A covariation analysis reveals elements of selectivity in quorum sensing systems
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Evaluation Summary:
Acyl-homoserine lactone (AHL) based quorum sensing systems are an important form of intercellular communication in bacteria. These systems, minimally comprised of a synthase and a receptor, often involve different types of AHLs. This paper demonstrates that amino acids in the active site of the synthase and the binding site of the receptor have co-evolved to provide specificity for different signaling molecules, even though the synthase and receptor do not directly interact. This type of information could potentially be used to rationally engineer synthases and receptors with different specificities, as well as predict the specificities of uncharacterized synthases and receptors.
(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 #1 and Reviewer #2 agreed to share their names with the authors.)
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Abstract
Many bacteria communicate with kin and coordinate group behaviors through a form of cell-cell signaling called acyl-homoserine lactone (AHL) quorum sensing (QS). In these systems, a signal synthase produces an AHL to which its paired receptor selectively responds. Selectivity is fundamental to cell signaling. Despite its importance, it has been challenging to determine how this selectivity is achieved and how AHL QS systems evolve and diversify. We hypothesized that we could use covariation within the protein sequences of AHL synthases and receptors to identify selectivity residues. We began by identifying about 6000 unique synthase-receptor pairs. We then used the protein sequences of these pairs to identify covariation patterns and mapped the patterns onto the LasI/R system from Pseudomonas aeruginosa PAO1. The covarying residues in both proteins cluster around the ligand-binding sites. We demonstrate that these residues are involved in system selectivity toward the cognate signal and go on to engineer the Las system to both produce and respond to an alternate AHL signal. We have thus demonstrated that covariation methods provide a powerful approach for investigating selectivity in protein-small molecule interactions and have deepened our understanding of how communication systems evolve and diversify.
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Author Response
We are glad to know others in the field find our approach and results valuable and appreciate the positive feedback.
Regarding protein stability, it was not our intention to determine the mechanism by which amino acid substitutions alter LasI/R activity. Indeed, sensitivity and selectivity could be altered for a number of reasons including changes to protein stability. This is especially true for LasR. For AHL receptors signal binding and protein stability are intricately linked. AHL receptors are usually unstable/insoluble in the absence of bound signal and changes in signal affinity frequently result in changes to receptor solubility. We have added a discussion of this to the text (lines 186-219, 252-254). We have also conducted experiments to measure LasR solubility in a subset of our variants (new Figure 4–figure …
Author Response
We are glad to know others in the field find our approach and results valuable and appreciate the positive feedback.
Regarding protein stability, it was not our intention to determine the mechanism by which amino acid substitutions alter LasI/R activity. Indeed, sensitivity and selectivity could be altered for a number of reasons including changes to protein stability. This is especially true for LasR. For AHL receptors signal binding and protein stability are intricately linked. AHL receptors are usually unstable/insoluble in the absence of bound signal and changes in signal affinity frequently result in changes to receptor solubility. We have added a discussion of this to the text (lines 186-219, 252-254). We have also conducted experiments to measure LasR solubility in a subset of our variants (new Figure 4–figure supplement 2; main text lines 219-224).
Reviewer #2 raises an interesting question about the consequences of LasI/R variants on P. aeruginosa sociality. We believe reporter assays are the most appropriate method by which to directly study LasR selectivity and sensitivity. Whether these changes to LasI/R sensitivity and selectivity result in altered sociality is a very intriguing, but separate, question and the subject of planned future research.
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Reviewer #3 (Public Review):
Some Gram-negative bacteria synthesize acyl-homoserine lactone molecules, which are secreted into the environment and then transported into nearby bacteria, where they are detected by receptors. Different species make acyl-homoserine lactones that differ in chain length and oxidation state at the C-3 position. The manuscript by Wellington et al. reports an elegant and compelling investigation of the specificity determinants involved in quorum sensing, using a combination of bioinformatics and experimental approaches.
Over the course of evolution, if an amino acid change occurs in one protein, then a compensating change can occur in a partner protein to restore/retain a functional interaction between the two. Analyses of evolutionarily covarying positions between two interacting proteins, or within a single …
Reviewer #3 (Public Review):
Some Gram-negative bacteria synthesize acyl-homoserine lactone molecules, which are secreted into the environment and then transported into nearby bacteria, where they are detected by receptors. Different species make acyl-homoserine lactones that differ in chain length and oxidation state at the C-3 position. The manuscript by Wellington et al. reports an elegant and compelling investigation of the specificity determinants involved in quorum sensing, using a combination of bioinformatics and experimental approaches.
Over the course of evolution, if an amino acid change occurs in one protein, then a compensating change can occur in a partner protein to restore/retain a functional interaction between the two. Analyses of evolutionarily covarying positions between two interacting proteins, or within a single protein, have long been used to identify positions that directly interact. Wellington et al. applied the same approach to two protein families (the synthases and receptors for acyl-homoserine lactones) to identify positions that are connected not by direct physical interaction between the two proteins but rather by interaction with the same acyl-homoserine lactone. The covariation analysis was made possible by the fortuitous case (and reasonable assumption) that genes encoding partner synthases and receptors are located close to one another within bacterial genomes.
The covarying residues turn out to be in the active site of the synthase and the binding site of the receptor, in positions that directly interact with the acyl-homoserine lactone. The authors made a variety of single amino acid substitutions at positions with high covariation scores in the Pseudomonas aeruginosa LasI synthase and LasR receptor proteins. The mutant proteins exhibited altered synthetic and detection specificities for acyl-homoserine lactones. Altering three residues simultaneously resulted in substantial changes in specificity.
