Evolution of regulatory interactions in bacteria

Mikhail GelfandResearch and Training Center “Bioinformatics”,

Institute for Information Transmission Problems, RASMoscow, Russia

Singapore, 17-18 July 2006

Comparative genomics of regulation• Why

– Functional annotation of genes– Metabolic modeling– Practical applications in genetic engineering, drug targeting etc.

• How– Close genomes: phylogenetic footprinting.

Regulatory sites are seen as conservation islands in alignments of gene upstream regions

– Distant genomes: consistency filtering. Candidate sites in one genome may be unreliable, but independent occurrence upstream of orthologous genes in many genomes yields reliable predictions

• Caveats– Presense of (predicted) binding sites does not immediately imply functional regulation– Operon structure– Need to verify presence of orthologous transcription factors in the studied genomes– Orthologous factors may have different binding motifs– One functional system may be regulated by different factors within and between

genomes• Many genomes

– Taxon-specific regulation– Evolution

• individual sites• transcription-fator families• transcription factors and their binding motifs• simple and complex regulatory systems

How it works: Two simple examples

• Biotin regulator of alpha-proteobacteria

• Universal regulator of ribonucleotide reductases: reconstruction of the regulatory system and the mechanism of regulation

BirA (biotin regulator in eubacteria and archaea): conserved signal, changed spacing

Profile 2: Gram-negative bacteriaProfile 1: Gram-positive bacteria, Archaea

BirA (biotin regulator in eubacteria and archaea): conserved signal, changed spacing

Profile 2: Gram-negative bacteriaProfile 1: Gram-positive bacteria, Archaea

BirA of alpha-proteobacteria: no DNA-binding domain

Identification of the candidate regulator (BioR) in alpha-

proteobacteria

1. Candidate binding sites: similar palindromes upstream of biotin biosynthesis and transport genes in different genomes

TTATAGATAATTATCTATAATTATAGATAgTTATCTATAATTATCTATAATTATAGATAgTTATCTATAATcATATATtATcATAGATAgTTATCTATAATTATCTATAATTATCTATtATTATCTAcAATTATCTATAATTATCTATAATTATCTATAATcATAGATtAcTATAGATAATTATCTAcAA

1. 2. Positional clustering:

candidate transcription factor from the GntR family is often found in the same loci (black arrows)

3. Phyletic patterns: phyletic distribution of candidate sites (red cirsles) exactly coincides with the phyletic distribution of the candidate regulator

4. Autoregulation: in many cases there are candidate sites upstream of the bioR gene itself

Conserved signal upstream of nrd genes

Identification of the candidate regulator by the analysis of phyletic patterns

• COG1327: the only COG with exactly the same phylogenetic pattern as the signal– “large scale” on the level of major taxa– “small scale” within major taxa:

• absent in small parasites among alpha- and gamma-proteobacteria

• absent in Desulfovibrio spp. among delta-proteobacteria

• absent in Nostoc sp. among cyanobacteria

• absent in Oenococcus and Leuconostoc among Firmicutes

• present only in Treponema denticola among four spirochetes

COG1327 “Predicted transcriptional regulator, consists of a Zn-ribbon and ATP-cone domains”: regulator of the riboflavin pathway?

Additional evidence – 1

• nrdR is sometimes clustered with nrd genes or with replication genes dnaB, dnaI, polA

Additional evidence – 2

• In some genomes, candidate NrdR-binding sites are found upstream of other replication-related genes– dNTP salvage– topoisomerase I,

replication initiator dnaA, chromosome partitioning, DNA helicase II

Multiple sites (nrd genes): FNR, DnaA, NrdR

Mode of regulation

• Repressor (overlaps with promoters)• Co-operative binding:

– most sites occur in tandem (> 90% cases)– the distance between the copies (centers of

palindromes) equals an integer number of DNA turns:• mainly (94%) 30-33 bp, in 84% 31-32 bp – 3 turns• 21 bp (2 turns) in Vibrio spp.• 41-42 bp (4 turns) in some Firmicutes

• experimental confirmation in Streptomyces (Borovok et al., 2004)

Evolutionary processes that shape regulatory systems

• Expansion and contraction of regulons• 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

Loss of regulators, and cryptic sites

Loss of the RbsR in Y. pestis (ABC-transporter also is lost)

