Metabolism

of

halophilic

archaea

In

spite

of

their

common

hypersaline

environment

halophilic

archaea

are

surprisingly

different

in

their

nutritional

demands

and

metabolic

pathways

The

metabolic

diversity

of

halophilic

archaea

was

investigated

at

the

genomic

level

through

systematic

metabolic

reconstruction

and

comparative

analysis

of

four

completely

sequenced

species

Halobacterium

salinarum

Haloarcula

marismortui

Haloquadratum

walsbyi

and

the

haloalkaliphile

Natronomonas

pharaonis

The

comparative

study

reveals

different

sets

of

enzyme

genes

amongst

halophilic

archaea

e.g.

in

glycerol

degradation

pentose

metabolism

and

folate

synthesis

The

carefully

assessed

metabolic

data

represent

a

reliable

resource

for

future

system

biology

approaches

as

it

also

links

to

current

experimental

data

on

-LRB-

halo

-RRB-

archaea

from

the

literature

Introduction

Extremely

halophilic

archaea

are

a

diverse

group

of

euryarchaeota

that

inhabit

hypersaline

environments

-LRB-

M

-RRB-

such

as

salt

lakes

salt

ponds

and

marine

salterns

They

are

often

referred

to

as

halobacteria

named

after

the

model

organism

Halobacterium

salinarum

whose

proton

pump

bacteriorhodopsin

is

one

of

the

best-studied

membrane

proteins

Although

haloarchaea

share

certain

features

in

order

to

adapt

to

their

extreme

environment

i.e.

acidic

protein

machineries

respiratory

chains

and

rhodopsins

their

metabolism

is

considerably

different

from

each

other

Although

there

are

carbohydrate-utilizing

species

such

as

Haloferax

mediterranei

Haloarcula

marismortui

and

Halococcus

saccharolyticus

which

catabolize

hexoses

-LRB-

glucose

fructose

-RRB-

pentoses

-LRB-

arabinose

xylulose

-RRB-

sucrose

and

lactose

-LRB-

Rawal

et

al.

Altekar

and

Rangaswamy

Johnsen

et

al.

Johnsen

and

Schonheit

-RRB-

other

haloarchaea

like

H.

salinarum

are

not

capable

of

sugar

degradation

-LRB-

Rawal

et

al.

-RRB-

Instead

non-carbohydrate-utilizing

species

thrive

on

amino

acids

and

typical

compounds

of

hypersaline

habitats

Haloferax

volcanii

for

example

is

able

to

grow

on

glycerol

and

organic

acids

-LRB-

Kauri

et

al.

-RRB-

excreted

by

primary

halophilic

producers

Dunaliella

salina

-LRB-

Elevi

Bardavid

et

al.

-RRB-

and

Microcoleus

chthonoplastes

-LRB-

Zviagintseva

et

al.

-RRB-

respectively

Haloarchaea

differ

not

only

in

their

catabolic

pathways

but

also

in

their

nutritional

requirements

While

simple

growth

media

were

described

for

Haloferax

volcanii

-LRB-

Kauri

et

al.

-RRB-

and

Natronomonas

pharaonis

-LRB-

Falb

et

al.

-RRB-

H.

salinarum

exhibits

complex

nutritional

demands

Growth

of

Halobacterium

cells

is

often

limited

in

synthetic

media

-LRB-

Oesterhelt

and

Krippahl

Grey

and

Fitt

-RRB-

in

spite

of

rich

amino

acid

-LRB-

at

least

amino

acids

-RRB-

and

cofactor

supplements

-LRB-

folate

biotin

thiamine

-RRB-

The

metabolic

diversity

of

halophilic

archaea

has

not

yet

been

investigated

at

the

genomic

level

by

metabolic

reconstruction

and

comparative

analysis

The

absence

of

enzyme

genes

for

numerous

metabolic

reactions

in

archaeal

genomes

has

limited

reconstruction

of

metabolic

pathways

so

far

However

many

of

these

pathway

gaps

have

been

elucidated

recently

with

the

discovery

of

novel

non-orthologous

enzymes

in

archaea

e.g.

for

the

de

novo

synthesis

of

mevalonate

-LRB-

Barkley

et

al.

