Evolution of regulatory interactions in bacteria

1
Evolution of regulatory interactions in bacteria

• Mikhail Gelfand
• Institute for Information Transmission Problems,
  RAS
• 4th Bertinoro Computational Biology (BCB) Meeting
  Evolution of and Comparative Approaches to
  Gene Regulation
• 24-30 June 2006

2
??? ??? ??????????. ? ???? ?????. ??????
????????? ?? ???? ????. ???? ???????????? ?????
??????. ??????? ????????, ??? ??? ??????.?????
????????

• A list of some observations. In a corner, its
  warm.
• A glance leaves an imprint on anything its dwelt
  on.
• Water is glasss most public form.
• Man is more frightening than its skeleton.
• Joseph Brodsky

3
Plan

• Evolution of individual sites
• Coevolution of transcription factors and their
  binding signals
• Distribution of transcription factor families in
  various genomes
• Evolution of simple and complex regulatory systems

4
Birth and death of sites is a very dynamic
process

• NadR-binding sites upstream of pnuB seem absent
  in Klebsiella pneumoniae and Serratia marcescens

5
but there are candidate sites further upstream
6
and they are clearly diferent (not simply
misaligned).
7
Loss of regulators and cryptic sites
Loss of the RbsR in Y. pestis (ABC-transporter
also is lost)
RbsR binding site
Start codon of rbsD
8
Unexpected conservation of non-consensus
positions in orthologous sites
regulatory site of LexA upstream of
lexAconsensus nucleotides are in caps
wrong consensus?
9
TF PurR, gene purL
TF PurR, gene purM
10
Non-consensus positions are more conserved than
synonymous codon positions
11
Relative conservation of non-consensus
nucleotides may be higher than conservation of
consensus nucleotides
12
Regulators and their signals

• Subtle changes at close evolutionary distances
• Cases of signal conservation at surprisingly
  large distances
• Changes in spacing / geometry of dimers
• Correlation between contacting nucleotides and
  amino acid residues

13
The LacI family subtle changes in signals at
close distances
G
n
A
CG
Gn
GC
14
NrdR (regulator of ribonucleotide reducases and
some other replication-related genes)
conservation at large distances
15
BirA (biotin regulator in eubacteria and
archaea) conserved signal, changed spacing
16
DNA signals and protein-DNA interactions
Entropy at aligned sites and the number of
contacts (heavy atoms in a base pair at a
distance ltcutoff from a protein atom)
CRP
PurR
IHF
TrpR
17
Specificity-determining positions in the LacI
family

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

44 SDPs
10 residues contact NPF (analog of the effector)
7 residues in the effector contact zone
(5?ltdminlt10?)
6 residues in the intersubunit contacts
5 residues in the intersubunit contact zone
(5?ltdminlt10?)
7 residues contact the operator sequence
6 residues in the operator contact zone
(5?ltdminlt10?)
LacI from E.coli
18
CRP/FNR family of regulators
19
Correlation between contacting nucleotides and
amino acid residues

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

Contacting residues REnnnR TG 1st arginine GA
glutamate and 2nd arginine
DD COOA ALTTEQLSLHMGATRQTVSTLLNNLVR DV COOA
ELTMEQLAGLVGTTRQTASTLLNDMIR EC CRP
KITRQEIGQIVGCSRETVGRILKMLED YP CRP
KXTRQEIGQIVGCSRETVGRILKMLED VC CRP
KITRQEIGQIVGCSRETVGRILKMLEE DD HCPR
DVSKSLLAGVLGTARETLSRALAKLVE DV HCPR
DVTKGLLAGLLGTARETLSRCLSRMVE EC FNR
TMTRGDIGNYLGLTVETISRLLGRFQK YP FNR
TMTRGDIGNYLGLTVETISRLLGRFQK VC FNR
TMTRGDIGNYLGLTVETISRLLGRFQK
TGTCGGCnnGCCGACA
TTGTGAnnnnnnTCACAA
TTGTgAnnnnnnTcACAA
TTGATnnnnATCAA
20
The correlation holds for other factors in the
family
21
Distribution of TF families in bacterial genomes
ExtraTrain database
Streptomyces coelicolor
LysR
Pseudomonas aeruginosa
TetR
AraC
LuxR
GntR
LacI
Agrobacterium tumefaciens
Escherichia coli
Bacillus subtilis
22
Strategies of successful TF families

