Comparative genomics and metabolic reconstruction of

bacterial pathogens

Mikhail GelfandInstitute 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

acpP

fabD

accA accD accB

accC

fabHfabF

fabG

fabZ

fabI

Gene fabI of Enoyl-ACP reductase (EC 1.3.1.9) is

missing in the genome 12B, and a number of Streptococci

fabI (Enoyl-ACP reductase, EC 1.3.1.9) target of triclosan.

Enzymatic activity, but no gene in Streptococci

Identification of a candidate by positional clustering

Genome XGenome X

TR? 6.3.4.15fabI hyp3.5.1.?hyp

TR? 2.1.1.79 FRNS

Genome YGenome Y

5.99.1.2

Clostridium acetobutylicum Clostridium acetobutylicum

TR?

Streptococcus pyogenesStreptococcus pyogenes

? hyp

TR? ?

fabH acpP

?

fabG fabF accAaccDaccCaccB fabZfabD

fabG fabF accBfabD accAaccDaccCfabZ

fabG fabF accAaccDaccCaccB fabZfabDfabH acpP

fabH acpP

fabH acpP fabG fabF accAaccDaccCaccB fabZfabD

Binding sites of FabR (“Tr?”, HTH)

HTH fabKfabH acpP fabG fabF accAaccDaccCaccB fabZfabDFad (42.1.17)

CONSENSUS acTTTGAtwaTCAAAgt

E. faecalis HTH-1 AgTTTGggTATCAAAGT

E. faecalis HTH-2 AgTTTGAacATCAAAtg

E. faecalis HTH-3 GtTTTGATAATCAAAGT

E. faecium HTH-1 ACTTTGATAATCAAAaT

E. faecium HTH-2 AgTTTGAacATCAAAag

E. faecium HTH-3 gaTTTGATAATCAAAcT

S. pyogenes 4.2.1.17 GaTTTGATTATCAAAtg 1

S. pyogenes HTH-1 AaTTTGATTgTCAAAGT 2

S. pyogenes fabK-1 CtTTTGATAtTCAAAtT 3

S. pyogenes fabK-2 AgTTTGATTATCAAAtT 4

S. pneumoniae 4.2.1.17 ACTTTGAcAgTgAAAta

S. pneumoniae HTH-1 gtTTTGATTgTaAAAGT

S. pneumoniae fabK-1 AgTTTGAcTgTCAAAtT

S. mutans 4.2.1.17-1 ACTTTGATTtTCAAAcT

S. mutans 4.2.1.17-2 AaTTTGATTATCttAaT

S. mutans HTH-1 ACTTTGATAgTCAAAGT

S. mutans fabK-1 AgTTTGAcAtTCAAAtc

S. mutans fabK-2 AgTTTGAcTgTCAAAtT

1 2 3 4

Metabolic reconstruction of the thiamin biosynthesis(new genes/functions shown in red)

thiN (confirmed)

(Gram-positive bacteria)

(Gram-negative bacteria)

Transport of HMPTransport of HET

Purine pathway

Carbohydrate metabolism in Streptococcus and Lactococcus spp.

S.

pneu

mon

iae

S.

pyog

enes

S.

equi

S.

uber

is

S.

agal

actia

e

S.

mut

ans

S.

ther

mop

hilu

s

S.

suis

L. la

ctis

L. c

asei

L. g

asse

ri

L. d

elbr

ueck

ii

P.

pent

osac

eus

L. b

revi

s L.

mes

ente

roid

esO

enoc

occu

s oe

ni

unknownarabinosearbutincellobiosedextranesculinfructosefucosegalactoseglucoseinulinlactosemaltosemannitolmannosemelibioseN-AcGluraffinoseribosesalicinsorbitolsorbosesucrosetagatosetrehalosexylose

Only biochemical data, genes unknown

Experimentally verified genes

Biochemical data and genomic predictions

Only genomic predictions

An uncharacterized locus in invasive speciesS

. pn

eum

onia

e

S.

pyog

enes

S.

equi

S.

uber

is

S.

agal

actia

e

S.

mut

ans

S.

ther

mop

hilu

s

S.

suis

L. la

ctis

L. c

asei

L. g

asse

ri

L. d

elbr

ueck

ii

P.

pent

osac

eus

L. b

revi

s L.

mes

ente

roid

esO

enoc

occu

s oe

ni

unknownarabinosearbutincellobiosedextranesculinfructosefucosegalactoseglucoseinulinlactosemaltosemannitolmannosemelibioseN-AcGluraffinoseribosesalicinsorbitolsorbosesucrosetagatosetrehalosexylose

S. pneumoniae

S. pyogenes

S. equi

S. agalactiae

S. suis

Structure of the genome loci

IS

IS

IS

S. pyogenes, S. agalactiae

S. equi

S. pneumoniae TIGR4

S. suis

S. pneumoniae R6

Gene functions

3-(4-deoxy-beta-D-gluc-4-enuronosyl)-N-acetyl-D-glucosamine

PTS transporterhydrolaseisomeraseoxidoreductasedehydrogenasekinasealdolasepyruvate +

D-glyceraldehyde 3-phosphate

hyaluronidase(hyaluronate lyase)

RegR

Candidate regulatory signal

Structure of the genome loci - 2

IS

IS

IS

S. pyogenes, S. agalactiae

S. equi

S. pneumoniae TIGR4

S. suis

S. pneumoniae R6

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

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 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

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 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)

• 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

• Andrei Osterman (Burnham Institute, San-Diego, USA) (fatty acids)