Comparative genomics and metabolic reconstruction of bacterial genomes

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Comparative genomics and metabolic reconstruction
of bacterial genomes

• Mikhail S. Gelfand
• Meeting of HHMI International Research Scholars
• Tallinn, 2004

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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

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Metabolic reconstruction of the lysine pathway

• 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

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Metabolic reconstruction of the methionine pathway

• Predictions
• Genes for theSAM-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

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Aromatic amino acid regulonsin Gram-positive
bacteria
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Prediction of transporter specificity via
analysis of regulation
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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 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
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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 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 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

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