1. Life without Fur

2. Mikhail Gelfand Research and Training Center of Bioinformatics, Institute for Information Transmission Problems, RAS Genome Dynamics: From Replication to Post-Translation and Turnover HHMI, 11-14 March 2007 Life without FUR: evolutionary reconstruction of transcriptional regulation of iron homeostasis in alpha-proteobacteria

3. 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

4. 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

5. Comparative genomics of regulatory systems Standard methods: –BLAST –Construction of phylogenetic trees to identify orthologs –Functional annotation by similarity –Co-localization patterns Analysis of regulation: –Phylogenetic footprinting (Conserved motifs upstream of orthologs) –Consistency filtering (true sites upstream of orthologs; false positives scattered at random)

6. Distribution of transcription factors in genomes

7. 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

8. 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

9. 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

10. Irr boxes Rhizobiaceae plus Bradyrhizobiaceae Rhodobacteriaceae Rhodospirillales

11. RirA/NsrR family (Rhizobiales)

12. IscR family

13. Summary: regulation of genes in functional subsystems Rhizobiales Bradyrhizobiaceae Rhodobacteriales The Zoo (likely ancestral state)

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

15. Experimental validation RirA: sites and binding motif in Rhisobium legumisaurum (site-directed mutagenesis). Andy Johnston lab (University of East Anglia) Microarray study if the Bradyrhizobium japonicum FUR – mutant: regulatory cascade FUR  irr: Mark O’Brian group (SUNY, Buffalo)

16. 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

17. More stories Regulation of methionine metabolism in Firmicutes (from S-boxes to T-boxes and transcriptional factors) T-box regulon in Firmicutes (duplications, bursts, changes of specificity) Regulation of respiration in gamma-proteobacteria (rewiring of regulatory cascades and shuffling of regulons) Emerging global regulators in Enterobacteriaceae (how FruR has become CRA, and how duplicated RbsR has become PurR)

18. Acknowledgements Dmitry Rodionov (IITP, now at Burnham Institute, La Jolla, CA) Andrew Johnston and Jonathan Todd (University of East Anglia, UK) Howard Hughes Medical Institute Russian Academy of Sciences program “Molecular and Cellular Biology”