Increased privatization of a public resource leads to spread of cooperation in a microbial population

ABSTRACT The phenomenon of cooperation is prevalent at all levels of life. In one such manifestation of cooperation in microbial communities, some cells produce costly extracellular resources that are freely available to others. These resources are referred to as public goods. Saccharomyces cerevisiae secretes invertase (public good) in the periplasm to hydrolyze sucrose into glucose and fructose, which are then imported by the cells. After hydrolysis of sucrose, a cooperator retains only 1% of the monosaccharides, while 99% of the monosaccharides diffuse into the environment and can be utilized by any cell. The non-producers of invertase (cheaters) exploit the invertase-producing cells (cooperators) by utilizing the monosaccharides and not paying the metabolic cost of producing the invertase. In this work, we investigate the evolutionary dynamics of this cheater-cooperator system. In a co-culture, if cheaters are selected for their higher fitness, the population will collapse. On the other hand, for cooperators to survive in the population, a strategy to increase fitness would likely be required. To understand the adaptation of cooperators in sucrose, we performed a coevolution experiment in sucrose. Our results show that cooperators increase in fitness as the experiment progresses. This phenomenon was not observed in environments which involved a non-public good system. Genome sequencing reveals duplication of several HXT transporters in the evolved cooperators. Based on these results, we hypothesize that increased privatization of the monosaccharides is the most likely explanation of spread of cooperators in the population. IMPORTANCE How is cooperation, as a trait, maintained in a population? In order to answer this question, we perform a coevolution experiment between two strains of yeast—one which produces a public good to release glucose and fructose in the media, thus generating a public resource, and the other which does not produce public resource and merely benefits from the presence of the cooperator strain. What is the outcome of this coevolution experiment? We demonstrate that after ~200 generations of coevolution, cooperators increase in frequency in the co-culture. Remarkably, in all parallel lines of our experiment, this is obtained via duplication of regions which likely allow greater privatization of glucose and fructose. Thus, increased privatization, which is intuitively thought to be a strategy against cooperation, enables spread of cooperation.

and i suggest the abstract should be modified.
Minor suggestions: Specify the species of yeast used in the text.
There are several places in the text with small missing words.e.g 'the' should be added before 'ability' on line 87.I suggest an extra round of proofreading.Specify the 'non-repressing medium' on line 109.I suggest adding the time 0 timepoint on FIgure 4, otherwise it looks like the two strains do not start at 1:1 in the experiment.Also, i suggest adding which strain is shown to the Y axis label.
The medium used in Figure 5 is not specified.
Line 259 I think references cultures after 1 day, but refers to this as the 'initial ratio,' which i found confusing.
There is a figure 7C referenced in the text, but not presented.
The claim (adaptation to maintain cooperation) started in the first full sentence on line 305 is not supported by the data.
Reviewer #2 (Comments for the Author): In this paper, Raj and Saini have used the yeast cheater-cooperator system to address how cooperators survive cheaters.In doing these evolutions, they identified cooperators which could outcompete cheaters due to some specific single mutations.I have a few comments and would like authors to address them: 1.It would have been better if the authors had attempted to engineer the mutations in parental strains to see if these single mutations had functional consequences.2. The authors should attempt to interpret their results of sequencing evolved strains.While the individual gene functions are not commensurate with the media in which evolution occurred, it would be useful to see if the genes function in either a coexpression network or functional modules.3. Unless it is the journal practice, it is better not to write methods in figure legends.The figure legend should describe the figure; that's it.

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Reviewer comments:
Reviewer #1 (Comments for the Author): The paper is improved but still requires significant revision.The figures were of very poor quality and were not labelled, which made them hard to read.That was likely a problem with uploading them.
The yeast nomenclature for referencing the gene deletions was still incorrect throughout the paper.
In Figure 8, I did not see what the authors described in the text.To me, it did not seem that the range of the data points changed much between 50 and 200 generations.It would also help to have a time 0 datapoint plotted on the graph.
The authors assign gene duplications as the mode of adaptation without testing this hypothesis.I agree that they are the likely cause, but the authors must directly show that via experimentation (e.g.make the duplications in the ancestral background or delete them in the evolved background) to make the conclusion as strongly as it is made in the text.Otherwise, they can only present the duplications as their best hypothesis for the cause of the adaptations.
Reviewer #2 (Comments for the Author):

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Thank you for submitting your paper to Microbiology Spectrum.
Importance: Remarkably, in all parallel lines of our experiment, this is obtained via duplication of regions which likely allow greater privatization of the public resources glucose and fructose.

Introduction:
The increased privatization of the public goods (glucose and fructose) resulting from the duplication event is the most likely explanation for an increase in cooperator fitness.
Results: A major likely determinant of the adaptation of the cooperator in all six co-evolved lines is amplification of regions containing one of HXT11, HXT14, HXT15 or HXT17 genes.
Discussion: Genome sequencing of the evolved cooperators strongly suggests that this increase in fitness was likely accomplished via duplication of genes which lead to increased privatization of the public good.Thank you for the privilege of reviewing your work.Below you will find my comments, instructions from the Spectrum editorial office, and the reviewer comments.
While in principle, the paper is accepted, consider the suggestions made by R#3 to improve clarity in the results and discussion sections.
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Thank you for submitting your paper to Spectrum.This revised version of the manuscript is much improved, but sections remain difficult to read (particularly in the organization of the figures).Some figures will do better if combined, since they have minimal information.
At places, some statements make assumptions without prior data, and have over interpreted results.So, again while reading, it becomes unclear what is known, what is new, and what is an actual conclusion.
The observations themselves are interesting, but somewhat anticipated, especially with the duplication of HXT transporters, enabling (possibly) more sugar transport.However, since the original hypothesis is clear, and the long-term evolution experiments are nice, and the observations solid, I would suggest a more moderate description of the results, and a more succinct discussion.
• Manuscript: A .DOC version of the revised manuscript • Figures: Editable, high-resolution, individual figure files are required at revision, TIFF or EPS files are preferred -23R2 (Increased privatization of a public resource leads to spread of cooperation in a microbial population.)Dear Dr. Supreet Saini: Comments for the Author):No comments Reviewer #3 (Comments for the Author):

Submissions of a paper that does not conform to Microbiology Spectrum guidelines will delay acceptance of your manuscript. "
• Manuscript: A .DOC version of the revised manuscript • Figures: Editable, high-resolution, individual figure files are required at revision, TIFF or EPS files are preferred For complete guidelines on revision requirements, please see the journal Submission and Review Process requirements at https://journals.asm.org/journal/Spectrum/submission-review-process.
Thank you for submitting your paper to Microbiology Spectrum.