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  1. Evaluation Summary:

    Santamaria et al. provide interesting insights into the complex regulation used by 31 Pseudomonas aeruginosa clinical strains to minimize the individual costs of cooperative phenotypes based on secondary metabolites. The data analysis is sound and of remarkable depth. Their results challenge the view that there is a tradeoff between primary and secondary metabolism in bacteria and that instead, secondary metabolites may be produced in low-stress conditions when excess carbon is available. However, the relevance of the laboratory growth conditions for these clinical strains requires additional justification.

    (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. The reviewers remained anonymous to the authors.)

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  2. Reviewer #1 (Public Review):

    Solving the puzzle of this paper was clearly not easy, and the authors used an impressive set of tools and statistical methods to get to the bottom of what they observed in a very creative way. However, the presentation of the manuscript and its relevance could perhaps be improved.

    First, I find the arguments in some parts of the manuscript to be a bit awkwardly formulated. For example, there is much discussion about social evolution and the paradox of why cells invest into rhamnolipid production, but this does not seem to be the topic of the paper, which focuses more on understanding P. aeruginosa's metabolism. Instead, there is very little discussion about the origin of these isolates and to what extent these findings may be relevant for P. aeruginosa's natural environment. I understand that this may be very speculative, but there could at least be more discussion on why glycerol was chosen as a growth medium, and what would happen if a more realistic growth medium were used instead. What environment does this bacterium experience and might it be surrounded by other species that could reduce oxidative stress?

    The overall message of the paper could be clarified: essentially, cells only produce rhamnolipids when they are not experiencing oxidative stress. I am sure the message is more nuanced, but this is not clear from the current abstract.

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  3. Reviewer #2 (Public Review):

    Santamaria et al. combined phenotypic analysis, growth curve analysis, metabolomics, flux balance modelling and transcriptomics in order to study the mechanisms behind the diverging levels of rhamnolipid secondary metabolite production in clinical Pseudomonas aeruginosa isolates when grown on glycerol as a sole carbon source. They show that lower levels of rhamnolipid production can be linked to a lower ability to reduce the oxidative stress caused by primary metabolism on glycerol as the sole carbon source. The data are consistent with a model of regulation where microbes can produce secondary metabolites -such as rhamnolipids- only after they meet the 'primary requirements of energy production, biomass synthesis and reduction of oxidative stress'. This adds another layer to the possible regulatory mechanisms microbes can use to minimize the individual cost of cooperation via secondary metabolites.

    Overall the data analysis is sound and of a remarkable depth. The findings are well-substantiated and the goals, methodologies, results and interpretations are clearly described.

    My main question is how relevant growth on glycerol as a sole carbon source is for clinical Pseudomonas aeruginosa strains. The key finding of this work is that rhamnolipid production depends on the ability of the strains to tolerate oxidative stress associated with growth on glycerol as a sole carbon source. Since growth on glycerol puts substantial strain on the primary metabolism it is conceivable that the oxidative stress associated with metabolism in this artificial condition will be higher or acting on different cellular components than in the natural habitats within the body. This raises the question as to whether the observed regulation is an evolutionary adaptation or rather an artifact of growing the isolates in this lab environment. The observation that a substantial part of the clinical strains did not have the ability to deal with the oxidative stress during growth on glycerol suggests that strains do experience lower levels of oxidative stress within the body. Please motivate.

    Several mechanisms of metabolic prudence have been described. It would be good to systematically compare similarities and differences between the proposed mechanism and earlier described mechanisms. This should provide a more nuanced view of the novelty of the proposed mechanism.

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