This paper constitutes a proof of principle for an approach that could be used to investigate other families of proteins connected by interactions with small molecules (e.g. metabolic pathways). Furthermore, it suggests a path toward rational engineering of quorum sensing systems for synthetic biology, as well as specificity prediction for uncharacterized quorum sensing pathways based simply on the primary amino acid sequences of the synthase and receptor proteins.
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Reviewer #2 (Public Review):
Wellington Miranda et al. investigated how acyl-homoserine lactone autoinducer mediated quorum sensing systems evolve. The authors used the statistical covariation method GREMLIN to identify key amino acids that have coevolved in the well-studied LasI/LasR quorum sensing AHL synthase/receptor pair from Pseudomonas aeruginosa PAO1. LasI produces and LasR detects 3OC12-HSL. The authors identify some new and some previously reported residues using the GREMLIN tool; they focus on L157 in LasI and G38, R61, A127, S129, and L130 in LasR as residues that determine selectivity of the acyl-homoserine lactone that is produced and detected, respectively. Quite expectedly, these residues are in or near the ligand-binding pocket of LasI and LasR. The authors further engineer the LasI/R system to produce and detect the …
Reviewer #2 (Public Review):
Wellington Miranda et al. investigated how acyl-homoserine lactone autoinducer mediated quorum sensing systems evolve. The authors used the statistical covariation method GREMLIN to identify key amino acids that have coevolved in the well-studied LasI/LasR quorum sensing AHL synthase/receptor pair from Pseudomonas aeruginosa PAO1. LasI produces and LasR detects 3OC12-HSL. The authors identify some new and some previously reported residues using the GREMLIN tool; they focus on L157 in LasI and G38, R61, A127, S129, and L130 in LasR as residues that determine selectivity of the acyl-homoserine lactone that is produced and detected, respectively. Quite expectedly, these residues are in or near the ligand-binding pocket of LasI and LasR. The authors further engineer the LasI/R system to produce and detect the non-native 3OC10-HSL autoinducer in addition to 3OC12-HSL, thereby broadening the specificity of LasI/R.
P. aeruginosa is an important pathogen and a powerful system for the study of quorum sensing. The use of GREMLIN to study how autoinducer synthase and receptor pairs coevolve in terms of sensitivity and specificity for a particular autoinducer is impressive. This paper adds an exciting approach to the growing literature on the evolution of sensitivity and promiscuity in quorum-sensing systems. Further, the authors have developed a thin layer chromatography based approach to separate and detect AHLs from nine samples simultaneously. This methodology should be widely useful to researchers.
The current manuscript does not provide any data about the solubitlity and/or stability of the LasI and LasR mutant proteins being studied. For instance, biochemical analyses would be needed to evaluate if the increased sensitivity of LasRA127L compared to wildtype is due to higher affinity for the autoinducer or because the variant is more stable. Further, this work relies solely on reporter assays and does not address the consequences of these LasI and LasR variants to quorum-sensing dependent P. aeruginosa group behaviors such as pyocyanin production and virulence.
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Reviewer #1 (Public Review):
Acyl-homoserine lactone (AHL) based quorum sensing systems are an important form of intercellular communication in bacteria. These systems, minimally comprised of a synthase and a receptor, often involve different types of AHLs. This paper uses covariation analyses to try and tease out the determinants of this AHL specificity. Using the GREMLIN pipeline, they identify a series of coevolving residues in LasR and LasI homologs. This is interesting in its own right, and a strength of the paper, as LasR and LasI don't physically interact, and instead interact and covary indirectly by virtue of sharing the same AHL. Through various reporter and biochemical assays, the authors then demonstrate that the residues identified are important for AHL recognition. In the last part of the manuscript, they attempt to use …
Reviewer #1 (Public Review):
Acyl-homoserine lactone (AHL) based quorum sensing systems are an important form of intercellular communication in bacteria. These systems, minimally comprised of a synthase and a receptor, often involve different types of AHLs. This paper uses covariation analyses to try and tease out the determinants of this AHL specificity. Using the GREMLIN pipeline, they identify a series of coevolving residues in LasR and LasI homologs. This is interesting in its own right, and a strength of the paper, as LasR and LasI don't physically interact, and instead interact and covary indirectly by virtue of sharing the same AHL. Through various reporter and biochemical assays, the authors then demonstrate that the residues identified are important for AHL recognition. In the last part of the manuscript, they attempt to use the covariation analysis to guide the 'rewiring' of LasR-LasI to behave like MupR-MupI. This is mostly successful, although LasR-LasI specificity hasn't been 'rewired' so much as simply broadened. The paper could also be improved by fleshing out the description of the results/data obtained - at times it is difficult to assess how the authors have arrived at certain rather sweeping conclusions.
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Evaluation Summary:
Acyl-homoserine lactone (AHL) based quorum sensing systems are an important form of intercellular communication in bacteria. These systems, minimally comprised of a synthase and a receptor, often involve different types of AHLs. This paper demonstrates that amino acids in the active site of the synthase and the binding site of the receptor have co-evolved to provide specificity for different signaling molecules, even though the synthase and receptor do not directly interact. This type of information could potentially be used to rationally engineer synthases and receptors with different specificities, as well as predict the specificities of uncharacterized synthases and receptors.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback …
Evaluation Summary:
Acyl-homoserine lactone (AHL) based quorum sensing systems are an important form of intercellular communication in bacteria. These systems, minimally comprised of a synthase and a receptor, often involve different types of AHLs. This paper demonstrates that amino acids in the active site of the synthase and the binding site of the receptor have co-evolved to provide specificity for different signaling molecules, even though the synthase and receptor do not directly interact. This type of information could potentially be used to rationally engineer synthases and receptors with different specificities, as well as predict the specificities of uncharacterized synthases and receptors.
(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 #1 and Reviewer #2 agreed to share their names with the authors.)
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