Evolution of the Quorum Sensing Regulon in Cooperating Populations of Pseudomonas aeruginosa

  1. Nicole E. Smalley
  2. Amy L. Schaefer
  3. Kyle L. Asfahl
  4. Crystal Perez
  5. E. Peter Greenberg
  6. Ajai A. Dandekar

  • Curated by eLife

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    Evaluation Summary:

    Pseudomonas aeruginosa regulates the production of many cooperative traits through quorum sensing cell-cell signaling. The authors carried out transcriptomic studies of experimental evolved populations of P. aeruginosa and observed that the size of the quorum sensing regulon decreases when only a few but not all the cooperative processes regulated quorum sensing are required for growth. Their findings are consistent with the hypothesis that quorum sensing regulated genes can be counter selected rapidly when not beneficial. This study is of interest for microbiologists studying quorum-sensing, and evolutionary biologists studying the evolution of cooperative behavior.

    (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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Abstract

Pseudomonas aeruginosa uses quorum sensing (QS) to activate expression of dozens of genes (the QS regulon). Because there is strain-to-strain variation in the size and content of the QS regulon, we asked how the regulon might evolve during long-term P. aeruginosa growth when cells require some but not all the functions activated by QS.

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  1. eLife

    Author Response:

    After reading the comments of all four of the reviewers and re-reading the submitted manuscript, it became apparent to us that the submission was inadequately prepared. As evidenced by the reviewers, we did not clearly explain our general rationale or our rationale for certain experimental approaches. In particular, we were unclear in our reasoning behind the media condition that we selected and why we chose focus on the two experimental lineages in which lasR mutants did not emerge. The manuscript has been rewritten in a way that should provide needed clarity. We have also added data requested by the reviewers. The revised manuscript was submitted to and has now been published in mBio (DOI: 10.1128/mbio.00161-22)

  2. eLife

    Evaluation Summary:

    Pseudomonas aeruginosa regulates the production of many cooperative traits through quorum sensing cell-cell signaling. The authors carried out transcriptomic studies of experimental evolved populations of P. aeruginosa and observed that the size of the quorum sensing regulon decreases when only a few but not all the cooperative processes regulated quorum sensing are required for growth. Their findings are consistent with the hypothesis that quorum sensing regulated genes can be counter selected rapidly when not beneficial. This study is of interest for microbiologists studying quorum-sensing, and evolutionary biologists studying the evolution of cooperative behavior.

    (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):

    Pseudomonas aeruginosa is currently widely used as good model bacterium to study the evolution and maintenance of cooperative behaviors in bacteria. This bacterium uses quorum sensing to regulate hundreds of different genes involved in group behaviors and its attractive because it is easy to manipulate, and because it is relevant in many environmental and clinical settings.

    In the manuscript "Evolution of the quorum sensing regulon in cooperating populations of Pseudomonas aeruginosa" the authors used experimental evolution to try to understand why some strains of Pseudomonas aeruginosa control hundreds of genes through quorum sensing while others have a much smaller number of genes regulated by quorum sensing. Here they evolved different P. aeruginosa populations in an environment where most of the of the cooperative processes regulated quorum sensing are not needed for growth. Evolution under this condition resulted an increase in fitness of the evolved populations and in the loss in the number of genes activated by quorum sensing. These results are consistent with the fact that many of the quorum sensing regulated traits are energetically costly.

    They used a transcriptomics population approach, where they measured genes expression of the evolved population in the quorum sensing active and inactive conditions, and they used these results to quantify the number of genes activated in the evolved populations versus the ancestral. This is a novel approach that it is useful to study the effects of evolution at the population level.

    The authors sequenced some of evolved clones and were able to obtain insights on the mechanist explanation for why one of the genes in the evolved population was no longer subject to quorum sensing regulation. By measuring promoter activity of a quorum sensing activated gene they could show that expression of this promoter was often decrease in some evolved clones while in others the promoter had delayed quorum sensing activation. These differences were shown to be related to different SNPs in the evolved clones in the regulator of this promoter. These results led to the conclusion that elimination of a gene from the quorum sensing can occur through different trajectories. I think it is interesting that reduction in the quorum sensing regulon can occur by small changes (SNPs) in the regulators belonging to the quorum sensing network because it might allow easy reversion of these losses. However, further characterization of other mutations from the evolved clones would be useful to understand how often these strategies occurred in opposition to others to understand the mechanistic explanation behind the loss of the genes from the quorum sensing regulon.

    The authors wanted to obtain insight regarding why some strains have a much larger number of quorum sensing regulated genes than others, and proposed that the number of genes regulated by quorum sensing might provide clues about the evolutionary history of different strains from different environments. I think this is an interesting hypothesis, but further work is needed to gain support for such hypothesis. Here the authors observed that populations evolved under conditions where very few quorum sensing regulated traits are needed could increase their fitness by reducing the quorum sensing regulon. This result is interesting, but it is expected that unnecessary traits are counter selected when not needed. Therefore, these results open the door for future experiments to determine if the system is flexible enough to revert these losses, in other words it would be interesting to perform similar experiments with the evolved clones under conditions where more quorum sensing regulated traits are beneficial to see if reversion can be achieved. If reversion indeed occurs, it will be interesting to determine if such reversion would occur through recovery of quorum sensing regulation or other mechanisms.

    Nonetheless, the current study provides novel and relevant insight on the characterization of quorum sensing genes activated by quorum sensing in Pseudomonas at the population level, and on the mechanisms involved during selection for reduction of the quorum sensing regulon.

