1. Evolution of regulatory interactions in bacteria Mikhail Gelfand Institute for Information Transmission Problems, RAS 4th Bertinoro Computational Biology (BCB) Meeting “Evolution of and Comparative Approaches to Gene Regulation” 24-30 June 2006

2. Это – ряд наблюдений. В углу – тепло. Взгляд оставляет на вещи след. Вода представляет собой стекло. Человек страшней, чем его скелет. Иосиф Бродский A list of some observations. In a corner, it’s warm. A glance leaves an imprint on anything it’s dwelt on. Water is glass’s most public form. Man is more frightening than its skeleton. Joseph Brodsky

3. Plan Evolution of individual sites Coevolution of transcription factors and their binding signals Distribution of transcription factor families in various genomes Evolution of simple and complex regulatory systems

4. Birth and death of sites is a very dynamic process NadR-binding sites upstream of pnuB seem absent in Klebsiella pneumoniae and Serratia marcescens

5. … but there are candidate sites further upstream …

6. … and they are clearly diferent (not simply misaligned).

7. Loss of regulators and cryptic sites Loss of the RbsR in Y. pestis (ABC-transporter also is lost) Start codon of rbsD RbsR binding site

8. Unexpected conservation of non-consensus positions in orthologous sites regulatory site of LexA upstream of lexA consensus nucleotides are in caps wrong consensus?

9. TF PurR, gene purL TF PurR, gene purM

10. Non-consensus positions are more conserved than synonymous codon positions

11. Relative conservation of non-consensus nucleotides may be higher than conservation of consensus nucleotides

12. Regulators and their signals Subtle changes at close evolutionary distances Cases of signal conservation at surprisingly large distances Changes in spacing / geometry of dimers Correlation between contacting nucleotides and amino acid residues

13. The LacI family: subtle changes in signals at close distances G A n CGCG GnGn GCGC

14. NrdR (regulator of ribonucleotide reducases and some other replication-related genes): conservation at large distances

15. BirA (biotin regulator in eubacteria and archaea): conserved signal, changed spacing Profile 2: Gram-negative bacteriaProfile 1: Gram-positive bacteria, Archaea

16. DNA signals and protein-DNA interactions CRPPurR IHFTrpR Entropy at aligned sites and the number of contacts (heavy atoms in a base pair at a distance <cutoff from a protein atom)

17. Specificity-determining positions in the LacI family Training set: 459 sequences, average length: 338 amino acids, 85 specificity groups 10 residues contact NPF (analog of the effector) 6 residues in the intersubunit contacts 7 residues contact the operator sequence 7 residues in the effector contact zone (5Ǻ<d min <10Ǻ) 5 residues in the intersubunit contact zone (5Ǻ<d min <10Ǻ) 6 residues in the operator contact zone (5Ǻ<d min <10Ǻ) – 44 SDPs LacI from E.coli

18. CRP/FNR family of regulators

19. Correlation between contacting nucleotides and amino acid residues CooA in Desulfovibrio spp. CRP in Gamma-proteobacteria HcpR in Desulfovibrio spp. FNR in Gamma-proteobacteria DD COOA ALTTEQLSLHMGATRQTVSTLLNNLVR DV COOA ELTMEQLAGLVGTTRQTASTLLNDMIR EC CRP KITRQEIGQIVGCSRETVGRILKMLED YP CRP KXTRQEIGQIVGCSRETVGRILKMLED VC CRP KITRQEIGQIVGCSRETVGRILKMLEE DD HCPR DVSKSLLAGVLGTARETLSRALAKLVE DV HCPR DVTKGLLAGLLGTARETLSRCLSRMVE EC FNR TMTRGDIGNYLGLTVETISRLLGRFQK YP FNR TMTRGDIGNYLGLTVETISRLLGRFQK VC FNR TMTRGDIGNYLGLTVETISRLLGRFQK TGTCGGCnnGCCGACA TTGTgAnnnnnnTcACAA TTGTGAnnnnnnTCACAA TTGATnnnnATCAA Contacting residues: REnnnR TG: 1 st arginine GA: glutamate and 2 nd arginine

