Life without Fur Life without FUR evolutionary reconstruction

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Life without Fur

Life without Fur

Life without FUR: evolutionary reconstruction of transcriptional regulation of iron homeostasis in alpha-proteobacteria Mikhail

Life without FUR: evolutionary reconstruction of transcriptional regulation of iron homeostasis in alpha-proteobacteria 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

Regulation of iron homeostasis (the Escherichia coli paradigm) Iron: • essential cofactor (limiting in

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

Regulation of iron homeostasis in α-proteobacteria [- Fe] [+Fe] [ - Fe] [+Fe] Rir.

Regulation of iron homeostasis in α-proteobacteria [- Fe] [+Fe] [ - Fe] [+Fe] Rir. A Irr Fe. S heme degraded Siderophore uptake 2+ 3+ Fe / Fe uptake Iron uptake systems Fur [- Fe] Iron storage ferritins Fe. S synthesis Heme synthesis Iron-requiring enzymes [iron cofactor] Fur Isc. R Fe Fe. S Transcription factors Fe. S status of cell [+Fe] Experimental studies: • FUR/MUR: Bradyrhizobium, Rhizobium and Sinorhizobium • Rir. A (Rrf 2 family): Rhizobium and Sinorhizobium • Irr (FUR family): Bradyrhizobium, Rhizobium and Brucella

Comparative genomics of regulatory systems • Standard methods: – – BLAST Construction of phylogenetic

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)

Distribution of transcription factors in genomes

Distribution of transcription factors in genomes

FUR/MUR branch of the FUR family Fur a sp| Escherichia coli: P 0 A

FUR/MUR branch of the FUR family Fur a sp| Escherichia coli: P 0 A 9 A 9 ECOLI Pseudomonas aeruginosa : sp|Q 03456 PSEAE NEIMA Fur in g- and b- proteobacteria Neisseria meningitidis : sp|P 0 A 0 S 7 HELPY Helicobacter pylori P 54574 BACSU Bacillus subtilis : sp|O 25671 SM mur Sinorhizobium meliloti Mesorhizobium sp. BNC 1 (I) MBNC 03003179 BQ fur 2 Bartonella quintana Brucella melitensis BMEI 0375 EE 36 12413 Sulfitobacter sp. EE-36 MBNC 03003593 Mesorhizobium sp. BNC 1 (II) Rhodobacterales bacterium HTCC 2654 RB 2654 19538 Agrobacterium tumefaciens AGR C 620 RHE_CH 00378 Rhizobium etli Rhizobium leguminosarum RL mur Nham 0990 Nitrobacter hamburgensis X 14 Nwi 0013 Nitrobacter winogradskyi Rhodopseudomonas palustris RPA 0450 Bradyrhizobium japonicum BJ fur Roseovarius sp. 217 ROS 217 18337 Jannaschia sp. CC 51 Jann 1799 Silicibacter pomeroyi SPO 2477 STM 1 w 01000993 Silicibacter sp. TM 1040 MED 193 22541 Roseobacter sp. MED 193 OB 2597 02997 Oceanicola batsensis. HTCC 2597 Loktanella vestfoldensis. SKA 53 03101 Rhodobacter sphaeroides Rsph 03000505 Roseovarius nubinhibens. ISM 15430 PU 1002 04436 Pelagibacter ubique. HTCC 1002 GOX 0771 Gluconobacter oxydans Zmomonas y mobilis ZM 01411 Saro 02001148 Novosphingobium aromaticivorans Sphinopyxis alaskensis RB 2256 Sala 1452 ELI 1325 Erythrobacter litoralis Oceanicaulis alexandrii HTCC 2633 OA 2633 10204 PB 2503 04877 Parvularcula bermudensis HTCC 2503 Caulobacter crescentus CC 0057 Rhodospirillum rubrum Rrub 02001143 Magnetospirillum magneticum (I) Amb 1009 Magnetospirillum magneticum(II) Amb 4460 Fur in e- proteobacteria Fur in Firmicutes Mur in a-proteobacteria Regulator of manganese uptake genes (sit, mnt. H) Fur a in a-proteobacteria Regulator of iron uptake and metabolism genes Irr a a-proteobacteria

Erythrobacter litoralis Caulobacter crescentus Novosphingobium aromaticivorans Zymomonas mobilis Oceanicaulis alexandrii Sphinopyxis alaskensis Gluconobacter oxydans

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

Irr branch of the FUR family Fur Escherichia coli : P 0 A 9

Irr branch of the FUR family Fur Escherichia coli : P 0 A 9 A 9 sp| ECOLI Pseudomonas aeruginosa : sp|Q 03456 PSEAE NEIMA Fur in g- and b- proteobacteria Neisseria meningitidis : sp|P 0 A 0 S 7 HELPY Helicobacter pylori sp| BACSU Bacillus subtilis : P 54574 : sp|O 25671 Fur in e- proteobacteria Fur in Firmicutes a-proteobacteria a Mur / Fur Irr- a Agrobacterium tumefaciens AGR C 249 Sinorhizobium meliloti SM irr Rhizobium etli RHE CH 00106 Rhizobium leguminosarum (I) RL irr 1 RL irr 2 Rhizobium leguminosarum (II) Mesorhizobium loti MLr 5570 MBNC 03003186 Mesorhizobium sp. BNC 1 BQ fur 1 Bartonella quintana Brucella melitensis (I) BMEI 1955 Brucella melitensis (II) BMEI 1563 BJ blr 1216 Bradyrhizobium japonicum (II) RB 2654 182 Rhodobacterales bacterium HTCC 2654 Loktanella vestfoldensis SKA 53 01126 Roseovarius sp. 217 ROS 217 15500 Roseovarius nubinhibens ISM 00785 OB 2597 14726 Oceanicola batsensis HTCC 2597 Jann 1652 Jannaschia sp. CC 51 Rsph 03001693 Rhodobacter sphaeroides Sulfitobacter sp. EE-36 EE 36 03493 STM 1 w 01001534 Silicibacter sp. TM 1040 Roseobacter sp. MED 193 17849 Silicibacter pomeroyi SPOA 0445 Rhodobacter capsulatus RC irr RPA 2339 Rhodopseudomonas palustris (I) RPA 0424* Rhodopseudomonas palustris (II) Bradyrhizobium japonicum (I) BJ irr* Nwi 0035* Nitrobacter winogradskyi Nham 1013* Nitrobacter hamburgensis X 14 PU 1002 04361 Pelagibacter ubique HTCC 1002 Irr in a-proteobacteria regulator of iron homeostasis

Irr boxes Rhizobiaceae plus Bradyrhizobiaceae Rhodobacteriaceae Rhodospirillales

Irr boxes Rhizobiaceae plus Bradyrhizobiaceae Rhodobacteriaceae Rhodospirillales

Rir. A/Nsr. R family (Rhizobiales)

Rir. A/Nsr. R family (Rhizobiales)

Isc. R family

Isc. R family

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

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

Reconstruction of history Frequent co-regulation with Irr Strict division of function with Irr Appearance

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

Experimental validation • Rir. A: sites and binding motif in Rhisobium legumisaurum (site-directed mutagenesis).

Experimental validation • Rir. A: 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)

All logos and Some Very Tempting Hypotheses: • Cross-recognition of FUR and Isc. R

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

More stories • Regulation of methionine metabolism in Firmicutes (from S-boxes to T-boxes and

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 Fru. R has become CRA, and how duplicated Rbs. R has become Pur. R)

Acknowledgements • Dmitry Rodionov (IITP, now at Burnham Institute, La Jolla, CA) • Andrew

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”