Download Comparative genomics and metabolic reconstruction of

Comparative genomics and
metabolic reconstruction of
bacterial genomes
Mikhail S. Gelfand
Meeting of HHMI International Research Scholars
Tallinn, 2004
Metabolic reconstruction
• Identification of missing genes in complete
genomes
• Search for candidates
– Analysis of individual genes to assign general
biochemical function:
• homology
• functional patterns
• structural features
– Comparative genomics to predict specificity:
•
•
•
•
analysis of regulation
positional clustering
gene fusions
phylogenetic patterns
L-aspartate
lysC,thrA,metL
Metabolic reconstruction
of the lysine pathway
lysC,dapG,yclM
-aspartyl-phosphate
asd
aspartate
semialdehyde
• Predictions:
– Genes for the acetylated
pathway in Gram-positive
bacteria
– Positive regulation of the
lysine catabolism genes in
Thermoanaerobacter and
Fusobacterium by LYSelements: 1st example of
activating riboswitches
– New transporters
dapA
hom
homoserine
thrA,
metL
dihydrodipicolinate
dapB
tetrahydrodipicolinate
dapD
N-succinyl-2-amino-6-ketopimelate
dapC(argD)
N-succinyl-L,L-diaminopimelate
dapE
dapD
N-acetyl-2-amino-6-ketopimelate
patA
N-acetyl-L,L-diaminopimelate
ykuR
L,L-diaminopimelate
dapF, dal
meso-diaminopimelate
Lysine transport
lysA
ddh
• Predictions:
–
A
Metabolic reconstruction
Genes for the
SAM-recycling pathway of the methionine pathway
– Transporters for
methionine and
methylthiribose
Threonine
– Other enzymes
– Transcriptional
regulation in
Streptococci
– Complicated S-box and
Cys-T-box regulation of
the ubiG-yrhBA operon
in C. acetobutylicum:
S-ribosylhomocysteine
activation via repression (SRH)
of the antisense
mtn
transcript
Aspartate semialdehyde
hom
Homoserine
cysH-...
metX
metB
O-acetylhomoserine
metI
Sulfide
metY
ubiG
yrhA
yrhB
S-box
sense transcript
Cystathionine
metC yrhA
Homocysteine
methyl-THF
betaine
yxjH*
metE , metH ,
dimethylglycine
S-adenosylmethionine
S-adenosylhomocysteine
(SAH)
(SAM)
THF
metK
CH3
Cysteine
Methionine
mtnKSUVWXYZ
antisense transcript
S-adenosylmethionine
methylene-THF
metF
A
Cys-T-box
yrhB
MTA
mtn
Methylthioribose (MTR)
Aromatic amino acid regulons
in Gram-positive bacteria
Prediction of transporter specificity via
analysis of regulation
Pasteurella ceae
NMB
S ON-2
S ON-1
VC-1
VC-2
BH
SON-3
clostridia
OB
FN 0978
OB1118
HP
C AC0744
LysW
CB
EF -nhaC 1 PPE
Archaea
LP-nha2
LGA
LME
LP -nha 1
LB
EF-nhaC2
TyrT
BC14 34
FN1414
BT1270
CB
NMB0536
FN0352
BC4121
TTE-nhaC
SA2117
CJ
OB2874
269.
47
CT C
CPE
DF
BL1111
MetT
BS-yh eL
FN0650 BC1709
CTC00901
FN062 4
CTC02520
BB 0637
CPE2317
FN1420
CTC0 2529
VCA0193
S O10 87
FN1422
BC0373
B B0 638
FN207 7
BH3946
BS-mleN
VC2037
SA2292
HI1107
V V 21061
MleN
Some confirmed predictions
PREDICTION
GENOME
REF – Prediction
REF – Verification
Mechanism of regulation of
riboflavin metabolism and transport
genes
Bacteria (Bacillus subtilis,
Escherichia coli)
Vitreschak et al.,
2002
Winkler et al.,
2002b; Mironov et
al., 2000
Mechanism of regulation of thiamin
metabolism and transport genes
Bacteria and archaea (Bacillus
subtilis, Escherichia coli)
Rodionov et al.,
2002b
Winkler et al.,
2002a
Transcription regulatory signal for
the nitrogen-fixation pathway
Methanogenic archaea
(Methanococcus maripaludis)
Gelfand et al.,
2000
Kessler and Leigh,
1999; Lie and
Leigh, 2003
Acyl-CoA-dehydrogenase FadE is
encoded by gene yafH
Gamma-proteobacteria
(Escherichia coli)
Sadovskaya et al.,
2001
Campbell and
Cronan, 2002
ThiN, an enzyme (MTH861) or
ThiD domain functionally
equivalent to ThiE
T. maritima, archaea
(Methanobacterium
thermoautotrophicum)
Rodionov et al.,
2002b
Morett et al., 2003
Riboflavin transporter YpaA:
specificity and regulation
Gram-positive bacteria (Bacillus
subtilis)
Gelfand et al.,
1999
Kreneva et al., 2000
Oligogalacturonide ABC-transporter Gamma-proteobacteria (Erwinia
ogtABCD (togMNAB)
chrysanthemi)
Rodionov et al.,
2000
Hugouvieux-CottePattat et al., 2001
Arginine ABC-transporter yqiXYZ:
specificity and regulation
Bacteria (Bacillus subtilis)
Makarova et al.,
2001
Sekowska et al.,
2001
Methionine transporter MetD
Bacillus subtilis, Escherichia coli Zhang et al., 2003
Zhang et al., 2003
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 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.)
• Andrei Mironov
•
•
•
•
•
•
•
•
•
•
•
•
Anna Gerasimova
Olga Kalinina
Alexei Kazakov
Ekaterina Kotelnikova
Galina Kovaleva
Pavel Novichkov
Olga Laikova
Ekaterina Panina
(now at UCLA, USA)
Elizabeth Permina
Dmitry Ravcheev
Dmitry Rodionov
Alexey Vitreschak
(on leave at 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 Academu of
Sciences