties together several reports of nutrient-dependent advantages of lasR like phenylalanine, including those reported recently (Scribner et al JBact 2021).

    The metabolomic study is interesting and offers a plausible correlation between the evolution of lasR mutants during infections of pwCF and the nutritional conditions that select these mutants. Naturally, these are not causative, which should be clarified. The summative figure describing a model of metabolic and hence genetic diversity of PA is also elegant.

    The figures and writing are clear and of high quality.

    Read the original source
    Was this evaluation helpful?
  4. Reviewer #2 (Public Review):

    In this paper, the authors thoroughly explore the selective advantage of LasR- mutants of Pseudomonas aeruginosa. As the authors state, selection of loss of function mutations in quorum sensing regulators, including LasR, is frequently observed during chronic infections and laboratory culture, but the drivers of this selection are poorly understood. Mould et al. utilize mathematical modeling, evolution experiments, and whole genome sequencing to show that metabolic advantages are sufficient for selection of LasR- mutants. Further, the authors use a reverse genetic screen paired with evolution experiments to identify the CbrA/CbrB pathway as necessary for this selection. Subsequently, the authors characterize the roles of genes within this pathway with regard to LasR- phenotypes. The authors also determined the nutrients enriched in bronchoalveolar lavage fluid from people with cystic fibrosis and show that LasR- strains have advantages in this nutrient environment. The authors' conclusions are well supported by their data and thoroughly verified using complementary approaches. In addition, the authors provide extensive supplementary data exploring alternative hypotheses related to their findings.

    There are several notable strengths of this work. For instance, the authors performed many experiments using both the PA14 laboratory strain and a cystic fibrosis isolate to illustrate the applicability of their findings to distinct genetic backgrounds. In addition, the authors' use of a mathematical model to test the hypothesis that metabolic advantages of LasR- mutants are sufficient to explain their selection and their application of a reverse genetic screen to evolution experiments are particularly clever approaches.

    The authors' finding that lasR mutations arise less frequently on a ∆cbrA or ∆cbrB mutant background is very interesting. Also, among the most compelling findings of this study was the parallel evolution of mutations in the downstream crc gene in ∆cbrB mutant cultures. Together, these findings strongly suggest that increased CbrB expression of lasR mutants plays an important role in their selection, as stated in the paper.

    Read the original source
    Was this evaluation helpful?
  5. Reviewer #3 (Public Review):

    The work of Mould et al. focuses on a protein LasR, which is a transcription factor involved in quorum sensing in Pseudomonas aeruginosa, which can frequently cause disease in patients with Cystic Fibrosis (CF). Isolates with loss-of-function mutations are frequently found in both environmental and clinical samples, and are associated with more severe outcomes during infection in people with CF. The authors set out to determine why strains with these loss-of-function have a seeming advantage over wild-type (WT) cells, both based on growth and mechanistically. They use mathematical modeling, experimental evolution, sequencing, and metabolome analysis to come to their well-supported conclusions. They determine that LasR- mutants can quickly take over cell populations when competing with WT cells using serial passage. Using reverse genetics, they then identify a pathway which contributes to this advantage. They ultimately determine that LasR- mutants alter metabolism in a way that they can grow on compounds most commonly found in the lungs of patients with CF via the CbrAB pathway.

    The conclusions in this paper well-supported, and the experiments mainly add to these conclusions. These methodologies and conclusions will add to the evolution field by helping to understand more about why certain genetic changes give an advantage to cells, even when there may also be disadvantages associated with those mutations. However, there are a few passages in the writing which confuse the conclusions a bit, and there a few places in the writing where it is unclear that the comparisons between cultures are done using the same methods. Specifically:

    1. It is not clear whether or why ∆anr or ∆rhlR strains are used to compare rates of LasR- mutations.
    2. The logic describing why the authors expect higher activity of the CbrA-CbrB-crcZ pathway in LasR- strains, and therefore more loss of function alleles in Crc or Hfq, and then confirm this theory with the data showing that they have mutations in crc or hfq in ∆cbrA and ∆cbrB mutants (but not in WT strains), where there should not be LasR- mutations, is not clear.
    3. It is not clear that all growth curves, which are compared to the mathematical model throughout the paper, are performed the same way (i.e. passaged every 48 hours).

    Read the original source
    Was this evaluation helpful?