Evolution of bacterial regulatory sytsems

Mikhail Gelfand

Research and Training Center “Bioinformatics”Institute for Information Transmission

ProblemsMoscow, Russia

Institute of Protein Research40th Anniversary Conference

June 2007

Comparative genomics of zinc regulons

Two major roles of zinc in bacteria: • Structural role in DNA polymerases,

primases, ribosomal proteins, etc.• Catalytic role in metal proteases and

other enzymesPoisonous in large concentrations=> the concentration of zinc is

tightly controlled

Regulators (zinc uptake) and motives nZUR-nZUR-

AdcRpZUR

TTAACYRGTTAA

GATATGTTATAACATATCGAAATGTTATANTATAACATTTC

GTAATGTAATAACATTAC

TAAATCGTAATNATTACGATTTA

Predictions • Known transporters – Orthologs of the AdcABC

and YciC transport systems– Paralogs of the

components of the AdcABC and YciC transport systems

• Candidate transporters with previously unknown specificity– ZinT is a new type of zinc-

binding component of zinc ABC transporter

• PHT (pneumococcal histidine triad) proteins of Streptococcus spp.– PHT proteins are adhesins

involved in the attachment of streptococci to epithelium cells, leading to invasion. This process is regulated by zinc concentration.

TM Zn adcA

zinT

S. pneumoniae S. equi

S. agalactiae

lmb phtD phtE

phtBphtA

lmb phtD

S. pyogenes

phtY

lmb phtD

zinc regulation shown in experiment

Zinc and (paralogs of) ribosomal proteins

L36 L33 L31 S14E. coli, S.typhi – – – + –K. pneumoniae – – – – –Y. pestis,V. cholerae – – – + –B subtilis – – + – – + – +S. aureus – – – – – – +Listeria spp. – – – – – +E. faecalis – – – – – – + –S. pne., S. mutans – – – – – –S. pyo., L. lactis – – – – – – +

nZ

UR

pZ

UR

Ad

cR

Zn-ribbon motif (Makarova-Ponomarev-Koonin, 2001)

L36 L33 L31 S14E. coli, S.typhi (–) – (–) + –K. pneumoniae (–) – (–) – –Y. pestis,V. cholerae (–) – (–) + –B subtilis (–) (–) + – (–) + (–) +S. aureus (–) (–) – – – (–) +Listeria spp. (–) (–) – – (–) +E. faecalis (–) (–) – – – (–) + –S. pne., S. mutans (–) (–) – – – (–)S. pyo., L. lactis (–) (–) – – – (–) +

nZ

UR

pZ

UR

Ad

cR

Summary of observations:

• Makarova-Ponomarev-Koonin, 2001:– L36, L33, L31, S14 are the only ribosomal proteins

duplicated in more than one species– L36, L33, L31, S14 are four out of seven ribosomal

proteins that contain the zinc-ribbon motif (four cysteines)

– Out of two (or more) copies of the L36, L33, L31, S14 proteins, one usually contains zinc-ribbon, while the other has eliminated it

• Among genes encoding paralogs of ribosomal proteins, there is (almost) always one gene regulated by a zinc repressor, and the corresponding protein never has a zinc ribbon motif

Zinc starvation and its consequences

Zn-rich conditions: sufficient Zn for the ribosomes and the enzymes

Good scenario: some ribosomes without Zn, some Zn left for the enzymes

Bad scenario: all Zn utilized by the ribosomes, no Zn for Zn-dependent enzymes

Regulatory mechanism

ribosomes

Zn-dependentenzymes

R

Sufficient Zn

Zn starvation

R

repressor

Prediction … (Proc Natl Acad Sci U S A. 2003 Aug 19;100(17):9912-7.)

… and confirmation (Mol Microbiol. 2004 Apr;52(1):273-83.)