Grochowski

et

al.

-RRB-

purines

-LRB-

Graupner

et

al.

Ownby

et

al.

-RRB-

and

cobamide

-LRB-

Woodson

et

al.

Woodson

and

Escalante-Semerena

Zayas

et

al.

-RRB-

Archaea

also

employ

novel

enzymes

and

precursors

for

pentose

formation

-LRB-

Grochowski

et

al.

-RRB-

and

aromatic

amino

acid

synthesis

-LRB-

White

Porat

et

al.

-RRB-

circumventing

absent

enzymes

of

the

classical

pentose-phosphate

pathway

For

this

detailed

review

of

haloarchaeal

metabolism

metabolic

pathways

of

halophilic

archaea

were

systematically

reconstructed

Currently

genome

sequences

of

four

diverse

haloarchaeal

species

are

available

for

comparative

analysis

-LRB-

Table

-RRB-

namely

that

of

the

model

organism

H.

salinarum

-LRB-

Ng

et

al.

Pfeiffer

et

al.

-RRB-

the

metabolic-versatile

H.

marismortui

-LRB-

Baliga

et

al.

-RRB-

the

haloalkaliphile

N.

pharaonis

-LRB-

Falb

et

al.

-RRB-

and

the

square-shaped

Haloquadratum

walsbyi

-LRB-

Bolhuis

et

al.

-RRB-

The

presented

metabolic

data

will

be

a

valuable

resource

for

future

system

biology

approaches

as

each

metabolic

reaction

has

been

carefully

assessed

and

linked

to

experimental

data

from

the

literature

Table

of

the

currently

sequenced

haloarchaeaH

salinarum

strains

marismortuiH

walsbyiN

pharaonisGene

identifierVNG/OErrn

pNGHQNPSalt

optimum

-LSB-

M

-RSB-

M

-LRB-

pH

-RRB-

IsolationSalted

fishDead

Sea

-LRB-

Israel

-RRB-

Solar

saltern

-LRB-

Spain

-RRB-

Soda

lake

-LRB-

Egypt

-RRB-

Main

research

interestsRhodopsins

signal

transductionVersatile

nitrogen

metabolismSquare-shaped

cells

halomucinHaloalkaliphilicity

respiratory

chainGenome

size

-LSB-

Mb

-RSB-

-LRB-

incl

CHRII

-RRB-

GC

rRNA

genes

-LRB-

motility

-RRB-

YesYesNoYes

Transducer

-LRB-

-RRB-

Rhodopsin

genes

-LRB-

bop

hop

sop

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

aTwo

strains

of

H.

salinarum

have

been

sequenced

strains

-LRB-

Ng

et

al.

-RRB-

and

-LRB-

Pfeiffer

et

al.

-RRB-

These

are

virtually

identical

and

differ

only

in

their

distribution

of

insertion

elements

and

their

plasmid

arrangements

Unless

mentioned

otherwise

the

two

strains

are

not

distinguished

in

this

review

because

they

exhibit

analogous

sets

of

enzyme

encoding

genes

Gene

identifiers

of

H.

salinarum

strain

-LRB-

e.g.

-RRB-

will

be

used

throughout

the

text

Materials

and

methods

Metabolic

reconstruction

procedure

Metabolic

pathways

of

H.

salinarum

were

reconstructed

in

a

two-step

procedure

At

first

relevant

reactions

that

take

part

in

a

given

metabolic

pathway

were

chosen

from

the

complete

set

of

reference

reactions

downloaded

from

the

KEGG

ligand

database

-LRB-

Kanehisa

et

al.

