Comparative genomics and metabolic reconstruction of bacterial pathogens

1
Comparative genomics and metabolic reconstruction
of bacterial pathogens

• Mikhail Gelfand
• Institute for Information Transmission Problems,
  RAS
• GPBM-2004

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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

3
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

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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

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Missing link in fatty acid biosynthesis in
Streptococci
accA
accD
accB
fabI (Enoyl-ACP reductase, EC 1.3.1.9) target of
triclosan. Enzymatic activity, but no gene in
Streptococci
Gene fabI of Enoyl-ACP reductase (EC 1.3.1.9) is
missing in the genome 12B, and a number of
Streptococci
accC
fabD
fabH
fabF
fabG
fabZ
fabI
acpP
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Identification of a candidate by positional
clustering
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Binding sites of FabR (Tr?, HTH)
HTH
acpP
Fad (42.1.17)
fabK
fabH
fabG
fabF
accA
accD
accC
fabZ
accB
fabD
1
2
3
4
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Metabolic reconstruction of the thiamin
biosynthesis(new genes/functions shown in red)
Purine pathway
thiN (confirmed)
Transport of HET
Transport of HMP
(Gram-positive bacteria)
(Gram-negative bacteria)
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Carbohydrate metabolism in Streptococcus and
Lactococcus spp.
Only biochemical data, genes unknown
Experimentally verified genes
Biochemical data and genomic predictions
Only genomic predictions
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An uncharacterized locus in invasive species
S. pneumoniae S. pyogenes S. equi S.
agalactiae S. suis
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Structure of the genome loci
S. pyogenes, S. agalactiae
S. equi
S. pneumoniae TIGR4
S. pneumoniae R6
S. suis
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Gene functions

• 3-(4-deoxy-beta-D-gluc-4-enuronosyl)-N-acetyl-D-gl
  ucosamine
• PTS transporter
• hydrolase
• isomerase
• oxidoreductase
• dehydrogenase
• kinase
• aldolase
• pyruvate D-glyceraldehyde 3-phosphate

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Candidate regulatory signal
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Structure of the genome loci - 2
S. pyogenes, S. agalactiae
S. equi
S. pneumoniae TIGR4
S. pneumoniae R6
S. suis
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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

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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

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Genomes and regulators
nZURFUR family
???
AdcR ?MarR family
pZURFUR family
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Regulators and signals
nZUR-?
nZUR-?
GAAATGTTATANTATAACATTTC
GATATGTTATAACATATC
GTAATGTAATAACATTAC
TTAACYRGTTAA
AdcR
pZUR
TAAATCGTAATNATTACGATTTA
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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

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zinT regulation
zinT is regulated by zinc repressors (nZUR-?,
nZUR-?, pZUR)
zinT is isolated
E. coli, S. typhi, K. pneumoniae
Gamma-proteobacteria
Alpha-proteobacteria
A. tumefaciens, R. sphaeroides
B. subtilis, S. aureus S. pneumoniae, S. mutans,
S. pyogenes, L. lactis, E. faecalis
Bacillus group
Streptococcus group
adcA-zinT is regulated by zinc repressors (pZUR,
AdcR) (ex. L.l.)
fusion adcA-zinT
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ZinT protein sequence analysis
TM
Zn
AdcA
Y. pestis, V. cholerae, B. halodurans
ZinT
S. aureus, E. faecalis, S. pneumoniae, S.
mutans, S. pyogenes
E. coli, S. typhi, K. pneumoniae, A. tumefaciens,
R. sphaeroides, B. subtilis
L. lactis
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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
  TM-segment)
• ZinT has a zinc-binding domain
• ZinT conclusions
• ZinT is a new type of zinc-binding component of
  zinc ABC transporter

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Zinc regulation of PHT (pneumococcal histidine
triad) proteins of Streptococci
S. pneumoniae
S. pyogenes
S. equi
S. agalactiae
phtE
lmb
phtD
lmb
phtD
phtB
phtA
phtY
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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

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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

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incorrect ?

• 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)

• Zinc-binding domains in zinc transporters
• EEEHEEHDHGEHEHSH
• HSHEEHGHEEDDHDHSH
• EEHGHEEDDHHHHHDED
• DEHGEGHEEEHGHEH
• (histidine-aspartate-glutamate-rich)

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Analyis of PHP proteins (contd)

• 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
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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

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Zinc and (paralogs of) ribosomal proteins
L36 L33 L31 S14
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
nZUR
pZUR
AdcR
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Zn-ribbon motif (Makarova-Ponomarev-Koonin, 2001)
L36 L33 L31 S14
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 () () ()
nZUR
pZUR
AdcR
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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

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Bad scenario
Zn-deplete conditions all Zn utilized by the
ribosomes, no Zn for Zn-dependent enzymes
Zn-rich conditions
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Regulatory mechanism
Sufficient Zn
ribosomes
R
repressor
Zn-dependentenzymes
Zn starvation
R
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Good scenario
Zn-deplete conditions some ribosomes without Zn,
some Zn left for the enzymes
Zn-rich conditions
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Prediction (Proc Natl Acad Sci U S A. 2003 Aug
19100(17)9912-7.)
and confirmation (Mol Microbiol. 2004
Apr52(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

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