Download Comparative genomics and metabolic reconstruction of

Comparative genomics and
metabolic reconstruction of
bacterial pathogens
Mikhail Gelfand
Institute for Information Transmission Problems, RAS
GPBM-2004
Metabolic reconstruction
• Identification of missing genes in complete
genomes
• Search for candidates
– Analysis of individual genes to assign general
function:
• homology
• functional patterns
• structural features
– Comparative genomics to predict specificity:
•
•
•
•
analysis of regulation
positional clustering
gene fusions
phylogenetic patterns
Enzymes
• Identification of a gap in a pathway (universal,
taxon-specific, or in individual genomes)
• Search for candidates assigned to the
pathway by co-localization and co-regulation
(in many genomes)
• Prediction of general biochemical function
from (distant) similarty and functional patterns
• Tentative filling of the gap
• Verification by analysis of phylogenetic
patterns:
– Absence in genomes without this pathway
– Complementary distribution with known enzymes
for the same function
Transporters
• Identification of candidates assigned to the pathway
by co-localization and co-regulation (in many
genomes)
• Prediction of general function by analysis of
transmembrane segments and similarty
• Prediction of specificity by analysis of phylogenetic
patterns:
– End product if present in genomes lacking this pathway
(substituting the biosynthetic pathway for an essential
compound)
– Input metabolite if absent in genomes without the
pathway (catabolic, also precursors in biosynthetic
pathways)
– Entry point in the middle if substituting an upper or side
part of the pathway in some genomes
Missing link in fatty acid biosynthesis in Streptococci
accA
accD
accB
Gene
fabI of Enoyl-ACP
fabI
(Enoyl-ACP
reductase,
(EC 1.3.1.9)
is
ECreductase
1.3.1.9) target
of triclosan.
missing
in the
genomebut
12B,
Enzymatic
activity,
no
and agene
number
of Streptococci
in Streptococci
accC
fab
D
fab
F
fab
G
fabZ
fabI
acp
P
fabH
Identification of a candidate by positional clustering
hyp
TR?
3.5.1.?
fabH
fabD
acpP
fabG
fabF
fabZ
accB
accC
accD
accA
fabI
hyp
6.3.4.15
Genome X
TR?
fabH
acpP
?
fabD
fabG
fabF
accB
fabZ
accC
accD
accA
fabZ
accC
accD
accA
2.1.1.79
FRNS
Genome Y
TR?
fabH
acpP
?
fabD
fabG
fabF
accB
5.99.1.2
Clostridium acetobutylicum
TR?
fabH
Streptococcus pyogenes
acpP
?
fabD
fabG
fabF
accB
fabZ
accC
accD
accA
hyp
Binding sites of FabR (“Tr?”, HTH)
1 Fad (42.1.17) 2
HTH fabH acpP
3 4 fabK
fabD
fabG
fabF
accB
fabZ accC
E. faecalis
E. faecalis
E. faecalis
CONSENSUS
HTH-1
HTH-2
HTH-3
acTTTGAtwaTCAAAgt
AgTTTGggTATCAAAGT
AgTTTGAacATCAAAtg
GtTTTGATAATCAAAGT
E. faecium
E. faecium
E. faecium
HTH-1
HTH-2
HTH-3
ACTTTGATAATCAAAaT
AgTTTGAacATCAAAag
gaTTTGATAATCAAAcT
S. pyogenes
S. pyogenes
S. pyogenes
S. pyogenes
4.2.1.17
HTH-1
fabK-1
fabK-2
GaTTTGATTATCAAAtg
AaTTTGATTgTCAAAGT
CtTTTGATAtTCAAAtT
AgTTTGATTATCAAAtT
S. pneumoniae
S. pneumoniae
S. pneumoniae
4.2.1.17
HTH-1
fabK-1
ACTTTGAcAgTgAAAta
gtTTTGATTgTaAAAGT
AgTTTGAcTgTCAAAtT
S. mutans
S. mutans
S. mutans
S. mutans
S. mutans
4.2.1.17-1
4.2.1.17-2
HTH-1
fabK-1
fabK-2
ACTTTGATTtTCAAAcT
AaTTTGATTATCttAaT
ACTTTGATAgTCAAAGT
AgTTTGAcAtTCAAAtc
AgTTTGAcTgTCAAAtT
1
2
3
4
accD
accA
Metabolic reconstruction of the thiamin biosynthesis
(new genes/functions shown in red)
Purine pathway
Transport of HMP
thiN
(confirmed)
Transport of HET
(Gram-positive bacteria)
(Gram-negative bacteria)
unknown
arabinose
arbutin
cellobiose
dextran
esculin
fructose
fucose
galactose
glucose
inulin
lactose
maltose
mannitol
mannose
melibiose
N-AcGlu
raffinose
ribose
salicin
sorbitol
sorbose
sucrose
tagatose
trehalose
xylose
L.