T-boxes: the mechanism (Grundy & Henkin; Putzer & Grunberg-

Manago)

Terminator(underlined) ===========> <===========

Antiterminator ==> ===> <===<== SA serS -> 26 CGTTA 51 AAATAGGGTGGCAACGCGTAGAC------------CACGTCCCTTGTAGGGATGTGGTCTTTTTTTA DHA tyrZ -> 47 CGTTA 65 AGGTAAGGTGGTAACACGGGAGCA-------TACTCTCGTCCTTCTGGCAATGAAGGACGGGAGTTTTTTGTTTT ST trpS -> 37 CCTTA 61 AATTGAGGTGGTACCGCGTATTACTT----GTAATAACGCCCTCACGTTTTAATAGCGTGGGGACTTTTTGCTAT CA aspS -> 39 CGTTA 34 ATAAAGGATGGCACCGTGAAAA----------GCCTTCACTCCTTACTGGAGTGGAGGCTTTTTTTATTTTAAATAAA DF valS -> 41 CGTTA 77 AATTAAGGTGGTAACGCGAGC------------TTTTCGTCCTTTTTAAAGAGGATGAAGAGCTCTTTTTTATTTCT PN thrS -> 30 CGTTA 38 AATGAAGGTGGAACCACGTTG-------------CGACGTCCTTTCGAGGATGTCGCATTTTTTTATTAG MN ileS -> 89 CGTTA 68 AATTAAGGTGGTACCACGAGC-------------TTTCGTCCTTTGATGAAAGTTCTTTTTTATTGAT DF leuS -> 28 AGCTA 29 AATTAGGGTGGTACCGCGAAGATT-------TATCCTCGTCCCTAAACGTAAGTTTAGTGACGAGGATTTTTTATTTTCA HD argS -> 41 CGTTA 27 AACGAGAGTGGTACCGCGGGTAA---------AAGCTCGCCTCTTTTTAGAAGAGGCGGGTTTTTTATTTT DF proS -> 33 CGTTA 30 AACTAGAGTGGTACCGCGGAAAT-----TAAACCTTTCGTCTCTATACTTGTATAGAGATGAGAGGTTTTTTATATTTTCAGGA ZC lysS -> 46 CGTTA 63 AACTGAGGTGGTACCGCGAAGCTAA-----CAACTCTCGTCCTCAAGATGAATAATCTTGGGGGTGGGAGTTTTTTTGTTGCAT BQ metS -> 55 CGTTA 66 AAATAAGGTGGTACCGCGACTGTTTA---TACAGCCCCGCCCTTATCTTTTTTAGATAAGGGCGGGGCTTTTTATATTTAA MN pheS -> 14 AATTA 20 AAAACGGATGGTACCGCGTGTC-------------AACGCTCCGCTTAAGGAGTTTTGGCACTTTTTTTGTTTT MN glyQ -> 14 AGCTA 23 AATTAGGGTGGAACCGCGTTT------------CAAACGCCCCTATGTCAGTTGGCATGGGAGTGATTGAGCGTGGCTCTTTT ST alaS -> 20 AATTA 18 AATAGAGGTGGTACCGCGGTT--------------TTCGCCCTCTGTGAGATGGACTTGTTTTGTATGGAGGACTATTTGAAA SA trpE -> 32 AATTA 4 AACTAAGGTGGCACCACGGTA-------------ACGCGTCCTTACAGGTATATGCGTTATGTGGTGTCTTTTT BS ilvB -> 50 CGTTA 47 AACAAGGGTGGTACCGCGGAAAGAAA---AGCCTTTTCGCCCCTTTTAGCTATCGCAGTTACTGCGCGGCTGATTGT CA ilvC -> 40 CGTTA 14 AATTTGGGTGGTACCGCGCGACCAAA-----AATTCTCGCCCCAAGCAGGGAATTTTGGCCGTTTTTTTATATAAATAAAT