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 CASB-20, UCDS, La Jolla, 13-14.III.2009

2. Plan • Co-evolution of transcription factors and their binding motifs • Evolution of regulatory systems and regulons

3. 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

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

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

6. The CRP/FNR family of regulators

7. Correlation between contacting nucleotides and amino acid residues Contacting residues: REnnnR TG: 1st arginine GA: glutamate and 2nd arginine • 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

8. The correlation holds for other factors in the family

9. The LacI family: subtle changes in motifs at close distances G n A CG Gn GC

10. The LacI family: systematic analysis • 1369 DNA-binding domains in 200 orthologous rows<Id>=35%, <L>=71 а.о. • 4484 binding sites, L=20н., <Id>=45% • Calculate mutual information between columns of TF and site alignments • Set threshold on mutual information of correlated pairs

11. Sites LAFDHDQILQMAQERLQGKVRYQP-IGFELLPEKFSLRQLQRMYETVLGRS---LDKRNF tTAaTGgCTTTAtGcCACTAT LAFDHNQILDYGYQRLRNKLEYSP-IAFEVLPELFTLNDLFQLYTTVLGED--FADYSNF TTAaaGTAAtAaTTACCATAA LSFDHNEILAYGHRRLRNKLEYSP-VAFEVLPEMFTLNDLYQLYTTVLGEN--FSDYSNFLSFDHNEILAYGHRRLRNKLEYSP-VAFEVLPEMFTLNDLYQLYTTVLGEN--FSDYSNF AaAtTGTCTTTAtGcCACTAT TTATGGTAAATTcTACCATAA LAFDHSKILAYGHRRLCNKLEYSP-VAFDVLPEYFTLNDLYQFYSTVLGAN--FSDYSNFLAFDHSKILAYGHRRLCNKLEYSP-VAFDVLPEYFTLNDLYQFYSTVLGAN--FSDYSNFLAFDHSKILAYGHRRLCNKLEYSP-VAFDVLPEYFTLNDLYQFYSTVLGAN--FSDYSNF TTATGGTAAATTcTACCATAA TTATgGTCAgTTTcACcAaAA TTaGTCgAAATAaccaACtAA LAFDHNQILDYGYQRLRNKLEYSP-IAFEVLPELFTLNDLFQLYTTVLGED--FADYSNF TTATCGTCAtCtcGACGACAA LSFDHNEILAYGHRRLRNKLEYSP-VAFEVLPEMFTLNDLYQLYTTVLGEN—-FSDYSNFLSFDHNEILAYGHRRLRNKLEYSP-VAFEVLPEMFTLNDLYQLYTTVLGEN--FSDYSNF TttAGGTAAgTTATACTTTTA tTAaTGgCTTTAtGcCACTAT Mutual information Z-score Definitions Protein alignment

12. Correlated pairs

13. -ATIKDVAKRANVSTTTV- AATTGTGAGCGCTCACT Higher order correlations SL SQ TL TQ

14. Not a phylogenetic trace

15. NrtR (regulator of NAD metabolism)

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

17. 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

18. 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

19. 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

20. 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

21. 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.

22. 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)

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

24. … caused by loss of transcription factor AhrC

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

26. Blow-up 1

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

28. … 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

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

30. T-boxes: Summary / History

31. Life without Fur

32. 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

33. [+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

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

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

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

37. 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

38. 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.

39. Andrei A. Mironov – software, algorithms Alexandra Rakhmaninova – SDP, protein-DNA correlations Anna Gerasimova (now at LBNL) – NadR 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