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Life without Fur
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Russian-German Systems Biology Workshop
Life without FUR: evolutionary reconstruction of transcriptional regulation of iron homeostasis in alpha-proteobacteria Mikhail Gelfand Research and Training Center “Bioinformatics”, Institute for Information Transmission Problems, RAS Russian-German Systems Biology Workshop Moscow, February 27-29, 2008
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Regulation of iron homeostasis (the Escherichia coli paradigm)
essential cofactor (limiting in many environments) dangerous at large concentrations FUR (Ferric Uptake Regulator: 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
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Regulation of iron homeostasis in α-proteobacteria
RirA Irr FeS heme degraded Fur Fe I r o n u p t a k e s y m S i d h F / T c f 2+ 3+ g H - q z [ ] IscR [- Fe] [+Fe] [ Fe] FeS status of cell Experimental studies: FUR/MUR: Bradyrhizobium, Rhizobium and Sinorhizobium RirA (Rrf2 family): Rhizobium and Sinorhizobium Irr (FUR family): Bradyrhizobium, Rhizobium and Brucella
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Comparative genomics of regulatory systems
Standard methods of comparative genomics: similarity search by BLAST Construction of phylogenetic trees to identify orthologs General functional annotation by similarity Assigning genes to functional subsystems using co-localization scores and phylogenetic profiles Analysis of regulation: Phylogenetic footprinting at short evolutionary distances: conserved motifs upstream of orthologs are likely sites Consistency filtering at longer distances: true sites occur upstream of orthologs; false positives scattered at random
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Distribution of transcription factors in genomes
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FUR/MUR branch of the FUR family
Fur in g- and b- proteobacteria Fur in e- proteobacteria Fur in Firmicutes in a-proteobacteria Fur MBNC RB AGR C 620 RL mur Nwi 0013 RPA0450 BJ fur ROS Jann 1799 SPO2477 STM1w MED OB SKA Rsph ISM 15430 GOX0771 ZM01411 Saro Sala 1452 ELI1325 OA PB CC0057 Rrub Amb1009 Amb4460 SM mur MBNC BQ fur2 BMEI0375 Mesorhizobium sp. BNC1 (I) Sinorhizobium meliloti Bartonella quintana Rhodopseudomonas palustris Bradyrhizobium japonicum Caulobacter crescentus Zmomonas mobilis y Rhodobacter sphaeroides Silicibacter sp. TM1040 Silicibacter pomeroyi Agrobacterium tumefaciens Rhizobium leguminosarum Brucella melitensis (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 bermudensis HTCC2503 Magnetospirillum magneticum EE Sulfitobacter sp. EE-36 ECOLI PSEAE NEIMA HELPY BACSU Helicobacter pylori : sp|O25671 Bacillus subtilis : P54574 sp| Neisseria meningitidis : sp|P0A0S7 Pseudomonas aeruginosa : sp|Q03456 Escherichia coli : P0A9A9 Mur a RHE_CH00378 Rhizobium etli PU Pelagibacter ubique HTCC1002 Irr a-proteobacteria Regulator of manganese uptake genes (sit, mntH) Regulator of iron uptake and metabolism genes
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FUR and MUR boxes Identified Mur-binding sites Sequence logos for Mur
Caulobacter crescentus Erythrobacter litoralis FUR and MUR boxes Zymomonas mobilis Novosphingobium aromaticivorans Oceanicaulis alexandrii Sphinopyxis alaskensis Gluconobacter oxydans Rhodospirillum rubrum Parvularcula bermudensis - Magnetospirillum magneticum Identified Mur-binding sites Bacillus subtilis Sequence logos for known Fur-binding sites in Escherichia coli and Bacillus subtilis Mur of - proteobacteria a Escherichia coli
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Irr branch of the FUR family
Fur in g- and b- proteobacteria Fur in e- proteobacteria Fur in Firmicutes Irr in a-proteo- bacteria regulator of iron homeostasis a-proteobacteria Fur ECOLI PSEAE NEIMA HELPY BACSU Helicobacter pylori : sp|O25671 Bacillus subtilis : P54574 sp| Neisseria meningitidis : sp|P0A0S7 Pseudomonas aeruginosa : sp|Q03456 Escherichia coli : P0A9A9 Mur / a I r - AGR C 249 SM irr RL irr1 RL irr2 MLr5570 MBNC BQ fur1 BMEI1955 BMEI1563 BJ blr1216 RB SKA ROS ISM 00785 OB Jann 1652 Rsph EE STM1w MED SPOA0445 RC irr RPA2339 RPA0424* BJ irr* Nwi 0035* Nham 1013* Nitrobacter hamburgensis X14 Nitrobacter winogradskyi Bradyrhizobium japonicum (I) Agrobacterium tumefaciens Rhizobium leguminosarum Mesorhizobium sp. BNC1 Sinorhizobium meliloti loti Bartonella quintana Brucella melitensis (II) Rhodobacter sphaeroides Rhodobacter capsulatus Silicibacter pomeroyi Silicibacter sp. TM1040 Roseobacter sp. MED193 Sulfitobacter sp. EE-36 Jannaschia sp. CC51 Oceanicola batsensis HTCC2597 Roseovarius nubinhibens ISM Roseovarius sp.217 Loktanella vestfoldensis SKA53 Rhodobacterales bacterium HTCC2654 Rhizobium etli RHE CH00106 Rhodopseudomonas palustris PU Pelagibacter ubique HTCC1002
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Irr boxes Rhizobiaceae plus Bradyrhizobiaceae Rhodobacteriaceae Rhodospirillales
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RirA/NsrR family (Rhizobiales)
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IscR family
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Summary: regulation of genes in functional subsystems
Rhizobiales Bradyrhizobiaceae Rhodobacteriales The Zoo (likely ancestral state)
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Reconstruction of history
Frequent co-regulation with Irr Strict division of function with Irr Appearance of the iron-Rhodo motif
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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)
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All logos and Some Very Tempting Hypotheses:
2 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
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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)
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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.
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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”
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