  4. eLife

    Reviewer #2 (Public Review):

    QS is a mechanism by which cells communicate with each other to coordinately regulate various behaviors. The question the authors attempt to address here is if these QS-regulated behaviors are no longer useful, will bacteria evolve to reduce the number of genes regulated by QS. Using the well-established P. aeruginosa model system, the authors show that after 1000 generations of evolution in a medium requiring QS for growth, the bacteria do reduce the number of genes induced by QS. They conclude this reduction increases the fitness of the evolved bacteria, although there is little data provided to support this claim. In addition, analysis of gene expression was carried out in a medium that is not reflective of the medium in which the evolution took place. This study is interesting as it combines long-term experimental evolution of a bacterial species in a medium that is dependent on maintaining QS with transcriptomic analysis of QS gene expression.

  5. eLife

    Reviewer #3 (Public Review):

    Pseudomonas aeruginosa is an important opportunistic pathogen causing life-threatening infections. Many of the virulence factor of this pathogen are regulated via chemical communication systems, referred to as quorum-sensing (QS) systems. There is great interest to understand QS evolution, as evolutionary changes affect the virulence potential of this pathogen. There are many studies that examined QS evolution in vitro and in hosts, and there is a consensus in the field that QS is under selection and that many QS-regulated traits are lost in long-term experiments and chronic infections.

    While previous studies typically focussed on specific QS-phenotypes, the current paper applies a transcriptomics approach, which allows to identify global changes in QS-regulons. This is an interesting and novel approach. However, the study also comes with two big limitations. First, sample size equals N=2, which is so small that no general conclusions can be made. Even more problematic is that there were initially 5 replicates, and two of those replicates clearly took a different evolutionary path. They were deliberately excluded, which further compromises any claims on generality. Second, there was only one treatment (growth in CAB medium). Thus, we gain no information on how QS-regulon size and composition evolves across environments. Is QS-shrinking a general phenomenon or is it a CAB-specific pattern? We simply cannot tell.

  6. eLife

    Reviewer #4 (Public Review):

    Smalley et al. investigated the evolutionary dynamics of the quorum-sensing regulon in 5 replicate populations of Pseudomonas aeruginosa (PA), in an environment maintaining selection for both cooperative and private QS-controlled traits. Building on previous studies illustrating differences in regulon size and content across PA isolates, the authors hypothesized that evolution over 1000 generations in a single defined environment would result in reduction in the size of the QS-controlled regulon. In agreement with this hypothesis, transcriptomic profiling of 2 of the 5 replicate lines indicated substantial (45% plus) reductions in QS regulon size (transcriptome data on the other 3 replicates were not shown). Molecular characterization of a gene lost from the QS regulon (pqsA) indicates this loss in some replicates was due to a mutation in it's QS-controlled transcriptional activator pqsR.

    Strengths:

    The primary strength of this paper is that it provides a direct attack on a series of basic questions of QS evolution - Can constant environments lead to the loss of genes from the QS regulon? On what timescale are they lost? What are the molecular mechanisms of loss? More broadly, the study sheds light on basic questions of the evolution of behavioral responses in bacteria.

    More specifically, the study provides a clear answer on the question of timescale (substantial change within 1000 generations), which has implications for inferring recent environmental history from regulon profiling. On the question of molecular mechanisms, the results are more anecdotal but do extend our understanding of how QS systems can be rewired during evolution.

    Methodologically, the study showcases and benchmarks an experimental design to query QS-regulons without constructing genetic QS knockouts, using instead a QS-quenching acylase treatment to turn QS regulons off, and QS signal supplementation to ensure QS regulons are on. Benchmarking this study on a prior QS regulon experiment is an important step to improve confidence in the results.

    Weaknesses:

    Limiting detailed mechanistic characterization to smaller number of evolutionary replicates is a standard step, yet it is important to make sure there is a clear rationale for the choice made, with careful consideration of how these choices could cause biased results. In the present study, 2 replicates from 5 were selected for detailed characterization, and I flag at this point that the rationale is not entirely evident. 2 replicates were discarded from consideration as they evolved to overcome the regulatory trap posed by the combination of a protein + adenosine environment with QS-control of protein (cooperative) + adenosine (private) metabolism. This was likely due to a regulatory rewiring to uncouple adenosine from QS-control, allowing the strain to then 'cheat' on protein degradation via an additional QS regulon loss. This sounds very much like a multi-step reduction in QS regulon, and it is not clear why this interesting path was discarded.

    The hypothesis as stated is already well supported by the common experimental evolution and clinical observation of rapid lasR mutant expansion. In mitigation of this point, the study takes steps to reduce selection for lasR mutants (via adenosine supplementation), and also seeks to directly characterize regulon size and content, which is a valuable and rarely-taken step.

    Likely impact:

    The study holds the potential for broad impact on research into regulatory control of behavior in bacteria, providing a key benchmark study profiling how QS regulons can rapidly shrink even when QS-controlled genes are under continued positive selection. Future studies will likely refine and build out the generality of this result, but examining evolutionary dynamics under different environments (e.g. requiring activity of different sets of QS genes, or fluctuating requirements), mechanisms of re-wiring for other genes and in different species and strains, and assessing convergence across replicates.

  7. Version published to 10.1128/mbio.00161-22
  8. Version published to 10.1101/2021.07.01.450773 on bioRxiv