20. The correlation holds for other factors in the family

21. Distribution of TF families in bacterial genomes Streptomyces coelicolor Pseudomonas aeruginosa ExtraTrain database AraC GntR LysR TetR LacI LuxR Agrobacterium tumefaciens Escherichia coli Bacillus subtilis

22. Strategies of successful TF families One ortholog per genome: –LexA, NrdR, HrcA, ArgR –present even in archaea: BirA (also enzyme), ModE Several (2-3) orthologs per genome –CRP/FNR, FUR Local explosions –LacI in alpha- and gamma-proteobacteria –2CS systems in delta-proteobacteria –sigma-factors in Streptomyces Because TF in a family tend to have related functions and these might depend on the lifestyle?

23. LacI family regulons in closely related strains (top: TFs, bottom: regulated genes) Seven Escherichia and Shigella spp. Five Salmonella spp. Four Bacillus cereus and B. anthracis strains

24. What are the driving forces for the present-day state? Expansion and contraction of regulons Duplications of regulators with or without regulated loci Loss of regulators with or without regulated loci Re-assortment of regulators and structural genes … especially in complex systems Horizontal transfer

25. Regulon expansion: how FruR has become CRA icdA aceA aceB aceEF pckA ppsApykF adhE gpmApgk tpiA gapA pfkA fbp Fructose fruKfruBA eda edd epd Glucose ptsHI-crr Mannose manXYZ mtlD mtlA Mannitol Gamma-proteobacteria

26. Common ancestor of Enterobacteriales icdA aceA aceB aceEF pckA ppsApykF adhE gpmApgk tpiA gapA pfkA fbp Fructose fruKfruBA eda edd epd Glucose ptsHI-crr Mannose manXYZ mtlD mtlA Mannitol Gamma-proteobacteria Enterobacteriales

27. Common ancestor of Escherichia and Salmonella icdA aceA aceB aceEF pckA ppsApykF adhE gpmApgk tpiA gapA pfkA fbp Fructose fruKfruBA eda edd epd Glucose ptsHI-crr Mannose manXYZ mtlD mtlA Mannitol Gamma-proteobacteria Enterobacteriales E. coli and Salmonella spp.

28. Trehalose/maltose catabolism in alpha-proteobacteria Duplicated LacI-family regulators: lineage- specific post-duplication loss

29. The binding signals are very similar (the blue branch is somewhat different: to avoid cross-recognition?)

30. Utilization of an unknown galactoside in gamma-proteobacteria Loss of regulator and merger of regulons: It seems that laci-X was present in the common ancestor (Klebsiella is an outgroup) Yersinia and Klebsiella: two regulons, GalR (not shown, includes genes galK and galT) and Laci-X Erwinia: one regulon, GalR

31. Utilization of maltose/maltodextrin in Firmicutes Two different ABC transporters (shades of red) PTS (pink) Glucoside hydrolases (shades of green) Two regulators (black and grey)

32. Modularity of the functional subsystem Two different ABC systems Three hydrolases in one operon (E. faecalis) or separately

33. Changes of regulation Two different ABC systems Displacement: invasion of a regulator from a different subfamily (horizontal transfer from a related species?) – blue sites

34. Orthologous TFs with completely different regulons

35. Utilization of xylose in alpha-proteobacteria xylBA Three different ABC transporters Three regulators: two from the LacI family and one from the ROK family

36. Changes in operon structure

37. Changes in regulation Duplication and displacement: Duplicated XylR-1a assumed the role of the ROK-family regulator Displacement: Operon regulation changed from XylR-1 to XylR-2 (different subfamily)

38. Catabolism of gluconate in proteobacteria

39. extreme variability of regulation of “marginal” regulon members γ Pseudomonas spp. β

40. Regulation of amino acid biosynthesis in Firmicutes Interplay between regulatory RNA elements and transcription factors Expansion of T-box systems (normally RNA structures regulating aminoacyl- tRNA-synthetases)

41. Aromatic amino acid regulons

42. Five regulatory systems for the methionine biosynthesis A.SAM- dependent RNA riboswitch B.Met-tRNA- dependent T-box (RNA) C,D,E. repressors of transcription

