Evolution of bacterial regulatory systems

Mikhail GelfandInstitute for Information Transmission Problems, RAS

BGRS-2004, Novosibirsk

Early analyses (BGRS’98, 00, 02)

“Making good predictions with bad rules”

Basic assumption: regulons are conserved =>

Consistency check: sites upstream of orthologous genes are correct; false positives are scattered at random

• Validation of individual sites• Validation of signals: candidate signals for

orthologous factors are correct if similar

Multiple genomes: taxon-specific regulation; multiple interacting systems; evolution of regulation

• Evolution of orthologous regulatory sites• Co-evolution of transcription factors

and their binding signals• Evolution of regulons

(sets of co-regulated genes)• Evolution of regulatory systems

Это – ряд наблюдений. В углу – тепло. Взгляд оставляет на вещи след. Вода представляет собой стекло. Человек страшней, чем его скелет.

Иосиф Бродский

A list of some observations. In a corner, it’s warm.A glance leaves an imprint on anything it’s dwelt on.Water is glass’s most public form.Man is more frightening than its skeleton.

Joseph Brodsky

Conservation of non-consensus positions in orthologous sites

regulatory site LexA lexAconsensus nucleotides are in caps

Escherichia coli TgCTGTATATActcACAGcA

Salmonella typhi aACTGTATATActcACAGcA

Yersinia pestis agCTGTATATActcACAGcA

Haemophilus influenzae atCTGTATAcAatacCAGTt

Pasteurella multocida TtCTGTATATAataACAGTt

Vibrio cholerae cACTGgATATActcACAGTc

wrong consensus?

PurR purLEscherichia coli ACGCAAACGgTTtCGT

Salmonella typhi ACGCAAACGgTTtCGT

Yersinia pestis ACGCAAACGgTTtCGT

Haemophilus influenzae AtGCAAACGTTTGCtT

Pasteurella multocida ACGCAAACGTTTtCGT

Vibrio cholerae ACGCAAACGgTTGCtT

PurR purMEscherichia coli tCGCAAACGTTTGCtT

Salmonella typhi tCGCAAACGTTTGCtT

Yersinia pestis tCGCAAACGTTTGCcT

Haemophilus influenzae tCGCAAACGTTTGCtT

Pasteurella multocida tCGCAAACGTTTGCtT

Vibrio cholerae ACGCAAACGTTTtCcT

Non-consensus positions are more conserved than synonymous codon positions

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Distance between genomes

Co

nse

rvat

ion

LexA non-cons.

Syn.2-fold

Syn. 3-fold

Syn. 4-fold

Non-consensus positions may be more conserved than consensus positions

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0,2

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Signal position (LexA)

Rel

ativ

e co

nser

vati

on

Non-consensus

Consensus

Regulators and their signals

• Subtle changes at close evolutionary distances

• Changes in spacing / geometry of dimers

• Correlation between contacting nucleotides and amino acid residues

• Cases of conservation at surprisingly large distances

Zinc repressorsnZUR-nZUR-

AdcRpZUR

TTAACYRGTTAA

GATATGTTATAACATATCGAAATGTTATANTATAACATTTC

GTAATGTAATAACATTAC

TAAATCGTAATNATTACGATTTA

Alignment of nZUR binding signals

GTAATGTAA TAACATTAC (alpha – most genera)GATATGTTA TAACATATC (alpha – Rhodobacter)GAAATGTTATANTATAACATTTC (gamma)

GaaATGTtA-----TAACATttC (consensus of consensi)

