REC protein family expansion by the emergence of a new signaling pathway

Megan E. Garber, Vered Frank, Alexey E. Kazakov, Matthew R. Incha, Alberto A. Nava, Hanqiao Zhang, Luis E. Valencia, Jay D. Keasling, Lara Rajeev, Aindrila Mukhopadhyay*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review


This report presents multi-genome evidence that REC protein family expansion occurs when the emergence of new pathways gives rise to functional discordance. Specificity between residues in REC domain containing response regulators with paired histidine kinases is under negative purifying selection, constrained by the presence of other bacterial two-component systems signaling cascades that share sequence and structural identity. Presuming that the two-component systems can evolve by neutral amino acid changes (neutral drift) when purifying evolutionary constraints are relaxed, how might the REC protein family expand by amino acid changes when these constraints remain intact? Using an unsupervised machine learning approach to observe the sequence landscape of REC domains across long phylogenetic distances, we find that within-gene recombination, a subcategory of gene conversion, switched the effector domain and, consequently, the regulatory context of a duplicated response regulator from transcriptional regulation by σ54 to that by σ70. We determined that the recombined response regulator diverged from its parent by episodic diversifying selection and neutral drift. Functional experiments of the parent of recombined response regulators in a model Pseudomonas putida KT2440 model system revealed that the parent and recombined response regulators sense and respond to different carboxylic acids. Finally, a residue-switching experiment using structural predictions and functional characterization suggests that the new residues in the recombined regulator could form a new interaction interface and mediate condition-specific phosphotransfer. Overall, our study finds that genetic perturbations can create conditions of functional discordance, whereby the REC protein family can evolve by episodic diversifying selection. IMPORTANCE We explore when and why large classes of proteins expand into new sequence space. We used an unsupervised machine learning approach to observe the sequence landscape of REC domains of bacterial response regulator proteins. We find that within-gene recombination can switch effector domains and, consequently, change the regulatory context of the duplicated protein.

Original languageAmerican English
Article number0262223
Issue number6
StatePublished - Dec 2023
Externally publishedYes

Bibliographical note

Publisher Copyright:
© 2023 American Society for Microbiology. All rights reserved.


  • domain swapping
  • evolution
  • gene regulation
  • genetic recombination
  • large protein families
  • response regulator
  • signal transduction
  • two-component regulatory systems


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