43. Methionine regulatory systems: loss of S-box regulons S-boxes (SAM-1 riboswitch) –Bacillales –Clostridiales –the Zoo: Petrotoga actinobacteria (Streptomyces, Thermobifida) Chlorobium, Chloroflexus, Cytophaga Fusobacterium Deinococcus proteobacteria (Xanthomonas, Geobacter) Met-T-boxes (Met-tRNA-dependent attenuator) + SAM-2 riboswitch for metK –Lactobacillales MET-boxes (candidate transcription signal) –Streptococcales Lact. Strep. Bac. Clostr. ZOO

44. Mapping the events to the phylogenetic tree Bacillus subtilis and related species Bacillus cereus and related species Strepto- coccus spp. Lacto- bacillus spp. Clostridium spp. Trp-T-boxes  TRAP Tyr-T-boxes  PCE emergence of MtaR Tyr-T-boxes  ARO expansion of Met-T-boxes, emergence of SAM-2 riboswitches loss of S-boxes (SAM-I riboswitches)

45. Combined regulatory network for iron homeostasis genes in in  -proteobacteria. [- Fe] [+Fe] [- Fe] [+Fe] [ Fe]- FeS FeS status of cell The connecting line denote regulatory interactions, which the thickness reflecting the frequency of the interaction in the analyzed genomes. The suggested negative or positive mode of operation is shown by dead-end and arrow-end of the line.

46. Rhizobiales Bradyrhizobiaceae Rhizobiaceae Rhodo- bacterales Hyphomonadaceae Rhodo- bacteraceae Rickettsiales Rhodo- spirillales Sphingomo- nadales - p r o t e o b a c t e r i a Organism Irr MntR Sinorhizobium meliloti Rhizobium leguminosarum Rhizobium etli Agrobacterium tumefaciens Mesorhizobium loti Mesorhizobiumsp. BNC1 Brucella melitensis Bartonellaquintana andspp. Bradyrhizobium japonicum Rhodopseudomonas palustris Nitrobacter hamburgensis Nitrobacter winogradskyi Rhodobacter capsulatus Rhodobacter sphaeroides Silicibacter sp. TM1040 Silicibacter pomeroyi Jannaschia sp.CC51 Rhodobacterales bacterium HTCC2654 Roseobactersp. MED193 Roseovarius nubinhibens ISM Roseovarius sp.217 Loktanella vestfoldensis SKA53 Sulfitobacter sp. EE-36 Oceanicola batsensis HTCC2597 Oceanicaulis alexandrii HTCC2633 Caulobacter crescentus Parvularcula bermudensis HTCC2503 Erythrobacter litoralis Novosphingobium aromaticivorans Sphinopyxis alaskensisgRB2256 Zymomonas mobilis Gluconobacter oxydans Rhodospirillum rubrum Magnetospirillum magneticum Pelagibacter ubique HTCC1002 SM + MUR / FUR RirAIscR RL RHE AGR ML MBNC BME BQ BJ RPA Nham Nwi RC Rsph STM SPO Jann RB2654 MED193 ISM ROS217 SKA53 EE36 OB2597 OA2633 CC PB2503 ELI Saro Sala ZM GOX Rrub Amb Abb. PU1002 ++ -- +++ -- +++ -- +++ -- ++ - +++ -- +++ -- +++ -- ++ -- + + + - - ++ --- ++ --- ++ --- + + +++ - + +++ - + +++ - + +++ - + ++ - +++ - + +++ - + ++ - + +++ - + ++ - + ++ - + ++ - + + - + #? -- + - + -- + - + -- + - + -- + - + -- + - + -- + - + -- ++ -- + - + -- + - + -- - + - + + + + Group Caulobacterales Parvularculales Rickettsia and Ehrlichia species - + --- + + SAR11 cluster A. B. C. D. Fe and Mn regulons Distribution of Irr, Fur/Mur, MntR, RirA, and IscR regulons in α-proteobacteria #?' in RirA column denotes the absence of the rirA gene in an unfinished genomic sequence and the presence of candidate RirA-binding sites upstream of the iron uptake genes.