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

Factor Organisms Consensus Specific aa Metabolic system Inducer

CRP Enterobacteria&Vibrio&PasteurellaceaeTTGTGAnnnnnnTCACAA R E R catabolic repression cAMPVFR Pseudomonas sp. TTGTGAnnnnnnTCACAA R E R virulence cAMPCLP Xanthomonas&Xylella sp. nTGTGAnnnnnnTCACAn R E R phytopathogenicity ? (not cAMP)FNR & ANR Gamma-proteobacteria nnTTGATnnnnATCAAnn V E R response to anaerobiosis O2,NOFNR Beta-proteobacteria nnTTGATnnnnATCAAnn L E R response to anaerobiosis O2FNR & FixK Alpha-proteobacteria nnTTGATnnnnATCAAnn I/L E R nitrogen fixation O2DNR & Nnr Pseudomonas &Paracoccus nnTTGATnnnnATCAAnn P E R denitrification NO, NO2FNR Bacillus sp. nTGTGAnnTAnnTCACAn R E R response to anaerobiosis O2-low conditionsPrfA Listeria nnTTAACAnnTGTTAAnn S S R virulence ?NtcA Cyanobacteria ntGTAnCnnnnGnTACan R V R nitrogen metabolism 2-oxoglutarateCysR Cyanobacteria ? R V R sulfate utilization sulfate?CooA Desulfovibrio sp. and R.rubrum nTGTCGGCnnGCCGACAn R Q T CO utilization COHcpR* Desulfovibrio sp. TTGTgAnnnnnnTcACAA R E R prismane & sulfate reduction ?HcpR* Desulfuromonas acetoxidans, Desulfotalea psychrophilaatTTGAccnnggTCAAat S/P E R prismane ?HcpR* Clostridia, Bacteroides, Thermotogales, Fusobacteria, TreponemactGTAACawwtCTTACag R P R prismane ?HcpR* ~P. gingivalis nTGTCGCnnnnGCGACAn R A R prismane ?HcpR* ~C. difficile nnGGATnnnnnnATCCnn R S R prismane ?HcpR* ~T.tengcongensis, D.halfniensa nTGTGAnnnnnnTCACAn R E R prismane ?HcpR* ~Acidithiobacillus ferrooxidans nCTTGATTnnAATCAAGn P E R prismane ?ArcR Bacillus, Enterococcus sp. nTGTGAnATATnTCACAn R E A/S arginine catabolism O2CprK Desulfitobacterium dehalogenas nnTTAnTGnnCAnTAAnn H V R/K halorespiration aromaticsFlpA&B Lactococcus lactis nnTTGATnnnnATCAAnn P E R ? Eh, O2

The LacI family of transcrip-

tional regulators (each branch represents a subfamily)

1**

T

2* 9

****

4****

10****

6**

7***

8**

T G

3**

5**** 12

*

13****

15****

16****

17***

14*

11****

24*

19**

T

25****

21*

22****

23**

18****

20*

G

27****

28**

30***

31****

T

32**

29****

26*

38****

C

39****

41****

A

42****

T43

****

40****

37*

34****

35****

36****

33**

A

Each ortho logous group is reduced to a s ing le representa ive.The branch colour denotes the feeder pathw ay regula ted.The experim enta l data ava ilab le for at least one regula tor o f an orthologous group is show n by the type-face of species designations:

, and the branch outline th icknessexperim entally confirm ed sites,

experim entally confirm ed regulation (the th icker line indica te experim enta lly confirm ed pathw ay).

The Logo's num bering corresponds to the branch num bers o f the tree.

regulon pred icted de novosignal p roposed de novo,new regu lon m em bers proposednew sites predicted.

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EC_RbsR

EC_PurR

PA1949

BS_CcpA

TTE0201

R EF00754

R EF00345

BS_R bsR

SACR_LACLA

M ALR_STAXYBS_YvdE

SM b21598

SM c04260

RAFR_ECOLI

SM b20324

R R C 03428

SM c03060

EC_MalI

SM c04401

SM c02975

SM b21272

m lr2242SCRR_SALTY

RKP03067

EC_FruR

R KP05215SM b21650

GALR_STRTR

EC_EbgR

EC_TreR

VC A0654

SCRR_STAXY

SM b21372

PA2259

BS_KdgR

XC C 2369

EC_AscGSTM 1555

EC_GalS

EC_GalREC_CytR

CSCR_ECOLI

SM c03165

R Sc1790

R KP05499

R PU 04121

PA2320

EC_IdnREC_GntR

R R C 03254

STM 2345

STM 3696

EC _YcjW

EC_LacI

D -galactose & galactosidesm altose & trehalose

sucroseD -fructose

D -riboseD -xylose

… and their signals

BirA: regulator of biotin biosynthesis and transport in eubacteria and archaea

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

Evolution of regulons and regulatory systems

• conserved cores

• taxon-specific marginal members

• migration of genes between interacting regulatory systems

• taxon-specific cross-regulation

• genome-specific operons and genomic loci

• complete change of regulatory mechanisms

Genome loci for hyaluronate utilization in invasive Streptococcus spp.