-RRB-

In

the

second

step

organism-specific

metabolic

pathways

were

reconstructed

by

flagging

each

reaction

existent

or

non-existent

in

H.

salinarum

with

a

certain

confidence

value

The

enzyme

gene

predicted

to

catalyze

this

reaction

in

H.

salinarum

was

linked

to

the

reaction

In

case

experimental

data

is

available

from

the

literature

a

comment

was

linked

to

the

reaction

or

enzyme

gene

which

includes

the

PubMed

identifier

of

the

external

reference

Each

reaction

was

manually

assessed

based

on

automatic

enzyme

assignments

derived

from

various

similarity

searches

-LRB-

i.e.

blast

search

with

UniProt

enzymes

COG

assignments

Pfam

search

-RRB-

genome

data

-LRB-

-RRB-

and

experimental

literature

data

for

H.

salinarum

In

case

a

reaction

or

enzyme

is

confirmed

by

experiments

but

no

genetic

evidence

was

found

in

the

genome

-LRB-

indicating

novel

yet

unknown

enzyme

variants

-RRB-

the

reaction

was

marked

existent

and

the

conflict

was

labeled

The

metabolic

data

for

H.

salinarum

given

in

Supplementary

Material

was

internally

stored

in

a

MySQL

database

and

managed

via

a

Web-interface

Comparative

analysis

of

the

complete

haloarchaeal

gene

sets

For

comparison

of

haloarchaeal

gene

sets

protein

databases

-LRB-

fasta

format

-RRB-

of

the

four

completely

sequenced

haloarchaeal

genomes

H.

salinarum

strain

N.

pharaonis

H.

walsbyi

and

H.

marismortui

were

blasted

against

each

other

and

against

the

non-redundant

-LRB-

nr

-RRB-

database

For

example

each

predicted

protein

sequence

of

N.

pharaonis

-LRB-

species

-RRB-

was

searched

against

the

H.

salinarum

database

-LRB-

species

-RRB-

and

the

nr

database

-LRB-

all

species

-RRB-

then

the

difference

between

the

best

blast

score

for

the

general

database

-LRB-

nr

-RRB-

and

for

the

related

species

H.

salinarum

was

calculated

Listing

N.

pharaonis

sequences

by

these

differences

in

descending

order

detects

genes

present

in

N.

pharaonis

but

absent

in

H.

salinarum

In

few

cases

the

N.

pharaonis

gene

had

a

close

homolog

in

the

general

database

but

only

a

more

distant

homolog

in

H.

salinarum

Such

a

gene

is

considered

present

in

H.

salinarum

when

the

blast

hit

is

highly

significant

-LRB-

E-value

better

than

e

-RRB-

were

considered

The

differential

blast

analysis

routine

was

applied

to

all

combinations

of

the

set

of

four

haloarchaeal

genomes

A

list

of

genes

present

in

only

a

subset

of

genomes

was

obtained

and

filtered

for

metabolic

function

-LRB-

Supplementary

Material

-RRB-

Results

and

discussion

The

metabolism

of

H.

salinarum

was

reconstructed

by

evaluating

metabolic

reactions

of

biochemical

reference

pathways

from

KEGG

-LRB-

Supplementary

Material

-RRB-

Enzyme

annotations

from

an

automatic

assignment

routine

and

from

curated

genome

data

-LRB-

-RRB-

were

carefully

assessed

in

order

to

avoid

misassignments

due

to

domain

rearrangements

-LRB-

e.g.

purine

biosynthesis

Fig.

-RRB-

or

overannotation

of

the

exact

substrate

specifity

-LRB-

e.g.

dehydrogenase

complex

-RRB-

Novel

enzyme

variants

closing

former

pathway

gaps

in

archaea

as

well

as

previously

published

experimental

data

of

H.

salinarum

were

also

considered

throughout

the

reconstruction

process

Comparative

analysis

of

the

currently

sequenced

haloarchaea

H.

marismortui

H.

walsbyi

N.

pharaonis

and

H.

salinarum

disclosed

major

metabolic

differences

i.e.

alternative

enzymes

and

metabolic

pathways

employed

by

haloarchaea

-LRB-

Supplementary

Material

-RRB-

In

the

following

reconstructed

metabolic

pathways

of

halophilic

archaea

will

be

reviewed

in

detail

with

emphasis

on

different

enzyme

genes

and

metabolic

pathways

which

lead

to

the

diverse

catabolic

and

anabolic

capabilities

of

halophilic

archaea

In

case

an

enzyme

gene

is

present

in

all

four

haloarchaeal

species

only

the

N.

pharaonis

identifier

-LRB-

e.g.