Start codon of rbsD

RbsR binding site

Regulon expansion: how FruR has become CRA

icdA

aceA

aceB

aceEF

pckA

ppsApykF

adhE

gpmApgk

tpiA

gapApfkAfbp

FructosefruKfruBA

eda

eddepd

Glucose

ptsHI-crr

Mannose

manXYZ

mtlDmtlAMannitol

Gamma-proteobacteria

Common ancestor of Enterobacteriales

icdA

aceA

aceB

aceEF

pckA

ppsApykF

adhE

gpmApgk

tpiA

gapApfkAfbp

FructosefruKfruBA

eda

eddepd

Glucose

ptsHI-crr

Mannose

manXYZ

mtlDmtlAMannitol

Gamma-proteobacteriaEnterobacteriales

Common ancestor of Escherichia and Salmonella

icdA

aceA

aceB

aceEF

pckA

ppsApykF

adhE

gpmApgk

tpiA

gapApfkAfbp

FructosefruKfruBA

eda

eddepd

Glucose

ptsHI-crr

Mannose

manXYZ

mtlDmtlAMannitol

Gamma-proteobacteriaEnterobacterialesE. coli and Salmonella spp.

Trehalose/maltose catabolism, alpha-proteobacteria

Duplicated LacI-family regulators: lineage-specific

post-duplication loss

The binding signals are very similar (the blue branch is somewhat different: to avoid cross-recognition?)

Utilization of an unknown galactoside, gamma-proteobacteria

Loss of regulator and merger of regulons: It seems that laci-X was present in the common ancestor (Klebsiella is an outgroup)

Yersinia and Klebsiella: two regulons, GalR (not shown, includes genes galK and galT) and Laci-X

Erwinia: one regulon, GalR

Utilization of maltose/maltodextrin, Firmicutes

Two different ABC transporters (shades of red)

PTS (pink)

Glucoside hydrolases (shades of green)

Two regulators (black and grey)

Modularity of the functional subsystem

Two different ABC systems

Three hydrolases in one operon (E. faecalis) or separately

Changes of regulation

Displacement: invasion of a regulator from a different subfamily (horizontal transfer

from a related species?) – blue sites

Orthologous TFs with completely different regulons

(alpha-proteobaceria and Xanthomonadales)

Catabolism of gluconate, proteobacteria

extreme variability of regulation of “marginal” regulon members

γ

Pseudomonas

spp

.

β

Combined regulatory network for iron homeostasis genes in -proteobacteria

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

The connecting line denote regulatory interactions, which the thickness reflecting the frequency of the interaction in the analyzed genomes. The suggested negative or positive mode of operation is shown by dead-end and arrow-end of the line.

Rhizobiales

Bradyrhizobiaceae

Rhizobiaceae

Rhodo-bacterales

Hyphomonadaceae

Rhodo-bacteraceae

Rickettsiales

Rhodo-spirillales

Sphingomo-nadales

- pro

teo

bacte

ria

Organism Irr MntR

Sinorhizobium meliloti

Rhizobium leguminosarum

Rhizobium etli

Agrobacterium tumefaciens

Mesorhizobium loti

Mesorhizobium sp. BNC1

Brucella melitensis

Bartonella quintana and spp.

Bradyrhizobium japonicum

Rhodopseudomonas palustris

Nitrobacter hamburgensis

Nitrobacter winogradskyi

Rhodobacter capsulatus

Rhodobacter sphaeroides

Silicibacter sp. TM1040

Silicibacter pomeroyi

Jannaschia sp.CC51

Rhodobacterales bacterium HTCC2654

Roseobacter sp. MED193

Roseovarius nubinhibens ISM

Roseovarius sp.217

Loktanella vestfoldensis SKA53

Sulfitobacter sp. EE-36

Oceanicola batsensis HTCC2597

Oceanicaulis alexandrii HTCC2633

Caulobacter crescentu s

Parvularcula bermudensis HTCC2503

Erythrobacter litoralis

Novosphingobium aromaticivorans

Sphinopyxis alaskensis g RB2256

Zymomonas mobilis

Gluconobacter oxydans

Rhodospirillum rubrum

Magnetospirillum magneticum

Pelagibacter ubique HTCC1002

SM +

MUR /

FUR RirA IscR

RL

RHE

AGR

ML

MBNC

BME

BQ

BJ

RPA

Nham

Nwi

RC

Rsph

STM

S PO

Jann

RB2654

MED193

ISM

ROS217

SKA53

EE36

OB2597

OA2633

CC

PB2503

ELI

Saro

Sala

ZM

GOX

Rrub

Amb

Abb.

PU1002

+ +- -

+ + +- -

+ + +- -

+ + +- -

+ + -

+ + +- -

+ + +- -

+ + +- -

+ + - -

+

+

+

-

-

+ + - --

+ + - --

+ + - --

+

+

+ ++- ++ ++ - +

+ ++ - +

+ ++ - +

+ + -

+ ++ - +

+ ++ - +

+ + - +

+ ++ - +

+ + - +

+ + - +

+ + - +

+ - +

#?

