Comparative genomics and metabolic reconstruction of bacterial genomes Mikhail S. Gelfand Meeting of...
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Transcript of Comparative genomics and metabolic reconstruction of bacterial genomes Mikhail S. Gelfand Meeting 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
Metabolic reconstruction of the lysine pathway
-aspartyl-phosphate
aspartate semialdehyde
homoserine
dihydrodipicolinate
tetrahydrodipicolinate
N-acetyl-2-amino-6-ketopimelateN-succinyl-2-amino-6-ketopimelate
N-acetyl-L,L-diaminopimelateN-succinyl-L,L-diaminopimelate
L,L-diaminopimelate
meso -diaminopimelate
Lysine transport
L-aspartate
lysC,dapG,yclMlysC,thrA,m etL
asd
hom
thrA,m etL
dapA
dapB
dapDdapD
ykuR
dapC(argD)
ddh
patA
dapE
dapF, dal
lysA
• Predictions:– Genes for the acetylated
pathway in Gram-positive bacteria
– Positive regulation of the lysine catabolism genes in Thermoanaerobacter and Fusobacterium by LYS-elements: 1st example of activating riboswitches
– New transporters
-aspartyl-phosphate
aspartate semialdehyde
homoserine
dihydrodipicolinate
tetrahydrodipicolinate
N-acetyl-2-amino-6-ketopimelateN-succinyl-2-amino-6-ketopimelate
N-acetyl-L,L-diaminopimelateN-succinyl-L,L-diaminopimelate
L,L-diaminopimelate
meso -diaminopimelate
Lysine transport
L-aspartate
lysC,dapG,yclMlysC,thrA,m etL
asd
hom
thrA,m etL
dapA
dapB
dapDdapD
ykuR
dapC(argD)
ddh
patA
dapE
dapF, dal
lysA
Metabolic reconstruction of the methionine pathway
• Predictions:– Genes for the
SAM-recycling pathway– Transporters for
methionine and methylthiribose
– Other enzymes– Transcriptional regulation
in Streptococci– Complicated S-box and
Cys-T-box regulation of the ubiG-yrhBA operon in C. acetobutylicum: activation via repression of the antisense transcript
Cystathionine
Homocysteinemethyl-THFbetaine
dim ethylglycine
Sulfide
CH
methylene-THF
THF
3
O-acetylhomoserine
Homoserine
Aspartate semialdehyde
Methionine
S-ribosyl-homocysteine
(SRH)
S-adenosyl-homocysteine
(SAH)
S-adenosyl-methionine
(SAM)
Methylthioribose (MTR)MTA
Threonine
metI yrhB
metC yrhA
metF
yxjH*
metK
mtnKSUVW XYZ
hom
cysH-...metB
metH ,
metX
metE ,mtn
mtn
metY
ubiG yrhA
antisense transcript
Cysteine
S-adenosylmethionine
yrhB
AA
Cys-T-box S-box
sense transcript
Aromatic amino acid regulonsin Gram-positive bacteria
Prediction of transporter specificity via analysis of regulation
BC14 34
FN062 4
SON-3
CJ
CPE
LysW
MetT
TyrT
MleN
DF
CT CCB
OB
S ON-2VC-2
NMB
S ON-1
VC-1
BHHP
C
TTE-nhaC
AC0744
FN 0978
BL1111
CTC00901
OB2874OB1118
NMB0536
FN0352BC4121
EF -nhaC 1
EF-nhaC2
PPE
LP-nha2
LP -nha 1 L
L
M
GA
ELB
BS-yh eL
BS-mleN
FN0650
VC2037
BC1709
SA2292
HI1107
V V 21061FN207 7
BH3946
BC0373
FN1422
B B0638
BB 0637
FN1420
CTC0 2529S O10 87
VCA0193
BT1270
C
C B
TC02520
CPE2317
FN1414
SA2117
Archaea
c lostr idia
Pasteurella ceae
Some confirmed predictionsPREDICTION 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 ogtABCD (togMNAB)
Gamma-proteobacteria (Erwinia chrysanthemi)
Rodionov et al., 2000
Hugouvieux-Cotte-Pattat 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
nZURFUR family
???
AdcR ?MarR family
pZURFUR family
Regulators and signals nZUR-nZUR-
AdcRpZUR
TTAACYRGTTAA
GATATGTTATAACATATCGAAATGTTATANTATAACATTTC
GTAATGTAATAACATTAC
TAAATCGTAATNATTACGATTTA
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
fusion: adcA-zinT
E. coli, S. typhi, K. pneumoniae Gamma-proteobacteria
Alpha-proteobacteria
B. subtilis, S. aureus
S. pneumoniae, S. mutans, S. pyogenes, L. lactis, E. faecalis
Bacillus group
Streptococcus group
zinT is regulated by zinc repressors (nZUR-, nZUR-, pZUR)
adcA-zinT is regulated by zinc repressors (pZUR, AdcR) (ex. L.l.)
A. tumefaciens, R. sphaeroides
ZinT: protein sequence analysis
E. coli, S. typhi, K. pneumoniae, A. tumefaciens, R. sphaeroides, B. subtilis
L. lactis
Y. pestis, V. cholerae, B. halodurans
TM Zn AdcA
S. aureus, E. faecalis, S. pneumoniae, S. mutans, S. pyogenes
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 TM-
segment)• 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. equiS. agalactiae
lmb phtD phtE
phtBphtA
lmb phtD
S. pyogenes
phtY
lmb phtD
zinc regulation shown in experiment
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:
EEEHEEHDHGEHEHSH
HSHEEHGHEEDDHDHSHEEHGHEEDDHHHHHDED
DEHGEGHEEEHGHEH
(histidine-aspartate-glutamate-rich)
• Histidine triads in streptococci:
HGDHYHY 7 out of 21
HGDHYHF 2 out of 21
HGNHYHF 2 out of 21
HYDHYHN 2 out of 21
HMTHSHW 2 out of 21
(specific pattern of histidines and aromatic amino acids)
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:
PHT internalin H-rich
4 HIS TRIADS LRR IRHDYNHNHTYEDEEGHAHEHRDKDDHDHEHED
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
HypothesisPHT proteins are adhesins involved in the attachment of
streptococci to epithelium cells, leading to invasion
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 – – – – – – +
nZU
RpZU
RAdc
R
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 (–) (–) – – – (–) +
nZU
RpZU
RAdc
R
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
ribosomes
Zn-dependentenzymes
R
Sufficient Zn
Zn starvation
R
repressor
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