BQ asnA -> 51 CGTTA 62 AATTTGGGTGGTACCGCGGAACC-----AAAGCCTTTCGTCCCAGTTTTTTGGGAAAGAAGGGCTTTTTTTGTTGGCTT BS proB -> 33 CGTTA 30 AATCAAGGTGGTACCACGGAAAC--------CCATTTCGTCCTTATGAATCAGGATGAAATGGGTTTTTTTATTGTAGA SA cysE -> 33 CATTA 62 ATTCAGAGTGGAACCGTGCGG-------------AAGCGCCTCTAACAATACAATTTGTATGTTAGTGGTGCTTTTTTG MN hisC -> 46 CGTTA 50 AATGAAGGTGGAACCACGTGTGT---------GTCAGCGTCCTTGCAAGTTTTTTGCAAGGGCGCTTTTTTGAATAGT DHA pheA -> 41 CGTTA 50 AAAAAGGGTGGTACCGCGTGAC---------TTAACTCGTCCCTTATTTGGGGGTGAGGTAAGTCTTTTTTTATTTA HD serA -> 42 cgtta 57 AATGAGGGTGGCACCGCGGTATG-------AACCTTCCGCCCCTCACGACAGTCGTCGTGTGGGCAGAAGGTTTTTTTACTATCA BQ phhA -> 51 CGTTA 34 AAATAGGGTGGTACCGCGATTC------------TTTCGCCCCTATCGGATTTTCCGATAGGGGCTTTTTCTATTTC EF yxjH -> 40 CGTTA 51 AAAAAAGGTGGTACCGCGATAA-----------TAATCGCCCTTTTACTAGTTACGGCTAGTAAAAGGGCGTTTTTTTATAAA CA yckK -> 38 CGTTA 57 AATTAGAGTGGTACCGTGGAATT-------CAACTTCTGCCTCTAACTATGAGGATAGAAGTTTTTTGTTTTTAT DF yqiX -> 41 CCTTA 30 AAAAAGAGTGGTAACGCGGATAT----------AATTCGTCTCTTAGCTGTAAAGCTAAGGGACTTTTTTGATTTA HD BH0807->74 TGTTA 56 AACTGGGGTGGCACCACGACAAG----------TGATCGTCCCCAAGACTTTTATCAGTCTTGGGGACGTTTTTTTGTTCAT EF yheL -> 8 AATTA 33 AATTAAGGTGGTACCGCGGAGA-----------GATTCGTCCTTATTCTTTAAGGATGAATCTCTCTTTTTATGTAGC BQ ykbA -> 46 CGTTA 45 AACAAGGGTGGAACCACGAATAT--------AACACTCGTCCCTTTTTTAGGGAGGAGTGTTTTTTTATT BQ sdt2 -> 40 CGTTA 56 AATTGAGGTGGTACCACGGTATTAACATTACATATATCGTCCTCTACATGCATATTTGCGTGTAGGGGACTTTTTTATTTTC EF yusC -> 42 CGTTA 60 AATTAAGGTGGTATCACGAAATGA-----CAAACTTTCGTCCTTTTTGCTGTAATAGCAAAAGGATGGAAGTTTTTTTGTTT CA yhaG -> 48 CGTTA 51 AATTTAGGTGGTACCGCGGAAGT---------ATCTCCGTCCTAATTAATAAGATTAGGGCGGAGTTTTTTATTTGC BQ brnQ -> 44 CGTTA 66 AATTAGGGTGGTATCGCGGGTAAA------TATAACTCGTCCCTTTCTTTAGGGACGAGTTTTTTGTGTTCTT REF01723 -> 44 CGTTA 55 AATTGAGGTGGCACCACGAATGC----------GATTCGTCCTCTTGGCTCACAGCCAAGAGGCTTTTTTGTTTTTTTAATA BS yvbW -> 56 CGTTA 32 AACAAGAGTGGTACCGCGGTCAGC--CGAAGGCTCGTCGTCTCTTTATCTATTAGATTAGGTAGGAGACGGCGGGCTTTTTT

