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PreprintCited by 3Bacterial Genetics and BiotechnologyGenomics and Phylogenetic StudiesGenomics and Chromatin Dynamics

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The evolution of function in the DNA binding domain of the CRP/FNR family

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Krishnaswamy, Madhumitha, Narain Seshasayee, Aswin Sai·2025·Genome Biology and Evolution
DOI 10.1101/2025.07.04.663149PMC PMC12531629

Preprint — published as 10.1093/gbe/evaf182 · Paper page

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Abstract

Transcriptional regulation, facilitated by transcription factors (TFs), contributes to bacterial response to environmental and cellular perturbations. How have TFs diversified and gained their functions? We use the CRP/FNR family to study this question across ∼6000 prokaryotic genomes. Characterized by homology to TFs CRP (cyclic AMP Receptor Protein) and FNR (Regulator of Fumarate and Nitrate Reductase) in Escherichia coli , the two functions of this family – sequence-specific DNA binding and transcriptional activation by direct contacts to RNA polymerase - are conferred by the helix-turn-helix containing DNA binding domain. We constructed a rooted phylogeny of this domain and performed residue conservation analysis on extant and in-silico reconstructed ancestral sequences of CRP/FNR family members. Residues that confer DNA binding specificity likely emerged in the overall ancestor of the sequence family. This ancestor does not fall into classes containing either E. coli CRP (Eco-CRP) or E. coli FNR (Eco-FNR) clades, but instead a large and diverse class that we call “CRP-like”. Residues key for base-specific DNA binding remain largely conserved in the family. Residues (together called AR1) which make direct contacts with αCTD of RNA Polymerase are conserved only in the Eco-CRP class and restricted to Gammaproteobacteria . The corresponding ‘287 determinant’ interface on αCTD is again fully conserved only in Gammaproteobacteria , which contain Eco-CRP members, following step-wise evolution initiated at the Proteobacterial common ancestor. These suggest suggesting co-emergence of the interface residues in Eco-CRP. Our analysis hence shows that Class I transcriptional activation through AR1 on CRP and the determinant of αCTD, as described in E. coli CRP, is phylogenetically restricted, while base-specific DNA binding has been present in the CRP/FNR family throughout its evolutionary history.