47. Genes Functions: Iron uptake Iron storage FeS synthesis Iron usage Heme biosynthesis Regulatory genes Manganese uptake Distribution of the conserved members of the Fe- and Mn-responsive regulons and the predicted RirA, Fur/Mur, Irr, and DtxR binding sites in  -proteobacteria

48. Phylogenetic tree of the Fur family of transcription factors in  -proteobacteria - I Fur in  - and  - proteobacteria Fur in  - proteobacteria Fur in Firmicutes in  proteobacteria Fur MBNC03003593 RB2654 19538 AGR C 620 RL mur Nwi 0013 RPA0450 BJ fur ROS217 18337 Jann 1799 SPO2477 STM1w01000993 MED193 22541 OB2597 02997 SKA53 03101 Rsph03000505 ISM 15430 GOX0771 ZM01411 Saro02001148 Sala 1452 ELI1325 OA2633 10204 PB2503 04877 CC0057 Rrub02001143 Amb1009 Amb4460 SM mur MBNC03003179 BQ fur2 BMEI0375 Mesorhizobium sp. BNC1(I) Sinorhizobium meliloti Bartonella quintana Rhodopseudomonas palustris Bradyrhizobium japonicum Caulobacter crescentus Zmomonas mobilisy Rhodobacter sphaeroides Silicibacter sp. TM1040 Silicibacter pomeroyi Agrobacterium tumefaciens Rhizobium leguminosarum Brucella melitensis Mesorhizobium sp. BNC1(II) Rhodobacterales bacterium HTCC2654 Nitrobacter winogradskyi Nham 0990 Nitrobacter hamburgensis X14 Jannaschia sp. CC51 Roseovarius sp.217 Roseobacter sp. MED193 Oceanicola batsensis HTCC2597 Loktanella vestfoldensis SKA53 Roseovarius nubinhibens ISM Gluconobacter oxydans Erythrobacter litoralis Novosphingobium aromaticivorans Sphinopyxis alaskensis RB2256 Oceanicaulis alexandrii HTCC2633 Rhodospirillum rubrum Parvularcula bermudensisHTCC2503 Magnetospirillum magneticum (I) EE36 12413 Sulfitobacter sp. EE-36 ECOLI PSEAE NEIMA HELPY BACSU Helicobacter pylori : sp|O25671 Bacillus subtilis: P54574sp| Neisseria meningitidis : sp|P0A0S7 Pseudomonas aeruginosa : sp|Q03456 Escherichia coli : P0A9A9sp| Mur Fur  Magnetospirillum magneticum (II) RHE_CH00378 Rhizobiumetli  PU1002 04436 Pelagibacter ubique HTCC1002  Irr in  proteobacteria  proteobacteria Regulator of manganese uptake genes (sit, mntH) Regulator of iron uptake and metabolism genes

49. The A, B, and C groups of - proteobacteria -  Mur Caulobacter crescentus Zymomonas mobilis Gluconobacter oxydans Erythrobacter litoralis Novosphingobium aromaticivorans Rhodospirillum rubrum Magnetospirillum magneticum Escherichia coli Sphinopyxis alaskensis Parvularcula bermudensis - Oceanicaulis alexandrii Bacillus subtilis Sequence logos for the identified Fur-binding sites in the D group of  proteobacteria Sequence logos for the known Fur-binding sites in Escherichia coli and Bacillus subtilis Identified Mur-binding sites

