Life without

Fur

Mikhail Gelfand

Research and Training Center “Bioinformatics”, Institute for Information Transmission Problems, RAS

Russian-German Systems Biology Workshop

Moscow, February 27-29, 2008

Life without FUR: evolutionary reconstruction of

transcriptional regulation of iron homeostasis in alpha-

proteobacteria

Regulation of iron homeostasis (the Escherichia coli paradigm)

Iron:• 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

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 IrrFeS heme

RirA

degraded

FurFe

Fur

Iron uptake systems

Siderophoreuptake

Fe / Feuptake Transcription

factors

2+ 3+

Iron storage ferritins

FeS synthesis

Heme synthesis

Iron-requiring enzymes

[iron cofactor]

IscR

Irr

[- Fe] [+Fe]

[+Fe][- Fe]

[+Fe][ Fe]-

FeS

FeS statusof cell

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

Distribution of transcription

factors in genomes

FUR/MUR branch of the FUR familyFur in - and - proteobacteria

Fur in - proteobacteria Fur in Firmicutes

in proteobacteria

Fur

MBNC03003593RB2654 19538

AGR C 620

RL mur

Nwi 0013RPA0450

BJ furROS217 18337

Jann 1799SPO2477

STM1w01000993MED193 22541

OB2597 02997SKA53 03101Rsph03000505ISM 15430

GOX0771ZM01411

Saro02001148Sala 1452

ELI1325OA2633 10204

PB2503 04877CC0057

Rrub02001143Amb1009Amb4460

SM murMBNC03003179

BQ fur2BMEI0375

Mesorhizobium sp. BNC1 (I)

Sinorhizobium meliloti

Bartonella quintana

Rhodopseudomonas palustris

Bradyrhizobium japonicum

Caulobacter crescentus

Zmomonas mobilisy

Rhodobacter sphaeroides

Silicibacter sp. TM1040Silicibacter pomeroyi

Agrobacterium tumefaciens

Rhizobium leguminosarum

Brucella melitensis

Mesorhizobium sp. BNC1 (II)

Rhodobacterales bacterium HTCC2654

Nitrobacter winogradskyiNham 0990 Nitrobacter hamburgensis X14

Jannaschia sp. CC51Roseovarius sp.217

Roseobacter sp. MED193Oceanicola batsensis HTCC2597

Loktanella vestfoldensis SKA53

Roseovarius nubinhibens ISM

Gluconobacter oxydans

Erythrobacter litoralis

Novosphingobium aromaticivoransSphinopyxis alaskensis RB2256

Oceanicaulis alexandrii HTCC2633

Rhodospirillum rubrum

Parvularcula bermudensis HTCC2503

Magnetospirillum magneticum (I)

EE36 12413 Sulfitobacter sp. EE-36

ECOLIPSEAE

NEIMAHELPY

BACSUHelicobacter pylori : sp|O25671

Bacillus subtilis : P54574sp|

Neisseria meningitidis : sp|P0A0S7Pseudomonas aeruginosa : sp|Q03456

Escherichia coli: P0A9A9sp|

Mur

Fur

Magnetospirillum magneticum (II)

RHE_CH00378Rhizobium etli

PU1002 04436Pelagibacter ubique HTCC1002

Irr

in proteobacteria

proteobacteria

Regulator of manganese uptake genes (sit, mntH)

Regulator of iron uptake and metabolism genes

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 known Fur-binding sites in Escherichia coli and Bacillus subtilis

Identified Mur-binding sites

FUR and MUR

boxes

Fur in - and - proteobacteria

Fur in - proteobacteria Fur in Firmicutes

Irr in proteo-bacteria regulator of ironhomeostasis

proteobacteria Fur

ECOLIPSEAE

NEIMAHELPY

BACSUHelicobacter pylori : sp|O25671

Bacillus subtilis : P54574sp|

Neisseria meningitidis : sp|P0A0S7

Pseudomonas aeruginosa : sp|Q03456Escherichia coli : P0A9A9sp|

Mur /

Fur

Irr-

AGR C 249SM irr

RL irr1RL irr2

MLr5570MBNC03003186

BQ fur1BMEI1955BMEI1563BJ blr1216

RB2654 182SKA53 01126

ROS217 15500ISM 00785

OB2597 14726Jann 1652

Rsph03001693EE36 03493

STM1w01001534MED193 17849

SPOA0445RC irr

RPA2339RPA0424*

BJ irr*Nwi 0035*Nham 1013* Nitrobacter hamburgensis X14

Nitrobacter winogradskyi

Bradyrhizobium japonicum (I)

Agrobacterium tumefaciens

Rhizobium leguminosarum (I)

Mesorhizobium sp. BNC1

Sinorhizobium meliloti

Mesorhizobium loti

Bartonella quintanaBrucella melitensis (I)

Bradyrhizobium japonicum (II)

Rhodobacter sphaeroides

Rhodobacter capsulatusSilicibacter pomeroyi

Silicibacter sp. TM1040Roseobacter sp. MED193

Sulfitobacter sp. EE-36

Jannaschia sp. CC51Oceanicola batsensis HTCC2597Roseovarius nubinhibens ISMRoseovarius sp.217Loktanella vestfoldensis SKA53

Rhodobacterales bacterium HTCC2654

Rhizobium etliRHE CH00106

Rhizobium leguminosarum (II)

Brucella melitensis (II)

Rhodopseudomonas palustris (II)Rhodopseudomonas palustris (I)

PU1002 04361 Pelagibacter ubique HTCC1002

Irr branch of the FUR family

Irr boxes

Rhizobiaceae plus

Bradyrhizobiaceae

Rhodobacteriaceae

Rhodospirillales

RirA/NsrR family (Rhizobiales)

IscR family

Summary: regulation of

genes in functional

subsystemsRhizobiales

Bradyrhizobiaceae

Rhodobacteriales

The Zoo (likely ancestral state)

Reconstruction of history

Appearance of theiron-Rhodo motif

Frequent co-regulation

with Irr

Strict division of function

with Irr

Experimental validation

• RirA: sites and binding motifin 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:

1. Cross-recognition of FUR and IscR motifs in the ancestor.

2. 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.

3. Iron-Rhodo boxes are recognized by IscR: directly testable

1

2

3

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)

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.

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 Sciencesprogram “Molecular and Cellular Biology”