• One ortholog per genome
• LexA, NrdR, HrcA, ArgR
• present even in archaea BirA (also enzyme), ModE
• Several (2-3) orthologs per genome
• CRP/FNR, FUR
• Local explosions
• LacI in alpha- and gamma-proteobacteria
• 2CS systems in delta-proteobacteria
• sigma-factors in Streptomyces
• Because TF in a family tend to have related
  functions and these might depend on the
  lifestyle?

23
LacI family regulons in closely related strains
(top TFs, bottom regulated genes)
Seven Escherichia and Shigella spp.
Four Bacillus cereus and B. anthracis strains
Five Salmonella spp.
24
What are the driving forces for the present-day
state?

• 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

25
Regulon expansion how FruR has become CRA
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
26
Common ancestor of 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
27
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.
28
Trehalose/maltose catabolism in
alpha-proteobacteria
Duplicated LacI-family regulators
lineage-specific post-duplication loss
29
The binding signals are very similar (the blue
branch is somewhat different to avoid
cross-recognition?)
30
Utilization of an unknown galactoside in
gamma-proteobacteria
Yersinia and Klebsiella two regulons, GalR (not
shown, includes genes galK and galT) 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)
31
Utilization of maltose/maltodextrin in Firmicutes
Two different ABC transporters (shades of
red) PTS (pink) Glucoside hydrolases (shades of
green) Two regulators (black and grey)
32
Modularity of the functional subsystem
Two different ABC systems Three hydrolases in one
operon (E. faecalis) or separately
33
Changes of regulation
Two different ABC systems
Displacement invasion of a regulator from a
different subfamily (horizontal transfer from a
related species?) blue sites
34
Orthologous TFs with completely different regulons
35
Utilization of xylose in alpha-proteobacteria
xylBA Three different ABC transporters Three
regulators two from the LacI family and one from
the ROK family
36
Changes in operon structure
37
Changes in regulation
Displacement Operon regulation changed from
XylR-1 to XylR-2 (different subfamily)
Duplication and displacement Duplicated XylR-1a
assumed the role of the ROK-family regulator
38
Catabolism of gluconate in proteobacteria
39
extreme variability of regulation of marginal
regulon members
ß
?
Pseudomonas spp.
40
Regulation of amino acid biosynthesis in
Firmicutes

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

41
Aromatic amino acid regulons
42
Five regulatory systems for the methionine
biosynthesis

• SAM-dependent RNA riboswitch
• Met-tRNA-dependent T-box (RNA)
• C,D,E. repressors of transcription

43
Methionine regulatory systems loss of S-box
regulons

• S-boxes (SAM-1 riboswitch)
• Bacillales
• Clostridiales
• the Zoo
• Petrotoga
• actinobacteria (Streptomyces, Thermobifida)
• Chlorobium, Chloroflexus, Cytophaga
• Fusobacterium
• Deinococcus
• proteobacteria (Xanthomonas, Geobacter)
• Met-T-boxes (Met-tRNA-dependent attenuator)
  SAM-2 riboswitch for metK
• Lactobacillales
• MET-boxes (candidate transcription signal)
• Streptococcales