-RRB-

will

be

exemplarily

mentioned

Glycolytic

pathways

Major

differences

in

sugar

degradation

capabilities

were

identified

amongst

halophilic

archaea

that

reflect

the

previous

classification

of

halophilic

archaea

into

carbohydrate

and

non-carbohydrate

utilizers

-LRB-

Fig.

Supplementary

Material

-RRB-

H.

salinarum

H.

marismortui

and

H.

walsbyi

possess

all

required

enzyme

genes

for

the

semi-phosphorylated

Entner-Douderoff

-LRB-

ED

-RRB-

pathway

previously

described

for

carbohydrate-utilizing

haloarchaea

-LRB-

Rawal

et

al.

Danson

and

Hough

Verhees

et

al.

-RRB-

However

only

H.

marismortui

and

H.

walsbyi

encode

a

classical

KDPG

aldolase

which

is

the

key

enzyme

of

the

pathway

in

addition

to

the

novel

archaeal

KD

-LRB-

P

-RRB-

G

aldolase

recently

described

for

thermophilic

archaea

-LRB-

Ahmed

et

al.

-RRB-

Thus

operation

of

different

ED

pathway

variants

between

H.

salinarum

and

the

other

two

species

is

indicated

It

should

further

be

noted

that

neither

the

ED

pathway

nor

growth

on

glucose

as

the

sole

carbon

source

has

been

established

for

Halobacterium

so

far

-LRB-

Gochnauer

and

Kushner

Rawal

et

al.

-RRB-

N.

pharaonis

is

likely

to

be

incapable

of

glucose

degradation

because

it

completely

lacks

all

enzyme

genes

of

the

ED

pathway

Fig.

central

intermediary

metabolism

in

halophilic

archaea

The

reaction

arrows

depict

the

reconstructed

metabolism

of

the

reference

species

H.

salinarum

-LRB-

green

reaction

exists

red

reaction

absent

-RRB-

The

four

geometric

symbols

illustrate

differences

in

enzyme

gene

sets

between

the

four

sequenced

haloarchaea

-LRB-

square

H.

marismortui

circle

H.

walsbyi

diamond

N.

pharaonis

triangle

H.

salinarum

green

gene

exists

red

gene

absent

-RRB-

Reactions

that

have

been

investigated

experimentally

through

NMR

studies

or

enzyme

activity

tests

in

H.

salinarum

are

highlighted

by

bold

arrows

-LRB-

green

reaction

exists

red

reaction

absent

-RRB-

For

some

of

the

experimentally

verified

reactions

there

are

currently

no

genetic

evidences

in

the

H.

salinarum

genome

-LRB-

green

arrows

with

red

border

-RRB-

Vice

versa

some

reactions

have

been

experimentally

excluded

but

probable

enzyme

genes

are

present

in

the

H.

salinarum

genome

-LRB-

red

arrows

with

green

border

-RRB-

Compounds

that

have

been

identified

through

labeling

studies

are

marked

by

asterisks

Proposed

essential

amino

acids

for

H.

salinarum

are

indicated

-LRB-

E

-RRB-

Compounds

AraHex

Frc

fructose

GAP

Glc

glucose

Gluc

gluconate

Glyn

glycerone

Glyc

glycerol

Glyox

glyoxylate

Icit

isocitrate

KDPG

Mal

malate

OA

oxalacetate

PEP

phosphoenolpyruvate

PGA

Pyr

pyruvate

Suc

succinate

sedoheptulose

AcCoA

acetyl-CoA

The

carbohydrate-utilizing

H.