Aminoacyl-tRNA synthetases

Amino acid biosynthetic genes

Amino acid transporters

TGG: T-box

Partial alignment of predicted T-boxes

Aminoacyl-tRNA synthetases

Amino acid biosynthetic genes

Amino acid transporters

… continued (in the 5’ direction)

anti-anti (specifier) codon specifier hairpin

===> ==> ===> <=== <== SC<=== SA SERS SER ---GTAGGACAAGTA 19 AGAGAGCTTGTGGTT---AGTGTGAACAAG--- 15 GAA--TCTACCTACTT -> DHA tyrZ Tyr ----AAGAACAAGTA 18 AGAAAGTTGCCGGCT---GATGAGAGGCGCTT 18 GAA--TACCTCTTTGA -> ST trpS Trp ---ATTAGAAGAGTA 16 AGAGAGTTAGTGGTT---GGTGCAAGCTAAC- 12 GAAA-TGGACTAATGA -> CA ASPS ASP -----GAGAAAAGTA 18 AGCGAATTGGGAAAT---GGTGTGAGCCCAA- 15 GAAA-GACATCTCGGA -> DF VALS VAL -GAAGAAGAGGAGTA 16 AGAGAGGAAAATTCACTGGCTGTAAGATTTTC 17 GAAT-GTAGCTTTGGA -> PN THRS THR ----AGAGACAAGTC 18 AGAGAGTGCGTGGTT---GCTGGAAACGCAT- 14 GAT--ACTACTCTTGA -> MN ileS Ile ----CAAAAACACAA 17 AGCGAATAGGTGAT----GGTGTAAGACCTATT 18 -----ATCATTTTGTT -> DF leuS Leu ----CTAGAGCAGTA 19 AGAGGAAGTGGAA-----GGTGAGAACTAATATT 10 GAA--CTTACTAGATT -> HD ARGS ARG -----TGGGAGAGTA 20 AGCGAGTCGGGAT-----GGTGGGAGCCGAT- 14 GAAA-CGCACCCATGA -> DF proS Pro ---AAAGAAATAGTA 18 AGAGAGAAAACGGT----GGTGAGAGTTTTC-- 14 GAA--CCTGTCTTTTA -> ZC lysS Lys ---AAGAGAAGAGTA 19 AGAGAGCTCTGGTA----GCTGAGAAAGAGC-- 15 GAAAAAAGACTTGGAG -> BQ metS Met ---AAAGGAAAAGTA 19 AGAGAGCTTCGGTA----GCTGAGAAGAAGC-- 14 GAACAATGGCCTTTGA -> MN pheS Phe ----TGAGATTAGTA 18 AGGGAATGCGGGGCGTG-ACTGGAAACCCGC- 16 GAA--TTCACTCAGAA -> MN glyQ Gly ---AGAAAGAGAGTT 15 AGCGAACCTGAGAG----AGTGTAAGTCAGGT 14 GACT-GGCACTTTCTC -> ST alaS Ala -AGTTAAGAATTGTT 17 AGAAAAGTGACGGTT---GCTGCGAGTCATT- 17 -----GCTACTTAACT ->

SA trpE Trp TCTAAAGAAATAGTA 22 AGAAAGCTAATGGGT---GATGGGAATTAGC-- 14 GAAT-TGGACTTTGGA -> BS ilvB Leu ---TGAGGATAAGTA 20 AGAGAACCGGGTTA----GCTGAGAACCGG--- 16 GAA--CTCGCCTCAGA -> CA ilvC Val -----AGGAAGAGTA 17 AGAGAGTGAGATACT---GGTGGGAACTCAT-- 13 GAAG-GTAGCCTTTGA -> BQ asnA Asn --AGGACGAGTAGTA 15 AGCGAGTCAGGGGT----GGTGTGAGCCTGA-- 15 GAAG-AACCTCCTGGA -> BS proB Pro -----AGGATTAGTA 18 AGAGAGCAAAATGAACC-GCTGAAACATTTTGC 15 GAA--CCTGCCTTGGA -> SA cysE Cys --CGAAGGATTAGTA 18 AGAGAGTGTACGGTT---GCTGTGAGTACA--- 14 GAA--TGCACCTTCGT -> MN hisC His -----AGAGAAAAAA 16 AGAGAGTATGGGAA----GCTGAAAACATAC-- 15 -----CACATTCTTGA -> DHA pheA Phe -----AAAGAGAGCA 19 AGGGAACTAAAGTCGGAGACTGAAAGCTTTAGT 14 GAGA-TTCACTCTGGA -> HD serA Ser ----GAAGATGAGGA 17 AGAGAGCTGGTGGTT---GCTGTGAACCAGCT- 18 -----AGCCCTTCTGA -> BQ phhA Tyr AGAATCGCAGTAGTA 17 AGAGAGCTAATGGTC---GGTGGAAATTGGC-- 14 GAAT-TACAATTCTGG -> EF yxjH Met -----TAGGAAAGTA 17 AGAGAGACTTTGGTT---GGTGAAAAAAGTT-- 13 GAAAAATGGCCTAGGA ->