mesenteroides
Oenococcus
oeni
L. brevis
P. pentosaceus
L. delbrueckii
L. gasseri
L. casei
L. lactis
S. suis
S. thermophilus
S. mutans
S. agalactiae
S. uberis
S. equi
S. pyogenes
S. pneumoniae
Carbohydrate metabolism in
Streptococcus and Lactococcus spp.
Only
biochemical
data, genes
unknown
Experimentally
verified genes
Biochemical
data and
genomic
predictions
Only genomic
predictions
unknown
arabinose
arbutin
cellobiose
dextran
esculin
fructose
fucose
galactose
glucose
inulin
lactose
maltose
mannitol
mannose
melibiose
N-AcGlu
raffinose
ribose
salicin
sorbitol
sorbose
sucrose
tagatose
trehalose
xylose
L.
mesenteroides
Oenococcus
oeni
L. brevis
P. pentosaceus
L. delbrueckii
L. gasseri
L. casei
L. lactis
S. suis
S. thermophilus
S. mutans
S. agalactiae
S. uberis
S. equi
S. pyogenes
S. pneumoniae
An uncharacterized locus in invasive species
S. pneumoniae
S. pyogenes
S. equi
S. agalactiae
S. suis
Structure of the genome loci
S. pyogenes, S. agalactiae
S. equi
S. pneumoniae TIGR4
IS
S. pneumoniae R6
IS
S. suis
IS
Gene functions
3-(4-deoxy-beta-D-gluc-4-enuronosyl)-Nacetyl-D-glucosamine
PTS transporter
hydrolase
isomerase
oxidoreductase
dehydrogenase
kinase
aldolase
pyruvate +
D-glyceraldehyde 3-phosphate
hyaluronidase
(hyaluronate
lyase)
RegR
Candidate regulatory signal
Structure of the genome loci - 2
S. pyogenes, S. agalactiae
S. equi
S. pneumoniae TIGR4
IS
S. pneumoniae R6
IS
S. suis
IS
Possible function
• Pathway exists in invasive species
• Sometimes co-localized with hyaluronidase
• Always co-regulated with hyaluronidase
Thus:
• Utilization of hyaluronate
• May be involved in pathogenesis
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
enzymes
Genomes and regulators
???
nZUR
FUR family
pZUR
AdcR ?
FUR family
MarR family
nZUR-
Regulators and signals
GATATGTTATAACATATC
nZUR-
GAAATGTTATANTATAACATTTC
GTAATGTAATAACATTAC
TTAACYRGTTAA
pZUR
TAAATCGTAATNATTACGATTTA
AdcR
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: regulation
zinT is isolated
zinT is regulated by zinc repressors
(nZUR-, nZUR-, pZUR)
E. coli, S. typhi, K. pneumoniae
Gamma-proteobacteria
A. tumefaciens, R. sphaeroides
Alpha-proteobacteria
B. subtilis, S. aureus
Bacillus group
S. pneumoniae, S. mutans,
S. pyogenes, L. lactis, E. faecalis
Streptococcus group
fusion: adcA-zinT
adcA-zinT is regulated by zinc
repressors (pZUR, AdcR) (ex. L.l.)