#?

#?

#?#?

- -

+ - +- -

+ - +- -

+ - +- -

+ - +- -

+ - +- -

+ - +- -

+ +- -

+ - +- -

+ - +- -

- +-

+

+

+

+

Group

Caulobacterales

Parvularculales

Rickettsia and Ehrlichia species - +- --

+ +SAR11 cluster

A.

B.

C.

D.

Fe and Mn regulons

Distribution of Irr,

Fur/Mur, MntR,

RirA, and IscR regulons

in α-proteobacteria

#?' in RirA column denotesthe absence of the rirA gene in an unfinished genomic sequence and the presence of candidate RirA-binding sites upstream of the iron uptake genes.

Phylogenetic tree of the Fur family of transcription factors in -proteobacteria - I

Fur in - and - proteobacteria

Fur in - proteobacteria Fur in Firmicutes

in proteobacteria

Fur

MBNC03003593

RB2654 19538AGR 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. TM1040

Silicibacter 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|P0A0S7

Pseudomonas aeruginosa : sp|Q03456Escherichia coli: P0A9A9sp|

Mur

Fur

Magnetospirillum magneticum (II)

RHE_CH00378 Rhizobium etli

PU1002 04436Pelagibacter ubique HTCC1002

Irr

in proteobacteria

proteobacteria

Regulator of manganese uptake genes (sit, mntH)

Regulator of iron uptake and metabolism genes

The A, B, and C groups

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 identified Fur-binding sites in the D group of proteobacteria

Sequence logos for the known Fur-binding sites in Escherichia coli and Bacillus subtilis

Identified Mur-binding sites

Phylogenetic tree of the Fur family of transcription factors in -proteobacteria - II

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 ISM

Roseovarius 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

Sequence logos for the identified Irr binding sites in -proteobacteria

(8 species) - IrrThe A group

The B group (4 species) - Irr

The C group (12 species) - Irr

Phylogenetic tree of the Rrf2 family of transcription factors in -proteobacteria

proteins with the conserved C-X(6-9)-C(4-6)-C motif within effector-responsive domain proteins without a cysteine triad motif

Iron repressor RirA (Rhizobium leguminosarum)

Nitrite/NO-sensing regulator NsrR (Nitrosomonas europeae, Escherichia coli)

Cysteine metabolism repressor CymR(Bacillus subtilis)

Iron-Sulfur cluster synthesis repressor IscR(Escherichia coli)

Positional clustering of rrf2-like genes with:iron uptake and storage genes;

Fe-S cluster synthesis operons;genes involved in nitrosative stress protection;

sulfate uptake/assimilation genes;thioredoxin reductase;

carboxymuconolactone decarboxylase-family genes;

hmc cytochrome operon

Cytochrome complex regulator Rrf2(Desulfovibrio vulgaris)

ZMO0116

GOX0099

Rrub02000219

ZMO0422

Sala_1236

ELI0458

Saro3534

DV Rrf2

OA2633_03246CC1866

Ricket.

Am

b3030

Rrub 02002540

PB2503_09884

STM_3629

MED193_04321

ISM_16015

OB2597_03589

RO

S2

17

_ 20

54

2RB

26

54

040

09

SKA53_

05183RC_0477

Rsph023725SPO2025

EE36_14302

EC IscR

RPA0663GOX1196

Amb0200Rrub_1115

Sa

la_2

595

Sa

r o02

00

1 62

0

CC

2 62

5

PB

250

3 _0

371 2

R rub02002859

RC_0031Rsph023756

AGR_C_1499

RHE_CH01133

RL_1316

AGR_L_2801SMb20994SMc02267

RHE_CH03364RL_3916

MLl4516MLr1674

Rrub02001767Amb1054

ROS217_16231STM_634

MED193_09800

SPO0432Rsph023178

RB2654_19993RC 0780

BQ04990MBNC02002196

MLr1147BMEII0707

AGR_C_344

RL RirA

SMc00785RHE CH00735

OA2633_11510

Nwi_0743

NE NsrR

Amb1318GOX0860RC NsrR

ROS217_15206Rsph03001477

EC

_Ns

rR

SPOA0186

Ricket.