ZOO
Lact.
Strep.
Bac.
Clostr.
44
Mapping the events to the phylogenetic tree
loss of S-boxes (SAM-I riboswitches)
expansion of Met-T-boxes, emergence of SAM-2
riboswitches
Trp-T-boxes ? TRAP Tyr-T-boxes ? PCE
emergence of MtaR Tyr-T-boxes ? ARO
Bacillus subtilis and related species
Bacillus cereus and related species
Strepto-coccus spp.
Lacto-bacillus spp.
Clostridium spp.
45
Combined regulatory network for iron homeostasis
genes in in a-proteobacteria.
Fe
Fe
- Fe
Fe
-
FeS status
of cell
FeS
- Fe
Fe
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.
46
Distribution of Irr, Fur/Mur, MntR, RirA,
and IscR regulons in a-proteobacteria
?' in RirA column denotes the absence of the
rirA gene in an unfinished genomic sequence and
the presence of candidate RirA-binding sites
upstream of the iron uptake genes.
47
Distribution of the conserved members of the Fe-
and Mn-responsive regulons and the predicted
RirA, Fur/Mur, Irr, and DtxR binding sites in
a-proteobacteria
Genes Functions Iron uptake Iron storage FeS
synthesis
Iron usage Heme biosynthesis Regulatory
genes Manganese uptake
48
Phylogenetic tree of the Fur family of
transcription factors in a-proteobacteria - I
Fur in g- and b- proteobacteria
Fur in e- proteobacteria
Fur in Firmicutes
in a-proteobacteria
Regulator of manganese uptake genes (sit, mntH)
in a-proteobacteria
Regulator of iron uptake and metabolism genes
a-proteobacteria
49
Erythrobacter litoralis
Caulobacter crescentus
Novosphingobium aromaticivorans
Zymomonas mobilis
Sequence logos for the identified Fur-binding
sites in the D group of a-proteobacteria
Sphinopyxis alaskensis
Oceanicaulis alexandrii
Rhodospirillum rubrum
Gluconobacter oxydans
Magnetospirillum magneticum
Parvularcula bermudensis -
Identified Mur-binding sites
Bacillus subtilis
The A, B, and C groups
Sequence logos for the known Fur-binding sites
in Escherichia coli and Bacillus subtilis
Mur
a
of - proteobacteria -
Escherichia coli
50
Phylogenetic tree of the Fur family of
transcription factors in a-proteobacteria - II
Fur in g- and b- proteobacteria
Fur in e- proteobacteria
Fur in Firmicutes
a-proteobacteria
Irr in a-proteo- bacteria regulator of
iron homeostasis
51
Sequence logos for the identified Irr binding
sites in a-proteobacteria.
(8 species) - Irr
The A group
The B group

(4 species) - Irr
The C group (12 species) - Irr
52
Phylogenetic tree of the Rrf2 family of
transcription factors in a-proteobacteria
Nitrite/NO-sensing regulator NsrR (Nitrosomonas
europeae, 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
Iron repressor RirA (Rhizobium leguminosarum)
Cysteine metabolism repressor CymR (Bacillus
subtilis)
Cytochrome complex regulator Rrf2 (Desulfovibrio
vulgaris)
Iron-Sulfur cluster synthesis repressor
IscR (Escherichia coli)
proteins with the conserved C-X(6-9)-C(4-6)-C
motif within effector-responsive domain proteins
without a cysteine triad motif
53
Sequence logos for the identified RirA-binding
sites in a-proteobacteria
The A group - RirA
(8 species)
(12 species)
The C group - RirA
54
An attempt to reconstruct the history
55
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
• Develop an evolutionary model that would converge
  to the present state (that is, have the same
  properties)
• Distribution of TF families sizes
• Distribution of regulon sizes
• Other graph-theoretical properties (node degrees
  etc.)
• General properties? E.g. stable cores and
  flexible margins of functional systems (in terms
  of gene presence and regulation)
• Microevolution (strains)
• metagenomic regulatory systems?
• 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

56
Acknowledgements

• Andrei A. Mironov
• Dmitry Rodionov (now at Burnham Institute)
• Olga Laikova
• Alexei Vitreschak
• Anna Gerasimova
• Ekateina Kotelnikova (now at Ariadne Genomics)
• Ekaterina Panina (now at UCLA)
• Leonid Mirny (MIT)

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