ZinT: protein sequence analysis
Y. pestis, V. cholerae,
B. halodurans
S. aureus, E. faecalis,
S. pneumoniae, S. mutans,
S. pyogenes
E. coli, S. typhi, K. pneumoniae,
A. tumefaciens, R. sphaeroides,
B. subtilis
L. lactis
TM Zn AdcA
ZinT
ZinT: summary
• zinT is sometimes fused to the gene of a zinc
transporter component adcA
• zinT is expressed only in zinc-deplete
conditions
• ZinT is attached to cell surface (has a TMsegment)
• ZinT has a zinc-binding domain
ZinT: conclusions:
• ZinT is a new type of zinc-binding
component of zinc ABC transporter
Zinc regulation of PHT
(pneumococcal histidine triad)
proteins of Streptococci
S. pneumoniae S. pyogenes S. equi
S. agalactiae
zinc regulation shown in
experiment
lmb phtD
phtA
phtE
phtB
lmb phtD
phtY
lmb phtD
Structural features of PHP proteins
• PHT proteins contain multiple HxxHxH
motifs
• PHT proteins of S. pneumoniae are paralogs
(65-95% id)
• Sec-dependent hydrophobic leader
sequences are present at the N-termini of
PHT proteins
• Localization of PHT proteins from S.
pneumoniae on bacterial cell surface has
been confirmed by flow cytometry
PHH proteins: summary
• PHT proteins are induced in zinc-deplete
conditions
• PHT proteins are localized at the cell
surface
• PHT proteins have zinc-binding motifs
A hypothesis:
• PHT proteins represent a new family of
zinc transporters
… incorrect 
• Zinc-binding domains
in zinc transporters:
• Histidine triads in
streptococci:
EEEHEEHDHGEHEHSH
DEHGEGHEEEHGHEH
HGDHYHY
HGDHYHF
HGNHYHF
HYDHYHN
HMTHSHW
(histidine-aspartateglutamate-rich)
(specific pattern of histidines
and aromatic amino acids)
HSHEEHGHEEDDHDHSH
EEHGHEEDDHHHHHDED
7 out of 21
2 out of 21
2 out of 21
2 out of 21
2 out of 21
Analyis of PHP proteins (cont’d)
• The phtD gene forms a candidate operon with the
lmb gene in all Streptococcus species
– Lmb: an adhesin involved in laminin binding,
adherence and internalization of streptococci into
epithelial cells
• PhtY of S. pyogenes:
– phtY regulated by AdcR
– PhtY consists of 3 domains:
4 HIS TRIADS
PHT
LRR IR
HDYNHNHTYEDEEGH
AHEHRDKDDHDHEHED
internalin
H-rich
PHH proteins: summary-2
•
•
•
•
•
PHT proteins are induced in zinc-deplete conditions
PHT proteins are localized at the cell surface
PHT proteins have structural zinc-binding motifs
phtD forms a candidate operon with an adhesin gene
PhtY contains an internalin domain responsible for the
streptococcal invasion
Hypothesis
PHT proteins are adhesins involved in the attachment of
streptococci to epithelium cells, leading to invasion
AdcR
pZUR
nZUR
Zinc and (paralogs of) ribosomal
proteins
L36
E. 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
–
L33
–
–
–
–+–
–––
––
–––
–––
–––
L31
–+
––
–+
–+
–
–
–
–
–
S14
–
–
–
–+
–+
–+
–+–
–
–+
Zn-ribbon motif
AdcR
pZUR
nZUR
(Makarova-Ponomarev-Koonin, 2001)
L36
E. 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
(–)
L33
–
–
–
(–) + –
(–) – –
(–) –
(–)  – –
(–) – –
(–) – –
L31
(–) +
(–) –
(–) +
(–) +
–
–
–
–
–
S14
–
–
–
(–) +
(–) +
(–) +
(–) + –
(–)
(–) +
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
Bad scenario
Zn-rich conditions
Zn-deplete conditions:
all Zn utilized by the
ribosomes, no Zn for
Zn-dependent enzymes
Regulatory mechanism
Sufficient Zn
ribosomes
repressor
R
Zn-dependent
enzymes
Zn starvation
R
Good scenario
Zn-rich conditions
Zn-deplete conditions:
some ribosomes
without Zn, some Zn
left for the enzymes
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.)
•
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Andrei A. Mironov
Anna Gerasimova
Olga Kalinina
Alexei Kazakov (hyaluronate)
Ekaterina Kotelnikova
Galina Kovaleva
Pavel Novichkov
Olga Laikova (hyaluronate)
Ekaterina Panina (zinc)
(now at UCLA, USA)
Elizabeth Permina
Dmitry Ravcheev
Alexandra B. Rakhmaninova
Dmitry Rodionov (thiamin)
Alexey Vitreschak (thiamin)
(on leave at LORIA, France)
• Andrei Osterman
(Burnham Institute,
San-Diego, USA)
(fatty acids)
• 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