Evolution of bacterial regulatory systems

1. Evolution of bacterial regulatory systems Mikhail Gelfand Research and Training Center “Bioinformatics” Institute for Information Transmission Problems Moscow, Russia ASM, Philadelphia, 18.IV.2009

2. Catalog of events • 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

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

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

5. Utilization of an unknown galactoside in gamma-proteobacteria Yersinia and Klebsiella: two regulons, GalR and Laci-X Erwinia: one regulon, GalR Loss of regulator and merger of regulons: It seems that laci-X was present in the common ancestor (Klebsiella is an outgroup)

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

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

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

9. 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

10. Common ancestor of gamma-proteobacteria Mannose Glucose ptsHI-crr manXYZ edd epd eda adhE aceEF icdA pykF ppsA mtlD mtlA Mannitol pgk gpmA pckA gapA fbp pfkA aceA tpiA fruBA fruK Fructose aceB Gamma-proteobacteria

11. Common ancestor of the Enterobacteriales Mannose Glucose ptsHI-crr manXYZ edd epd eda adhE aceEF icdA pykF ppsA mtlD mtlA Mannitol pgk gpmA pckA gapA fbp pfkA aceA tpiA fruBA fruK Fructose aceB Gamma-proteobacteria Enterobacteriales

12. Common ancestor of Escherichia and Salmonella Mannose Glucose ptsHI-crr manXYZ edd epd eda adhE aceEF icdA pykF ppsA mtlD mtlA Mannitol pgk gpmA pckA gapA fbp pfkA aceA tpiA fruBA fruK Fructose aceB Gamma-proteobacteria Enterobacteriales E.coli and Salmonella spp.

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

14. Why T-boxes? • May be easily identified • In most cases functional specificity may be reliably predicted by the analysis of the specifier codons (anti-anti-codons) • Sufficiently long to retain phylogenetic signal => T-boxes are a good model of regulatory evolution

15. Partial alignment of predicted T-boxes TGG: T-box Aminoacyl-tRNA synthetases Amino acid biosynthetic genes Amino acid transporters

16. … continued (in the 5’ direction) anti-anti (specifier) codon Aminoacyl-tRNA synthetases Amino acid biosynthetic genes Amino acid transporters

17. 805 T-boxes in 96 bacteria • Firmicutes • aa-tRNA synthetases • enzymes • transporters • all amino acids excluding glutamate • Actinobacteria (regulation of translation – predicted) • branched chain (ileS) • aromatic (Atopobium minutum) • Delta-proteobacteria • branched chain (leu – enzymes) • Thermus/Deinococcus group (aa-tRNA synthases) • branched chain (ileS, valS) • glycine • Chloroflexi, Dictyoglomi • aromatic (trp – enzymes) • branched chain (ileS) • threonine

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

19. … caused by loss of transcription factor AhrC

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

21. Blow-up 1

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

23. … and validation Bacillales(his operon) • 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 Clostridiales Thermoanaerobacteriales Halanaerobiales Bacillales

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

25. More duplications: THR-T-box in C. difficileand B. cereus

26. T-boxes: Summary / History

27. Life without Fur

28. 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, Fe2+, Fe3+) • storage • iron-dependent enzymes • synthesis of heme • synthesis of Fe-S clusters Similar in Bacillus subtilis

29. [+Fe] [+Fe] [- Fe] [ Fe] - Irr Irr RirA RirA FeS heme degraded 2+ 3+ S i d e r o p h o r e F e / F e I r o n - r e q u i r i n g I r o n s t o r a g e F e S H e m e T r a n s c r i p t i o n u p t a k e u p t a k e e n z y m e s f e r r i t i n s s y n t h e s i s s y n t h e s i s f a c t o r s I r o n u p t a k [ i r o n c o f a c t o r ] e s y s t e m s FeS status IscR Fur Fur of cell Fe FeS [- Fe] [+Fe] 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