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Fig. 1. Classification and phylogeny of the c-f-HTH domain. a) Heatmap showing the presence/absence (black/white) of the different classes across all taxa which showed the presence of the c-f-HTH domain (N = 4,114), represented at the phylum level (class level for certain diverse phyla). Note: grayscale heatmap with thin binary values. b) Collapsed gene tree (N = 10,224) of the c-f-HTH domain sequences rooted using the DNA-binding domain of a methyltransferase enzyme from Fungi. The size of collapsed nodes indicates the number of extant tips, and colored based on classification. Collapsed and visualized using iTOL. c) Collapsed Neighbor Joining (NJ) tree (N = 10,227) of c-f-HTH domain sequences along with four key ancestral nodes, as indicated in legend. Clades containing nodes of interest are shaded on the basis of overall regulators present in the clade. Collapsed and visualized using iTOL.
Fig. 1. Classification and phylogeny of the c-f-HTH domain. a) Heatmap showing the presence/absence (black/white) of the different classes across all taxa which showed the presence of the c-f-HTH domain (N = 4,114), represented at the phylum level (class level for certain diverse phyla). Note: grayscale heatmap with thin binary values. b) Collapsed gene tree (N = 10,224) of the c-f-HTH domain sequences rooted using the DNA-binding domain of a methyltransferase enzyme from Fungi. The size of collapsed nodes indicates the number of extant tips, and colored based on classification. Collapsed and visualized using iTOL. c) Collapsed Neighbor Joining (NJ) tree (N = 10,227) of c-f-HTH domain sequences along with four key ancestral nodes, as indicated in legend. Clades containing nodes of interest are shaded on the basis of overall regulators present in the clade. Collapsed and visualized using iTOL.CC BY · PubMed Central
Fig. 2. Conservation of key functional residues. Heatmaps for each key residue of interest across the different domain classes, ordered by physicochemical properties. Color of intersecting boxes is scaled according to the amino acid frequency from low (light) to high (dark) and indicates the occurrence of a particular amino acid for the specified key residue in all members of a class. The distributions for Eco-CRP are indicated in the green dashed boxes.
Fig. 2. Conservation of key functional residues. Heatmaps for each key residue of interest across the different domain classes, ordered by physicochemical properties. Color of intersecting boxes is scaled according to the amino acid frequency from low (light) to high (dark) and indicates the occurrence of a particular amino acid for the specified key residue in all members of a class. The distributions for Eco-CRP are indicated in the green dashed boxes.CC BY · PubMed Central
Fig. 3. Conservation and emergence in the gene tree. Collapsed outgroup rooted gene trees of the c-f-HTH domain (N = 10,224) colored on a grayscale gradient (low to high) on the basis of PSSM scores calculated for a) activating region 1 (AR1) residues, b) Residues involved in making phosphate backbone contacts, c) Residues involved in making specific base contacts with DNA. The size of collapsed nodes corresponds to the number of extant tips. Legend in gray box is Fig. 1b replicated for reference to identity of different clades in the gene tree. Collapsed and visualized using iTOL.
Fig. 3. Conservation and emergence in the gene tree. Collapsed outgroup rooted gene trees of the c-f-HTH domain (N = 10,224) colored on a grayscale gradient (low to high) on the basis of PSSM scores calculated for a) activating region 1 (AR1) residues, b) Residues involved in making phosphate backbone contacts, c) Residues involved in making specific base contacts with DNA. The size of collapsed nodes corresponds to the number of extant tips. Legend in gray box is Fig. 1b replicated for reference to identity of different clades in the gene tree. Collapsed and visualized using iTOL.CC BY · PubMed Central
Fig. 4. Patterns of conservation of αCTD. a) Smoothscatter plot and scatterplot of PSSM scores corresponding to 287 determinants of αCTD versus AR1 scores of Eco-CRP and CRP-like regulators. The density of the points is shown in the smooth scatter plot while the points are colored on the basis of class in the scatter plot. Regression lines are plotted in gray for CRP-like and Eco-CRP. b) Conservation of the 287 determinant mapped onto the 16S rRNA species tree (N = 1,454). PSSM scores of the 287 determinants divided into four categories as indicated in legend—extremely low, low, medium, and high, mapped onto their corresponding genomes on the species tree. Colourstrip annotations show key classes present and the taxonomic annotation of the genome. Note, for simplicity, extremely low and low categories are combined into one in the text. Colored and visualized using iTOL.
Fig. 4. Patterns of conservation of αCTD. a) Smoothscatter plot and scatterplot of PSSM scores corresponding to 287 determinants of αCTD versus AR1 scores of Eco-CRP and CRP-like regulators. The density of the points is shown in the smooth scatter plot while the points are colored on the basis of class in the scatter plot. Regression lines are plotted in gray for CRP-like and Eco-CRP. b) Conservation of the 287 determinant mapped onto the 16S rRNA species tree (N = 1,454). PSSM scores of the 287 determinants divided into four categories as indicated in legend—extremely low, low, medium, and high, mapped onto their corresponding genomes on the species tree. Colourstrip annotations show key classes present and the taxonomic annotation of the genome. Note, for simplicity, extremely low and low categories are combined into one in the text. Colored and visualized using iTOL.CC BY · PubMed Central
Fig. 5. The evolutionary trajectory of functions in the c-f-HTH domain of the CRP/FNR family. Conservation of the key residues identified in the study marked the 16S rRNA species tree. Colorstrip annotations (innermost to outermost) correspond to the range in PSSM scores observed for the 287 determinant, AR1, and specific base contact residues. For genomes coding for multiple CRP/FNR family proteins, AR1 and specific base contact scores for the best-scoring protein are shown. Branches are colored on the basis of taxonomic annotation, and Gammaproteobacteria, showing the highest scores for 287 determinants of αCTD, along with AR1 and specific base contact scores, is highlighted. Colored and visualized using iTOL.
Fig. 5. The evolutionary trajectory of functions in the c-f-HTH domain of the CRP/FNR family. Conservation of the key residues identified in the study marked the 16S rRNA species tree. Colorstrip annotations (innermost to outermost) correspond to the range in PSSM scores observed for the 287 determinant, AR1, and specific base contact residues. For genomes coding for multiple CRP/FNR family proteins, AR1 and specific base contact scores for the best-scoring protein are shown. Branches are colored on the basis of taxonomic annotation, and Gammaproteobacteria, showing the highest scores for 287 determinants of αCTD, along with AR1 and specific base contact scores, is highlighted. Colored and visualized using iTOL.CC BY · PubMed Central