CA yckK Cys ----AAGAACCAGTA 17 AGAGAAAAATCTCCAAG-GCTGAAAGGGATTTT 15 GAA--TGCATCTTTGA -> DF yqiX Arg -----AGAGAAAGTA 16 AGCGAGTTAGGGGTT---GGTGTAAGCCTAGC- 14 GAAG-AGAGCTCTGGA -> HD BH0807 Lys ----AGAGAAGAGTA 19 AGAAAGCCTGTAGTT---GCTGAGAACGGGT-- 14 GAAGCAAGACTCTGAG -> EF yheL Tyr -TTATTAGCCCAGTA 19 AGAAAGTCGATGGTT---GCTGCGAATCGAT-- 13 GAAT-TACACTAATAA -> BQ ykbA Thr --GAGGACACGATCA 16 AGAGAGGGAAGCCTTTG-GCTGTGAGCTTCCT- 14 GATT-ACCACCTCTGA -> BQ sdt2 Trp ---GCAAGAAGAGTA 18 AGAGAGCTGGGGGAA---GGTGTGAGCCCGGT- 15 GAA--TGGGCTTGCGA -> EF yusC Met ----AAAGAAGAGTA 18 AGAGAGCCCTGTTT----GCTGAGAATGGG--- 16 GAAG-ATGGTCTTTGA -> CA yhaG Trp ----AAGGAAGAGTA 18 AGAGAGCTGAGGGT----GGTGTGATCTCAGT- 15 GAA--TGGACCTTTTA -> BQ brnQ Ile ----GAGAACGAGTA 19 AGAGAGTTGGCGATTT--GCTGAAAGCCAAC-- 15 GAAA-ATCATCTCCGA -> REF01723 His --TTAGGACATAGTA 18 AGAGACTTTTTCATTG--GCTGAAAGAAAAAG- 17 -----CACACCTAAAA -> BS yvbW Leu -----GGGAGCAGTA 18 AGAGAGCTGCGGGGT---GGTGCGACGCAGC-- 13 GAA--CTCGCCCGGGA ->

Why T-boxes?

• May be easily identified• In most cases functional specificity may

be reliably predicted by the analysis of specifier codons (anti-anti-codons)

• Sufficiently long to retain phylogenetic signal

• Thus T-boxes are a good model of regulatory evolution

~800 T-boxes in ~90 bacteria• Firmicutes

– aa-tRNA synthetases– enzymes– transporters– all amino acids excluding glutamate

(lysine and glutamine – rare)

• Actinobacteria (regulation of translation) – branched chain (ileS)– aromatic (Atopobium minutum)

• Delta-proteobacteria – branched chain (leu – enzymes)

• Thermus/Deinococcus group (aa-tRNA synthases)– branched chain (ileS, valS)– glycine

• Chloroflexi, Dictyoglomi– aromatic (trp – enzymes)– branched chain (ileS)– threonine

Double and one-and-a-half T-boxes• TRP: trp operon (Bacillales,

C. beijerincki, D. hafniense)• TYR: pah (B. cereus)• THR: thrZ (Bacillales);

hom (C. difficile)• ILE: ilv operon (B. cereus)• LEU: leuA (C. thermocellum)

• ILE-LEU: ilvDBNCB-leuACDBA (Desulfotomaculum reducens)

