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