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References 45 · Cited by 3

Research works printed but not linked: 41

  1. 10.1093/bioinformatics/bty314 · DOI
  2. 10.1126/science.1076376 · DOI
  3. 10.1073/pnas.0408579102 · DOI · Paper page
  4. 10.1038/nrmicro787 · DOI · Paper page
  5. 10.1006/jmbi.1999.3161 · DOI · Paper page
  6. 10.1126/science.6372090 · DOI · Paper page
  7. 10.1111/j.1365-2958.1993.tb01626.x · DOI · Paper page
  8. 10.1371/journal.pcbi.1002195 · DOI · Paper page
  9. 10.1126/science.aaf4417 · DOI
  10. 10.1128/MMBR.68.3.518-537.2004 · DOI · Paper page
  11. 10.1073/pnas.0506687102 · DOI · Paper page
  12. 10.1093/molbev/msx281 · DOI · Paper page
  13. 10.1038/s41596-024-01011-0 · DOI · Paper page
  14. 10.1038/nmeth.4285 · DOI · Paper page
  15. 10.1093/molbev/mst010 · DOI · Paper page
  16. 10.1146/annurev.bi.62.070193.003533 · DOI · Paper page
  17. 10.1038/s41598-019-55253-0 · DOI
  18. 10.1016/S0168-6445(03)00066-4 · DOI · Paper page
  19. 10.1093/femsml/uqad024 · DOI
  20. 10.1046/j.1365-2958.2000.02172.x · DOI
  21. 10.1016/j.sbi.2004.01.012 · DOI · Paper page
  22. 10.1146/annurev-micro-092611-150012 · DOI · Paper page
  23. 10.1046/j.1365-2958.2000.02086.x · DOI
  24. 10.1093/nar/gkae268 · DOI · Paper page
  25. 10.1186/s40064-016-2526-5 · DOI
  26. 10.1093/gbe/evt004 · DOI
  27. 10.1007/BF02817665 · DOI
  28. 10.1093/molbev/msaa015 · DOI · Paper page
  29. 10.1371/journal.pgen.1003565 · DOI · Paper page
  30. 10.1016/j.jmb.2025.169177 · DOI
  31. 10.1006/jmbi.1996.0409 · DOI · Paper page
  32. 10.1371/journal.pone.0009490 · DOI · Paper page
  33. 10.1093/nar/25.2.326 · DOI
  34. 10.3389/fmicb.2017.00959 · DOI
  35. 10.1007/BF00871630 · DOI · Paper page
  36. 10.1093/nar/gkl315 · DOI · Paper page
  37. 10.1016/j.tim.2015.04.009 · DOI
  38. 10.1093/bioinformatics/btp033 · DOI · Paper page
  39. 10.1111/j.1365-2958.1993.tb00949.x · DOI
  40. 10.1093/nar/19.24.6705 · DOI
  41. 10.1093/nar/25.20.4028 · DOI

Metadata

Authors
Krishnaswamy, M., & Narain Seshasayee, A. S.
Affiliations
Tata Institute of Fundamental Research (IN)
ORCID
Krishnaswamy, Madhumitha
Venue
Genome Biology and Evolution
Publisher
openRxiv
Published
2025-07-07
Volume
17
Issue
10
ISSN
1759-6653 (ISSN-L)
DOI
10.1101/2025.07.04.663149
PMC
PMC12531629
OpenAlex
W4414500780
Cited by
3
References
45
Citations by year
2026: 3
FWCI
1.75
Type
journal article
Language
en
Open access
Licence
Locations
  • Genome Biology and Evolution (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed (publishedVersion)
Funders
Department of Atomic Energy, Government of India (RTI-4006)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Bacterial Genetics and Biotechnology
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Phylogenetic Studies
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Chromatin Dynamics
Keywords
transcription factors, DNA-binding domain, helix-turn-helix, transcriptional activation, ancestral sequence reconstruction, protein evolution, Gammaproteobacteria, bacterial transcriptional regulation, phylogenetic analysis, amino acid conservation
MeSH terms
Protein Domains, Cyclic AMP Receptor Protein, DNA-Binding Proteins, Escherichia coli, Iron-Sulfur Proteins, Phylogeny, Prokaryotic Cells, Sporothrix, Transcription Factors, Evolution, Molecular

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Krishnaswamy, M., & Narain Seshasayee, A. S. (2025). The evolution of function in the DNA binding domain of the CRP/FNR family. Genome Biology and Evolution. https://doi.org/10.1101/2025.07.04.663149

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