• TRP: trp operon (T. tengcongensis)• PHE: arpLA-pheA (D. reducens, S. wolfei) • PHE: trpXY2 (D. reducens) • PHE: yngI (D. reducens) • TYR: yheL (B. cereus) • SER: serCA (D. hafniense)• THR: thrZ (S. uberis)• THR: brnQ-braB1 (C. thermocellum)• HIS: hisXYZ (Lactobacillales)• ARG: yqiXYZ (C. difficile)

Predicted regulation of translation:

ileS in many Actinobacteria• Instead of the terminator, the sequester

hairpin (hides the translation initiation site)• Same mechanism regulates different

processes – cf. riboswitches

Same enzymes – different regulators (common part of the aromatic amino acids biosynthesis pathway)

P H E T Y R

trpE

P E P E 4 P

D A H P

S H IK IM AT E

C H O R IS M AT E

trpDCFBA

tyrA hisC aspB

phhA

aroF

aroI aroE

aroA

aroD

aroB

aroC

aroA pheB aroH

yhaG

T R P

T R P

k in u ren in e p a th w ay

A N T H R A N IL AT E

F O L AT E

pabA pabB

A D C

trpG

TRP trpXYZ

TRP\PHE yocR fam ily

TYR yheL

aro:Regulated by TYR (BC)Regulated by PHE (SW O, DRE, HMO, CH, MTH, CTH)Regulated by TRP (DE, DEH)

cf. E.coli: aroF,G,H: feedback inhibition by TRP, TYR, PHE; transcriptional regulation by TrpR, TyrR

Recent duplications and bursts: ARG-T-box in Clostridium difficile

LJ_ARGS

LME_ARGS

LR_ARGS

LP_ARGS

CBE_ARGS

CPE_ARGSCB_ARGS

CTC_ARGS

CAC_ARGS

CDF_YQIXYZ

RDF02391

СDF_ARGC

CDF_ARGH

BC_ARGS2EF_ARGS

BH_ARGS

LSA_ARGSPPE_ARGS

LGA_ARGS

Bacillales

argSyqiXYZ

RDF02391

argCJBDF

predictedamino acidtransporters

NEW

argG

argH

Clostridiumdifficile

amino acidbiosynthetic genes

: ARG-specific T-box regulatory site

aminoacyl-tRNA synthetase

biosynthetic genes

amino acid transporters

NEW

Lactobacillales Clostridiales

argS argS

others

Expansion of T-box regulon

regulation of expression of arginine biosynthetic and transport genes by T-box antitermination

: ARG-specific T-box regulatory site

Binding to 5’ UTR gene region regulation of gene expression

Other clostridia spp. (CA, CTC, CTH, CPE, CB, CPE)

yqiXYZ

argC

argH

yqiXYZ

argC

argG

argH

AhrC regulatory protein (negative regulation of arginine metabolism positive regulation of arginine catabolism)

...AhrC site

: AhrC binding site

Gram+ bacteria: Clostridiumdifficile:

AhrC is lost

5’

More duplications: THR-T-box in C. difficile

MMY_THRS

OOE_THRS

HMO_YNGICAC_THRZ

BC_THRZ*

BC_THRZ

BC_HOM

BH_THRS

BE_THRSBCE_BRNQ2

BC_THRS

BL_THRZ

BCL_THRZ*

BS_THRZ*

BCL_THRZ

BS_THRZ

BL_THRSBS_THRS

BCL_THRSLMO_THRS

LB_THRSPPE_THRS

LJ_THRS

LP_THRS

TR_THRZ

EX_THRS

CBE_THRZ CTH_THRZCPE_THRS

TTE_THRZ

CDF_THRZ

CDF_HOMCDF_THRC

CDF_HOM*

С _THRZBCTE_THRZ

CBE_THRS

CTC_BRNQ1

LL_THRS

SUI_THRS

STH_THRS

SG_THRS

SMI_THRSSPN_THRS

SMU_THRSSAG_THRSSUB_THRS

SEQ_THRSSPY_THRS

SA_THRS

LME_THRS

MFL_THRS

: THR-specific T-box regulatory site

Bacillales

Clostridiales

LactobacillaceaeLeuconostocaceae

thrS

hom

thrS

thrZ

hom

thrS

thrZ

thrCB

С. difficile

hom

thrS

thrZ

brnQ

hom

thrS

thrZ

brnQB. cereus

brnQ

thrS

thrZ

thrS

thrZ

others

aminoacyl-tRNA synthetase

biosynthetic genes

amino acid transporters

Streptococcaecae

thrCB

Duplications and changes in specificity:

ASN/ASP/HIS T-boxes

CB_ASNS2

CDF_ASNA

EF_HISS

EX_HISS

BCL_HISSBH_HISS

OB_HISS

BC_HISS

TTE_HISS

DRE_HISS

CH_HISSCTH_HISS

PL_HISS

BE_HISSBL_HISS

BS_HISS

LME_HISXYZCDF_HISZX

LRE_HISXYZLSA_HISXYZ

OOE_HISXYZ

LP_HISXYZ

SGO_HISC

SMU_HISC

EF_HISXYZ

LMO_HISXYZ

EF_HISXYZ

LME_HIS(Z G\ )

LL_HISCLP_HISZ

LCA_HISZCB_ASNS3

CAC_ASNS32

BC_ASNS2

PPE_HISXYZ

PPE_ASNS

LB_ASNA

LD_ASNALJ_ QHMPgln

LJ_ASNA

PPE_ASNALP_ASNA

EX_ASNA

LB_ASNS2

CTC_ASNS2

PPE_HISSLP_HISS

LB_HISS

LJ_HISS

LRE_HISS

LRE_ASPS

LCA_HISS

CPE_ASNA

BC_ASNACBE_ASNS2

CTC_ASNACDF_ASNS2

CPE_ASNS2

his operon

his XYZ

Lactobacilla les

NEW

hisS

Other Gram +

ASP\ASN

HIS

Bacillales

HIS

aspS

SMU_ASPS2SG_ASPS2glnQHMP

L. johnsoniiasnA

ASP

ASN

asnAASN

Lac acillalestobasnS

ASN

aspS

hisXYZ

P. pentosaceus

asnS

HIS

ASP

Clostridiales

asnAASN

ASN

asnA

asnS

asnA

ASP

Rapid m utation of regulatory codons

ASN

AACGAC

hisSASP

Lac acillalestob

HIS

ASPhisS

L. reuteriaspS

ASN

ASN

ASN

ASN

Blow-up

PPE_ASNS2

LB_ASNA

LD_ASNALJ_GLNQHMP

LJ_ASNA

PPE_ASNALP_ASNA

PPE_HISSLP_HISS

LB_HISS

LJ_HISS

LRE_HISS

LRE_ASPS

LCA_HISS

aspShisSASP

Lac acillalestob

HIS ASPhisS

L. reuteri

aspS

ASP HIS

CACGAC

asnAASN

Lac acillalestob

disruption of hisS-aspS operonmutation of regulatory codon

L. johnsonii

asnA

ASP

ASN

glnQHMP

PPE_HISXYZ

ASN

AAC

P. pentosaceus

HIS

ASPhisXYZ

asnS

HIS

CAC

ASPASN

AAC GAC

Duplications and changes in specificity : branched-chain amino

acids

DG_VALS EX_VALS BCL_VALS

CTH_VALS

BC_VALS

BH_VALSBE_VALS

CH_VALS LMO_VALS

CA_ILVC

SA_VALS

OOE_LEUS

PPE_LEUS

LB_LEUS EF_LEUS

LJ_LEUSLGA_LEUS

OB_ILVB

LP_LEUS

LSA_LEUS

OB_LEUSSW O_029_0008

SWO_LEUS BS_YVBW

BL_YVBW

DRE_070_0004CH_LEUS

LM

O_L

EU

S

BL_LEUS

BS_LEUS

BE_LEUSBH_LEUS

BC_LEUS

BCL_LEUS

DTH_ILVB

BS_ILVB

PL_ILVB

BH_ILVB

BE_ILVBBL_ILVB

BCL_ILVB

GSU_LEUA

DH

A_L

EU

A

TTE_LEUS

CTH_148_0001

DF_LEUS

CDF_LEUA

CPE_LEUS

CBE_LEUS

CTC_LEUS

CB_LEUSCA_LEUS

EX_LEUS

DAC_LEUA

BC_YOCR3

OB1271

LP3666

STH_ILES

LP_BRNQ1_ile

SUB_ILES

LL_ILES

LCR_ILES

SPY_ILES

SZ_ILES

SEQ_ILES

SAG_ILES

SMU_ILES

SOB_ILES SMI_ILESSP_ILES

SG_ILES

EF_ILES LME_ILES

LJ_ILES

LD_ILES SA_ILES

LB_ILES

OOE_LP3666LRE_PANE

LP_BRNQ2_val