50. Phylogenetic tree of the Fur family of transcription factors in  -proteobacteria - II Fur in  - and  - proteobacteria Fur in  - proteobacteria Fur in Firmicutes Irr in  proteo- bacteria regulator of iron homeostasis  proteobacteria Fur ECOLI PSEAE NEIMA HELPY BACSU Helicobacter pylori: sp|O25671 Bacillus subtilis: P54574sp| Neisseria meningitidis : sp|P0A0S7 Pseudomonas aeruginosa : sp|Q03456 Escherichia coli : P0A9A9sp| Mur / Fur  I rr- AGR C 249 SM irr RL irr1 RL irr2 MLr5570 MBNC03003186 BQ fur1 BMEI1955 BMEI1563 BJ blr1216 RB2654 182 SKA53 01126 ROS217 15500 ISM 00785 OB2597 14726 Jann 1652 Rsph03001693 EE36 03493 STM1w01001534 MED193 17849 SPOA0445 RC irr RPA2339 RPA0424* BJ irr* Nwi 0035* Nham 1013* Nitrobacter hamburgensisX14 Nitrobacter winogradskyi Bradyrhizobium japonicum (I) Agrobacterium tumefaciens Rhizobium leguminosarum (I) Mesorhizobium sp. BNC1 Sinorhizobium meliloti Mesorhizobiumloti Bartonella quintana Brucella melitensis (I) Bradyrhizobium japonicum (II) Rhodobacter sphaeroides Rhodobactercapsulatus Silicibacter pomeroyi Silicibacter sp. TM1040 Roseobacter sp. MED193 Sulfitobacter sp. EE-36 Jannaschia sp. CC51 Oceanicola batsensis HTCC2597 Roseovarius nubinhibens ISM Roseovariussp.217 Loktanella vestfoldensis SKA53 Rhodobacterales bacterium HTCC2654  Rhizobiumetli RHE CH00106 Rhizobium leguminosarum (II) Brucella melitensis (II) Rhodopseudomonas palustris (II) Rhodopseudomonas palustris (I) PU1002 04361 Pelagibacter ubique HTCC1002

51. Sequence logos for the identified Irr binding sites in  -proteobacteria. (8 species) - IrrThe A group The B group (4 species) - Irr The C group (12 species) - Irr

52. Phylogenetic tree of the Rrf2 family of transcription factors in  -proteobacteria proteins with the conserved C-X(6-9)-C(4-6)-C motif within effector-responsive domain proteins without a cysteine triad motif Iron repressor RirA (Rhizobium leguminosarum) Nitrite/NO-sensing regulator NsrR (Nitrosomonas europeae, Escherichia coli) Cysteine metabolism repressor CymR (Bacillus subtilis) Iron-Sulfur cluster synthesis repressor IscR (Escherichia coli) Positional clustering of rrf2-like genes with: iron uptake and storage genes; Fe-S cluster synthesis operons; genes involved in nitrosative stress protection; sulfate uptake/assimilation genes; thioredoxin reductase; carboxymuconolactone decarboxylase-family genes; hmc cytochrome operon Cytochrome complex regulator Rrf2 (Desulfovibrio vulgaris)

53. The A group - RirA(8 species) (12 species)The C group - RirA Sequence logos for the identified RirA-binding sites in  -proteobacteria

54. An attempt to reconstruct the history

55. Open problems Model the evolution of regulatory systems (a catalog of elementary events, estimates of probabilities) –Birth of a binding site; what are the mechanisms? –Loss of a binding site –Duplication of a regulated gene and/or a regulator –Horizontal transfer of a regulated gene and/or a regulator –Loss of structural a gene and/or a regulator Develop an evolutionary model that would converge to the present state (that is, have the same properties) –Distribution of TF families sizes –Distribution of regulon sizes –Other graph-theoretical properties (node degrees etc.) –General properties? E.g. stable cores and flexible margins of functional systems (in terms of gene presence and regulation) “Microevolution” (strains): –“metagenomic” regulatory systems? Co-evolution of TFs and DNA sites: –“Neutral” model for the evolution of binding sites (with invariant functional pressure from the bound protein) –How do the signals evolve? What is the driving force – changes in TFs? –TF-family, position-specific protein-DNA recognition code? All that needs to take into account the incompleteness and noise in the data

56. Acknowledgements Andrei A. Mironov Dmitry Rodionov (now at Burnham Institute) Olga Laikova Alexei Vitreschak Anna Gerasimova Ekateina Kotelnikova (now at Ariadne Genomics) Ekaterina Panina (now at UCLA) Leonid Mirny (MIT) Howard Hughes Medical Institute Russian Fund of Basic Research Russian Academy of Sciences, program “Molecular and Cellular Biology” INTAS