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

31. Fur in g- and b- proteobacteria Escherichia coli : P0A9A9 sp| ECOLI Fur Pseudomonas aeruginosa : sp|Q03456 PSEAE Fur in e- proteobacteria Neisseria meningitidis : sp|P0A0S7 NEIMA HELPY : sp|O25671 Helicobacter pylori Fur in Firmicutes BACSU Bacillus subtilis : P54574 sp| SM mur Sinorhizobium meliloti MBNC03003179 Mesorhizobium sp. BNC1 (I) BQ fur2 Bartonella quintana BMEI0375 Brucella melitensis EE36 12413 sp. EE-36 Sulfitobacter a MBNC03003593 sp. BNC1 (II) Mesorhizobium RB2654 19538 HTCC2654 Rhodobacterales bacterium AGR C 620 Agrobacterium tumefaciens RHE_CH00378 Rhizobium etli RL mur Rhizobium leguminosarum Nham 0990 Mur Nitrobacter hamburgensis X14 in a-proteobacteria Nwi 0013 Nitrobacter winogradskyi RPA0450 Rhodopseudomonas palustris Regulator of manganese uptake genes (sit, mntH) BJ fur Bradyrhizobium japonicum ROS217 18337 Roseovarius sp.217 Jann 1799 Jannaschia sp. CC51 SPO2477 Silicibacter pomeroyi STM1w01000993 Silicibacter sp. TM1040 MED193 22541 sp. MED193 Roseobacter OB2597 02997 HTCC2597 Oceanicola batsensis SKA53 03101 Loktanella vestfoldensis SKA53 Rsph03000505 Rhodobacter sphaeroides ISM 15430 Roseovarius nubinhibens ISM PU1002 04436 Pelagibacter ubique HTCC1002 GOX0771 Gluconobacter oxydans ZM01411 Zmomonas mobilis y Saro02001148 Novosphingobium aromaticivorans a Sala 1452 RB2256 Sphinopyxis alaskensis Fur ELI1325 in a-proteobacteria Erythrobacter litoralis OA2633 10204 Oceanicaulis alexandrii HTCC2633 PB2503 04877 Parvularcula bermudensis HTCC2503 Regulator of iron uptake and metabolism genes CC0057 Caulobacter crescentus Rrub02001143 Rhodospirillum rubrum Amb1009 (I) Magnetospirillum magneticum a Amb4460 Magnetospirillum magneticum (II) Irr a-proteobacteria FUR/MUR branch of the FUR family

32. FUR and MUR boxes Erythrobacter litoralis Caulobacter crescentus Novosphingobium aromaticivorans Zymomonas mobilis Oceanicaulis alexandrii Sphinopyxis alaskensis Rhodospirillum rubrum Gluconobacter oxydans Parvularcula bermudensis - Magnetospirillum magneticum Identified Mur-binding sites Bacillus subtilis Sequence logos for the known Fur-binding sites in Escherichia coli and Bacillus subtilis Mur a of - proteobacteria - Escherichia coli

33. Irr branch of the FUR family Fur in g- and b- proteobacteria Escherichia coli ECOLI : P0A9A9 sp| Fur Pseudomonas aeruginosa : sp|Q03456 PSEAE Neisseria meningitidis : sp|P0A0S7 NEIMA Fur in e- proteobacteria HELPY Helicobacter pylori : sp|O25671 Fur in Firmicutes BACSU Bacillus subtilis : P54574 sp| a a-proteobacteria Mur / Fur AGR C 249 Agrobacterium tumefaciens SM irr Sinorhizobium meliloti RHE CH00106 Rhizobium etli RL irr1 Rhizobium leguminosarum (I) RL irr2 Rhizobium leguminosarum (II) MLr5570 Mesorhizobium loti MBNC03003186 sp. BNC1 Mesorhizobium BQ fur1 Bartonella quintana BMEI1955 Irrina-proteo- bacteria: regulator of iron homeostasis Brucella melitensis (I) BMEI1563 Brucella melitensis (II) BJ blr1216 (II) Bradyrhizobium japonicum RB2654 182 Rhodobacterales bacterium HTCC2654 SKA53 01126 Loktanella vestfoldensis SKA53 ROS217 15500 Roseovarius sp.217 ISM 00785 ISM Roseovarius nubinhibens OB2597 14726 Oceanicola batsensis HTCC2597 Jann 1652 sp. CC51 Jannaschia a I r r - Rsph03001693 Rhodobacter sphaeroides EE36 03493 Sulfitobacter sp. EE-36 STM1w01001534 sp. TM1040 Silicibacter MED193 17849 Roseobacter sp. MED193 SPOA0445 Silicibacter pomeroyi RC irr Rhodobacter capsulatus RPA2339 (I) Rhodopseudomonas palustris RPA0424* Rhodopseudomonas palustris (II) BJ irr* (I) Bradyrhizobium japonicum Nwi 0035* Nitrobacter winogradskyi Nham 1013* Nitrobacter hamburgensis X14 PU1002 04361 Pelagibacter ubique HTCC1002

34. Irr boxes Rhizobiaceae plus Bradyrhizobiaceae Rhodobacteriaceae Rhodospirillales

35. RirA/NsrR family (Rhizobiales)

36. IscR family

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

38. Reconstruction of history Frequent co-regulation with Irr Strict division of function with Irr Appearance of theiron-Rhodo motif

39. 2 All logos and Some Very Tempting Hypotheses: • Cross-recognition of FUR and IscR motifs in the ancestor. • 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. • Iron-Rhodo boxes are recognized by IscR: directly testable 1 3

40. 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.

41. Andrei A. Mironov – software, algorithms Alexandra Rakhmaninova – SDP, protein-DNA correlations Olga Kalinina (on loan to EMBL) – SDP Yuri Korostelev – protein-DNA correlations 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” People