Evolution and regulation of microbial secondary metabolism

  1. Guillem Santamaria
  2. Chen Liao
  3. Zhe Wang
  4. Kyu Rhee
  5. Francisco Pinto
  6. Jinyuan Yan
  7. Joao B. Xavier
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Abstract

Microbes have disproportionate impacts on the macroscopic world. This is in part due to their ability to grow to large groups and cooperatively secrete massive amounts of secondary metabolites that impact their environment. Yet, the conditions enabling secondary metabolism without compromising primary needs remain unclear. Here we investigated the biosynthesis of rhamnolipids, a secondary metabolite that Pseudomonas aeruginosa makes to decrease the surface tension of surrounding liquid. Using a combination of genomics, metabolomics, transcriptomics, and mathematical modeling we show that biosynthesis of rhamnolipids from glycerol varies inconsistently across the phylogenetic tree; instead, non-producer lineages are also those worse at reducing the oxidative stress of primary glycerol metabolism. The link to oxidative stress explains the inconsistent distribution across the P. aeruginosa tree, adding a new layer to the regulation of rhamnolipids—a microbial secondary metabolite important for fitness in natural and clinical settings.

Significance

The bacterium Pseudomonas aeruginosa is a major source of hospital-acquired infections. This pathogen’s knack for virulence relies on its ability to multiply and secrete massive amounts of substances that overwhelm microbial competitors and weaken host defenses. It remains unclear how the bacteria conciliate their need to grow and multiply—a need at the individual-level— with their ability to secrete products—a need of the population. Here we combined genomics, metabolomics and mathematical modeling to study the biosynthesis of rhamnolipids, a surfactant that P. aeruginosa makes to reduce the surface tension of surrounding liquids. Our study reveals a new link between oxidative stress and rhamnolipid synthesis, which helps explain how this important bacterial product has evolved and how it persists in many lineages of pathogens.

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  1. eLife

    Author Response

    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.

    We are pleased to see that the referee was favorably impressed. We hope that this revision has improved the presentation of the manuscript and has clarified its relevance.

    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?

    We understand that the referee would like a broader analysis of how the growth environment impacts surfactant secretion. We have added an entirely new section titled “Mathematical model predicts impact of carbon sources on surfactant production” at the end of the results section to address this issue (p. 16-19, l. 358-435). In the new section we present new data on how a range of carbon sources, beyond glycerol, impact P. aeruginosa growth and biosurfactant secretion. Then we use our model to determine carbon sources that favor secretion and we identify D-glucose as being better than glycerol. These new experimental and computational work refine our model to explain surfactant secretion more broadly than in glycerol. The biosynthesis of this secondary metabolite is favored when the carbon and energy source imposes a low burden on the primary metabolism. We also investigated the rhlAB expression dynamics in PA14 in glucose to further support our results.

    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.

    We have changed the abstract to clarify our main point: that cells only produce surfactants when they are not experiencing oxidative stress and they more carbon source than needed for growth.

  2. eLife

    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.)

  3. eLife

    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.

  4. eLife

    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.

  5. Version 2 published on bioRxiv
  6. Version 1 published on bioRxiv