LCA_BRNQ1_val

LCA_BRNQ2_ileLRE_BRNQ _ile

LJ_BRNQ _ile

LSA_ILES

LJ_OPP

CTC_ILES

CB_ILES

CPE_ILES

TTE_ILES

PPE_ILES

LRE_3666_1

LMO_ILES

DF_ILES

EX_ILES

BC_ILES

BS_ILESBL_ILES

BH_ILES

BCL_ILES

OOE_ILES

DRE_ILVD*_leu

DRE_ILVD _ile

CH_YBGE

BC_ILVB

BE_ILES

DRE_ILES

HMO_ILES

CH_ILES

LRE_3666_2

DHA_ILES

OB_ILES

CTC_BRNQ2CPE_BRNQ

CDF_ILVCCTC_BRNQ1

CAC_BRNQ

BC_ILES2

BCE_BRNQ1

LP_ILES

CTH_ILESLR_LEUS

HMO_ILVB

BC_YBGE*BC_YBGE

DF_VALS

CB_VALS

CBE_VALS

CAC_VALS

CTC_VALS

LL_VALSLCR_VALS LR_VALS

LP_VALS

LSA_VALS

LME_VALS

EF_VALS

PPE_VALS

LD_VALS

LJ_VALS

CPE_VALS

DHA_VALS

BS_VALS

BL_VALS

DRE_VALS

TTE_VALS

HMO_VALS

LEU

VAL ILE

valSVAL

leuSLEU

Firmicutes

Ilv operon

LEU

Bacillales

.......

leu operon

LEU

δ-proteobacteriaClostridium difficileDesulfitobacteriumhafniense

.......

148_0001

LEU

C therm ocellum.

029_0008

LEU

Syntrophom onaswolfei

yvbW

LEU

B. Subtilis.B licheniform is

YOCR3

LEU

B. cereus

OB1271

LEU

Oceanobacillusiheyensis

C acetobutylicum .ilvC

VAL

FirmicutesileS

ILE

LactobacillaceaeClostridiaceaeBacillus cereus

brnQILE

Firmicutes

Lactobacillus casei Lactobacillus plantarum

brnQ

VAL

Ilv operon

LEU

Desulfotomaculum reducens

.......

IlvBN

ILE

Heliobacillus mobilis

Ilv operon2

ILE.......

Ilv operon

ILE

Carboxydothermushydrogenoformans

.......

IlvCB

ILE

.C difficile

ILE

ILE

Recent T-box duplication and mutation of regulatory codon

LEU

ATCCTC

lp3666

ILE

Lactobacillales

opp

ILE

Lactobacillus johnsonii

panE

ILE

Lactobacillus reuteri

Ilv operon2

ILE.......

B. cereus

ILE VAL

GTCCTC

T-box duplication and mutation of regulatory codon

ATC

ATC CTC

Blow-up

transporter:

dual regulation of common enzymes:

ATC CTC

ATC GTC

Summary / History

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

Distribution of

transcription factors in genomes

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

• Andrey Mironov (software): – genome analysis– conserved RNA patterns

• Ekaterina Panina (now at UCLA, USA)– zinc and ribosomes

• Alexey Vitreschak– T-boxes

• Dmitry Rodionov– iron homeostasis

• Support:– Howard Hughes Medical Institute– INTAS– Russian Fund of Basic Research– Russian Academy of Sciences

(“Molecular and Cellular Biology”)