IS

IS

IS

S. pyogenes, S. agalactiae

S. equi

S. pneumoniae TIGR4

S. suis

S. pneumoniae R6

Respiration in gamma-proteobacteria1. Three regulators, different regulatory cascades

Haemophilus ducreyi, Vibrio spp.

Haemophilus influenzae, Pasteurella multocida, A. actinomycetemcomitans

Escherichia coli(experimental data)

Respiration in gamma-proteobacteria2. New genome/taxon-specific regulon members

Escherichia coli (known) New, non-homologous regulon member

Yersinia pestis FnrFnr ArcAArcA ——Yersinia entercolitica FnrFnr —— — —Pasteurella multocida FnrFnr ArcAArcA NarPNarPActinobacillus actinomycetemcomitans —— ArcAArcA NarPNarP Haemophilus influenzae FnrFnr ArcAArcA —— Haemophilus ducreyi FnrFnr ArcAArcA NarPNarPVibrio vulnificus —— ArcAArcA ——Vibrio parahaemolyticus —— ArcAArcA ——Vibrio cholerae FnrFnr ArcAArcA ——Vibrio fischeri —— ArcAArcA ——

Respiration in gamma-proteobacteria3. New genome/taxon-specific regulon members, cont’d

Synthesis of molybdate cofactor

Yersinia pestis FnrFnr —— — —Yersinia entercolitica FnrFnr ArcAArcA — —Pasteurella multocida FnrFnr ArcAArcA ——Actinobacillus actinomycetemcomitans FnrFnr —— NarPNarP Haemophilus influenzae FnrFnr —— NarPNarP Haemophilus ducreyi FnrFnr ArcAArcA NarPNarP Vibrio vulnificus —— —— NarPNarP Vibrio parahaemolyticus —— —— NarPNarP Vibrio cholerae —— —— NarPNarP Vibrio fischeri —— ArcAArcA NarPNarP

Zinc repressors - recapitulationnZUR-nZUR-

AdcRpZUR

TTAACYRGTTAA

GATATGTTATAACATATCGAAATGTTATANTATAACATTTC

GTAATGTAATAACATTAC

TAAATCGTAATNATTACGATTTA

Five regulatory

systems for methionine

biosynthesis

A. SAM-dependent RNA riboswitch

B. Met-tRNA-dependent T-box (RNA)

C,D,E. repressors of transcription

Three methionine regulatory systems in Gram-positive bacteria: loss of S-box regulons

• S-boxes (riboswitch)– Bacillales– Clostridiales– the Zoo:

• Petrotoga

• actinobacteria (Streptomyces, Thermobifida)

• Chlorobium, Chloroflexus, Cytophaga

• Fusobacterium

• Deinococcus

• proteobacteria (Xanthomonas, Geobacter)

• Met-T-boxes (Met-tRNA-dependent attenuator)– Lactobacillales

• MET-boxes (candidate transcription signal)– Streptococcales

Lact. Strep. Bac. Clostr.

ZOO

Catabolism of gluconate in proteobacteria

Three regulatory systemsone global (FruR), two taxon-specific (GntR, PtxS)

β

γ1

Pse

udom

onas

spp

.

Instead of conclusions…

• Andrei A. Mironov (BGRS’98,00,02,04)• Anna Gerasimova (BGRS’02,04)• Olga Kalinina (BGRS’02,04)• Alexei Kazakov (BGRS’02,04)• Ekaterina Kotelnikova (BGRS’02,04)• Galina Kovaleva (BGRS’04)• Pavel Novichkov (BGRS’00,02,04)• Olga Laikova (BGRS’02,04)• Ekaterina Panina (BGRS’00)

(now at UCLA, USA)• Elizabeth Permina (BGRS’02,04)• Dmitry Ravcheev (BGRS’02,04)• Alexandra B. Rakhmaninova (BGRS’00)• Dmitry Rodionov (BGRS’00)• Alexey Vitreschak (BGRS’00,04)

(visiting LORIA, France)

• Howard Hughes Medical Institute

• Ludwig Institute of Cancer Research

• Russian Fund of Basic Research

• Programs “Origin and Evolution of the Biosphere” and “Molecular and Cellular Biology”, Russian Academy of Sciences