Evolution of bacterial regulatory systems

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Evolution of bacterial regulatory systems

• Mikhail Gelfand
• Research and Training Center Bioinformatics
• Institute for Information Transmission Problems
• Moscow, Russia

ASM, Philadelphia, 18.IV.2009
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Catalog of events

• Expansion and contraction of regulons
• New regulators (where from?)
• 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

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Trehalose/maltose catabolism in
alpha-proteobacteria
Duplicated LacI-family regulators
lineage-specific post-duplication loss
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The binding motifs are very similar (the blue
branch is somewhat different to avoid
cross-recognition?)
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Utilization of an unknown galactoside in
gamma-proteobacteria
Yersinia and Klebsiella two regulons, GalR and
Laci-X
Erwinia one regulon, GalR
Loss of regulator and merger of regulons It
seems that laci-X was present in the common
ancestor (Klebsiella is an outgroup)
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Utilization of maltose/maltodextrin in Firmicutes
Displacement invasion of a regulator from a
different subfamily (horizontal transfer from a
related species?) blue sites
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Orthologous TFs with completely different
regulons (alpha-proteobaceria and Xanthomonadales)
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Cryptic sites and loss of regulators
Loss of RbsR in Y. pestis (ABC-transporter also
is lost)
RbsR binding site
Start codon of rbsD
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Regulon expansion, or how FruR has become CRA

• CRA (a.k.a. FruR) in Escherichia coli
• global regulator
• well-studied in experiment (many regulated genes
  known)
• Going back in time looking for candidate
  CRA/FruR sites upstream of (orthologs of) genes
  known to be regulated in E.coli

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Common ancestor of gamma-proteobacteria
Mannose
Glucose
ptsHI-crr
manXYZ
edd
epd
eda
adhE
aceEF
icdA
ppsA
pykF
mtlD
mtlA
Mannitol
pckA
gpmA
pgk
gapA
fbp
pfkA
aceA
tpiA
fruK
fruBA
Fructose
aceB
Gamma-proteobacteria
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Common ancestor of the Enterobacteriales
Mannose
Glucose
ptsHI-crr
manXYZ
edd
epd
eda
adhE
aceEF
icdA
ppsA
pykF
mtlD
mtlA
Mannitol
pckA
gpmA
pgk
gapA
fbp
pfkA
aceA
tpiA
fruK
fruBA
Fructose
aceB
Gamma-proteobacteria Enterobacteriales
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Common ancestor of Escherichia and Salmonella
Mannose
Glucose
ptsHI-crr
manXYZ
edd
epd
eda
adhE
aceEF
icdA
ppsA
pykF
mtlD
mtlA
Mannitol
pckA
gpmA
pgk
gapA
fbp
pfkA
aceA
tpiA
fruK
fruBA
Fructose
aceB
Gamma-proteobacteria Enterobacteriales E. coli
and Salmonella spp.
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Regulation of amino acid biosynthesis in the
Firmicutes

• Interplay between regulatory RNA elements and
  transcription factors
• Expansion of T-box systems (normally RNA
  structures regulating aminoacyl-tRNA-synthetases)

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Why T-boxes?

• May be easily identified
• In most cases functional specificity may be
  reliably predicted by the analysis of the
  specifier codons (anti-anti-codons)
• Sufficiently long to retain phylogenetic signal
• gt T-boxes are a good model of regulatory
  evolution

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Partial alignment of predicted T-boxes
TGG T-box
Aminoacyl-tRNA synthetases
Amino acid biosynthetic genes
Amino acid transporters
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continued (in the 5 direction)
anti-anti (specifier) codon
Aminoacyl-tRNA synthetases
Amino acid biosynthetic genes
Amino acid transporters
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805 T-boxes in 96 bacteria

• Firmicutes
• aa-tRNA synthetases
• enzymes
• transporters
• all amino acids excluding glutamate
• Actinobacteria (regulation of translation
  predicted)
• 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

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Recent duplications and bursts ARG-T-box in
Clostridium difficile
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caused by loss of transcription factor AhrC
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Duplications and changes in specificity
ASN/ASP/HIS T-boxes
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Blow-up 1
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Blow-up 2. Prediction

• Regulators lost in lineages with expanded
  HIS-T-box regulon??

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and validation

• conserved motifs upstream of HIS biosynthesis
  genes
• candidate transcription factor yerC co-localized
  with the his genes
• present only in genomes with the motifs upstream
  of the his genes
• genomes with neither YerC motif nor HIS-T-boxes
  attenuators

Bacillales (his operon)
Clostridiales Thermoanaerobacteriales Halanaerobia
les Bacillales
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The evolutionary history of the his genes
regulation in the Firmicutes
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More duplications THR-T-box in C. difficile and
B. cereus
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T-boxes Summary / History
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Life without Fur
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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, Fe2, Fe3)
• storage
• iron-dependent enzymes
• synthesis of heme
• synthesis of Fe-S clusters
• Similar in Bacillus subtilis

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Regulation of iron homeostasis in a-proteobacteria

• Experimental studies
• FUR/MUR Bradyrhizobium, Rhizobium and
  Sinorhizobium
• RirA (Rrf2 family) Rhizobium and Sinorhizobium
• Irr (FUR family) Bradyrhizobium, Rhizobium and
  Brucella

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Distribution of transcription factors in genomes
Search for candidate motifs and binding sites
using standard comparative genomic techniques
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FUR/MUR branch of the FUR family
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FUR and MUR boxes
Erythrobacter litoralis
Caulobacter crescentus
Novosphingobium aromaticivorans
Zymomonas mobilis
Sphinopyxis alaskensis
Oceanicaulis alexandrii
Rhodospirillum rubrum
Gluconobacter oxydans
Magnetospirillum magneticum
Parvularcula bermudensis -
Identified Mur-binding sites
Bacillus subtilis
Sequence logos for the known Fur-binding sites
in Escherichia coli and Bacillus subtilis
Mur
a
of - proteobacteria -
Escherichia coli
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Irr branch of the FUR family
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Irr boxes

• Rhizobiaceae plus
• Bradyrhizobiaceae
• Rhodobacteriaceae
• Rhodospirillales

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RirA/NsrR family (Rhizobiales)
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IscR family
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Regulation of genes in functional subsystems
Rhizobiales
Bradyrhizobiaceae
Rhodobacteriales
The Zoo (likely ancestral state)
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Reconstruction of history
Frequent co-regulation with Irr
Strict division of function with Irr
Appearance of theiron-Rhodo motif
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All logos and Some Very Tempting Hypotheses
2

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
3
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Summary and 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.

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People

• Andrei A. Mironov software, algorithms
• Alexandra Rakhmaninova SDP, protein-DNA
  correlations
• Olga Kalinina (on loan to EMBL) SDP
• Yuri Korostelev protein-DNA correlations
• Olga Laikova LacI
• Dmitry Ravcheev CRA/FruR
• Dmitry Rodionov (on loan to Burnham Institute)
  iron etc.
• Alexei Vitreschak T-boxes and riboswitches
• Andy Jonson (U. of East Anglia) experimental
  validation (iron)
• Leonid Mirny (MIT) protein-DNA, SDP
• Andrei Osterman (Burnham Institute)
  experimental validation

• Howard Hughes Medical Institute
• Russian Foundation of Basic Research
• Russian Academy of Sciences, program Molecular
  and Cellular Biology

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