Sala_1049Saro02000305

OB2597_05195ROS217_02155

ROS217_14291

CC0132

SMc01160

BJ blr7974

RL_5159AGR_L_2343

AGR_C_402

AGR_L_1131

SPO3722RHE_CH02777RL_3336

SPO1393

MBNC02000669MLl1642

SMc02238AGR_C_872

RL_619RHE_CH00547

MBNC03004487

RirA

NsrR

IscR

IscR-II

Rhizo biales

Rh o dob acterales

Jann_2366

BS CymR

The A group - RirA (8 species)

(12 species)The C group - RirA

Sequence logos for the identified RirA-binding sites in -proteobacteria

Genes Functions:Iron uptakeIron storageFeS synthesis

Iron usageHeme biosynthesisRegulatory genesManganese uptake

Distribution of the conserved members of the Fe- and Mn-responsive regulons and the predicted RirA, Fur/Mur, Irr, and DtxR binding sites in -proteobacteria

An attempt to reconstruct the history

Regulators and their signals

• Subtle changes at close evolutionary distances

• Cases of motif conservation at surprisingly large distances

• Correlation between contacting nucleotides and amino acid residues

DNA signals and protein-DNA interactions

CRP PurR

IHF TrpR

Entropy at aligned sites and the number of contacts (heavy atoms in a base pair at a distance <cutoff from a protein atom)

Specificity-determining positions in the LacI family

• Training set: 459 sequences, average length: 338 amino acids, 85 specificity groups

10 residues contact NPF (analog of the effector)

6 residues in the intersubunit contacts

7 residues contact the operator sequence

7 residues in the effector contact zone (5Ǻ<dmin<10Ǻ)

5 residues in the intersubunit contact zone (5Ǻ<dmin<10Ǻ)

6 residues in the operator contact zone (5Ǻ<dmin<10Ǻ)

– 44 SDPs

LacI from E.coli

The LacI family: subtle changes in signals at close distances

G

An

CGGn GC

CRP/FNR family of regulators

FNR

HcpR

CooA

Gam ma

Desulfovibrio

Desulfovibrio

TGTCGGCnnGCCGACA

TTGTgAnnnnnnTcACAA

TTGTGAnnnnnnTCACAA

TTGATnnnnATCAA

Correlation between contacting nucleotides and amino acid residues

• CooA in Desulfovibrio spp.• CRP in Gamma-proteobacteria• HcpR in Desulfovibrio spp. • FNR in Gamma-proteobacteria

DD COOA ALTTEQLSLHMGATRQTVSTLLNNLVRDV COOA ELTMEQLAGLVGTTRQTASTLLNDMIREC CRP KITRQEIGQIVGCSRETVGRILKMLEDYP CRP KXTRQEIGQIVGCSRETVGRILKMLEDVC CRP KITRQEIGQIVGCSRETVGRILKMLEEDD HCPR DVSKSLLAGVLGTARETLSRALAKLVEDV HCPR DVTKGLLAGLLGTARETLSRCLSRMVEEC FNR TMTRGDIGNYLGLTVETISRLLGRFQKYP FNR TMTRGDIGNYLGLTVETISRLLGRFQKVC FNR TMTRGDIGNYLGLTVETISRLLGRFQK

TGTCGGCnnGCCGACA

TTGTgAnnnnnnTcACAA

TTGTGAnnnnnnTCACAA

TTGATnnnnATCAA

Contacting residues: REnnnRTG: 1st arginineGA: glutamate and 2nd arginine

The correlation holds for

other factors in the family

Open problems

• Model the evolution of regulatory systems (a catalog of elementary events, estimates of probabilities)– Birth of a binding site; what are the mechanisms?– Loss of a binding site– Duplication of a regulated gene and/or a regulator– Horizontal transfer of a regulated gene and/or a regulator– Loss of structural a gene and/or a regulator– General properties?

• Distribution of TF family and regulon sizes• Stable cores and flexible margins of functional systems (in terms of gene

presence and regulation)

• Co-evolution of TFs and DNA sites: – “Neutral” model for the evolution of binding sites (with invariant functional

pressure from the bound protein)– How do the signals evolve? What is the driving force – changes in TFs?– TF-family, position-specific protein-DNA recognition code?

All that needs to take into account the incompleteness and noise in the data

Acknowledgements

• Andrei A. Mironov (algorithms and software)• Alexandra B. Rakhmaninova (SDPs)• Dmitry Rodionov (now at Burnham Institute) (BioR,

NrdR, iron)• Olga Laikova (LacI, sugars)• Dmitry Ravcheev (FruR)• Olga Kalinina (SDPs/LacI)

• Leonid Mirny, MIT (protein/DNA contacts, SDPs)• Andy Johnston, University of East Anglia (iron)

• Howard Hughes Medical Institute • Russian Fund of Basic Research• Russian Academy of Sciences, program “Molecular and Cellular Biology”• INTAS