1. Evolution of bacterial regulatory systems Mikhail Gelfand Research and Training Center “Bioinformatics” Institute for Information Transmission Problems Moscow, Russia Berlin, June 2008

2. Plan Individual sites Transcription factors and their binding motifs Regulatory systems and regulons

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

4. … but there are candidate sites further upstream …

5. … and they are clearly different (not simply misaligned).

6. Cryptic sites and loss of regulators Loss of RbsR in Y. pestis (ABC-transporter also is lost) Start codon of rbsD RbsR binding site

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

8. TF PurR, gene purL TF PurR, gene purM

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

10. Regulators and their motifs Cases of motif conservation at surprisingly large distances Subtle changes at close evolutionary distances Correlation between contacting nucleotides and amino acid residues Changes in symmetry patterns

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

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

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

14. 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

15. The CRP/FNR family of regulators

16. 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

17. The correlation holds for other factors in the family

18. NrtR (regulator of NAD metabolism): systematic search for correlated positions analysis of correlated positions in proteins and sites analysis of specificity determining positions the same positions in one alpha-helix identified plans for experimental verification

19. Comparison with the recently solved structure: correlated positions indeed bind the DNA (more exactly, form a hydrophobic cluster)

20. NiaR: changed dimer structure? The GalR family and C- proteins of RM- systems: direct and inverted repeats BirA: changed spacing

21. What are the events leading to the present-day state? Expansion and contraction of regulons New regulators (where from?) 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

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

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

24. 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 and Laci-X Erwinia: one regulon, GalR

25. Utilization of maltose/maltodextrin in Firmicutes Displacement: invasion of a regulator from a different subfamily (horizontal transfer from a related species?) – blue sites

26. Orthologous TFs with completely different regulons (alpha-proteobaceria and Xanthomonadales)

27. Catabolism of gluconate in proteobacteria

28. Extreme variability of the regulation of “marginal” regulon members γ Pseudomonas spp. β

29. Regulon expansion, or how FruR has become CRA CRA (a.k.a. FruR) in Escherichia coli: –global regulator –well-studied in experiment (many regulated genes known) Going back in time: looking for candidate CRA/FruR sites upstream of (orthologs of) genes known to be regulated in E.coli

30. Common ancestor of gamma-proteobacteria 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

31. Common ancestor of the 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

32. 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.

33. 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)

34. Three regulatory systems for the methionine bio- synthesis A.SAM- dependent riboswitch B.Met-T-box C. MtaR: repressor of transcription MtaR

35. 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 candidate TF-binding motif: MtaR –Streptococcales Lact. Strep. Bac. Clostr. ZOO

36. Recent duplications and bursts: ARG-T-box in Clostridium difficile

37. … caused by loss of transcription factor AhrC

38. Duplications and changes in specificity: ASN/ASP/HIS T-boxes

39. Blow-up 1

40. Blow-up 2. Prediction Regulators lost in lineages with expanded HIS-T-box regulon??

41. … and validation conserved motifs upstream of HIS biosynthesis genes candidate transcription factor yerC co-localized with the his genes present only in genomes with the motifs upstream of the his genes genomes with neither YerC motif nor HIS-T-boxes: attenuators Bacillales (his operon) Clostridiales Thermoanaerobacteriales Halanaerobiales Bacillales

42. The evolutionary history of the his genes regulation in the Firmicutes

43. T-boxes: Summary / History

44. Life without Fur

45. Regulation of iron homeostasis (the Escherichia coli paradigm) Iron: essential cofactor (limiting in many environments) dangerous at large concentrations FUR (responds to iron): synthesis of siderophores transport (siderophores, heme, Fe 2+, Fe 3+ ) storage iron-dependent enzymes synthesis of heme synthesis of Fe-S clusters Similar in Bacillus subtilis

46. Regulation of iron homeostasis in α-proteobacteria Experimental studies: FUR/MUR: Bradyrhizobium, Rhizobium and Sinorhizobium RirA (Rrf2 family): Rhizobium and Sinorhizobium Irr (FUR family): Bradyrhizobium, Rhizobium and Brucella RirA Irr FeSheme RirA degraded Fur Fe Fur Iron uptak e systems Sideroph ore uptake Fe / Fe uptake Transcription factors 2+3+ Iron storage ferritins FeS synthesis Heme synthesis Iron-requiring enzymes [iron cofactor] IscR Irr [- Fe] [+Fe] [- Fe] [+Fe] [ Fe]- FeS FeS status of cell

47. Distribution of transcription factors in genomes Search for candidate motifs and binding sites using standard comparative genomic techniques

48. FUR/MUR branch of the FUR family 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. 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 known Fur-binding sites in Escherichia coli and Bacillus subtilis Identified Mur-binding sites FUR and MUR boxes

50. 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 Irr branch of the FUR family

51. Irr boxes Rhizobiaceae plus Bradyrhizobiaceae Rhodobacteriaceae Rhodospirillales

52. RirA/NsrR family (Rhizobiales)

53. IscR family

54. Regulation of genes in functional subsystems Rhizobiales Bradyrhizobiaceae Rhodobacteriales The Zoo (likely ancestral state)

55. Reconstruction of history Appearance of the iron-Rhodo motif Frequent co-regulation with Irr Strict division of function with Irr

56. All logos and Some Very Tempting Hypotheses: 1.Cross-recognition of FUR and IscR motifs in the ancestor. 2.When FUR had become MUR, and IscR had been lost in Rhizobiales, emerging RirA (from the Rrf2 family, with a rather different general consensus) took over their sites. 3.Iron-Rhodo boxes are recognized by IscR: directly testable 1 2 3

57. Summary and open problems Regulatory systems are very flexible –easily lost –easily expanded (in particular, by duplication) –may change specificity –rapid turnover of regulatory sites With more stories like these, we can start thinking about a general theory –catalog of elementary events; how frequent? –mechanisms (duplication, birth e.g. from enzymes, horizontal transfer) –conserved (regulon cores) and non-conserved (marginal regulon members) genes in relation to metabolic and functional subsystems/roles –(TF family-specific) protein-DNA recognition code –distribution of TF families in genomes; distribution of regulon sizes; etc.

58. People Andrei A. Mironov – software, algorithms Alexandra Rakhmaninova – SDP, protein-DNA correlations Anna Gerasimova (now at U. Michigan) – NadR Olga Kalinina (on loan to EMBL) – SDP Yuri Korostelev – protein-DNA correlations Ekateina Kotelnikova (now at Ariadne Genomics) – evolution of sites Olga Laikova – LacI Dmitry Ravcheev– CRA/FruR Dmitry Rodionov (on loan to Burnham Institute) – iron etc. Alexei Vitreschak – T-boxes and riboswitches Andy Jonson (U. of East Anglia) – experimental validation (iron) Leonid Mirny (MIT) – protein-DNA, SDP Andrei Osterman (Burnham Institute) – experimental validation Howard Hughes Medical Institute Russian Foundation of Basic Research Russian Academy of Sciences, program “Molecular and Cellular Biology”