marismortui

has

acquired

a

wide

range

of

enzyme

genes

involved

in

the

uptake

and

degradation

for

various

sugars

Fructose

and

sucrose

are

likely

degraded

via

the

modified

Embden-Meyerhof

-LRB-

EM

-RRB-

pathway

which

has

been

described

for

haloarchaea

-LRB-

Altekar

and

Rangaswamy

-RRB-

The

pathway

involves

the

key

enzyme

-LRB-

EC

-RRB-

and

sucrose

hydrolase

-LRB-

EC

-RRB-

Haloarcula

also

contains

probable

gene

clusters

for

maltose

uptake

-LRB-

maltose

ABC

transporter

-RRB-

and

metabolism

-LRB-

several

α-glucosidases

e.g.

-RRB-

as

well

as

a

gene

cluster

for

D-xylulose

oxidation

-LRB-

-RRB-

The

latter

encodes

the

previously

characterized

D-xylulose

dehydrogenase

-LRB-

-RRB-

-LRB-

Johnsen

and

Schonheit

-RRB-

Embden-Meyerhof

pathway

and

gluconeogenesis

Consistent

with

the

previous

biochemical

findings

-LRB-

Rawal

et

al.

Altekar

and

Rangaswamy

-RRB-

genes

for

-LRB-

EC

-RRB-

the

key

enzyme

of

the

classical

Embden-Meyerhof

pathway

are

absent

in

all

four

haloarchaeal

genomes

Alternative

archaeal

types

of

that

depend

on

ADP

-LRB-

Thermococcus

Pyrococcus

-RRB-

or

PP

-LRB-

i

-RRB-

-LRB-

Thermoproteus

-RRB-

-LRB-

Kengen

et

al.

Selig

et

al.

-RRB-

as

co-substrates

have

not

been

found

either

However

a

gene

for

fructose

degradation

via

the

haloarchaeal

variant

of

the

EM

pathway

-LRB-

Altekar

and

Rangaswamy

Rangaswamy

and

Altekar

b

-RRB-

is

present

in

H.

marismortui

-LRB-

-RRB-

Although

the

upper

hexose

part

of

the

classical

EM

pathway

is

missing

in

haloarchaea

the

lower

triose

part

of

the

pathway

that

leads

to

pyruvate

is

likely

to

be

functional

in

all

species

as

required

enzyme

genes

are

encoded

in

their

genomes

In

fact

several

enzyme

activities

have

already

been

proven

in

H.

salinarum

-LRB-

Rawal

et

al.

-RRB-

The

final

glycolysis

step

from

phosphoenolpyruvate

to

pyruvate

might

be

catalyzed

by

pyruvate

kinase

-LRB-

EC

e.g.

-RRB-

and

pyruvate

water

dikinase

-LRB-

EC

e.g.

-RRB-

in

haloarchaea

In

a

thermophilic

archaeon

both

enzymes

participated

in

glycolysis

but

only

deletion

of

the

pyruvate

water

dikinase

gene

completely

omitted

growth

on

sugars

-LRB-

Imanaka

et

al.

-RRB-

Complete

gene

sets

for

the

reverse

EM

pathway

-LRB-

gluconeogenesis

-RRB-

from

pyruvate

to

phosphorylated

glucose

were

identified

in

all

four

haloarchaeal

genomes

Consistently

labeling

experiments

-LRB-

Sonawat

et

al.

Ghosh

and

Sonawat

-RRB-

and

enzyme

activity

studies

-LRB-

Rawal

et

al.

-RRB-

have

confirmed

gluconeogenesis

in

H.

salinarum

which

is

required

to

synthesize

hexoses

for

membrane

constituents

For

example

glucose

was

found

to

be

incorporated

into

different

sugar

moieties

-LRB-

glucose

mannose

galactose

-RRB-

of

halobacterial

glycolipids

-LRB-

Weik

et

al.

-RRB-

Furthermore

saccharide

units

are

attached

to

surface

proteins

of

H.

salinarum

such

as

the

S-layer

protein

and

flagellins

-LRB-

Sumper

-RRB-

Sugar

moieties

of

lipids

and

proteins

are

likely

synthesized

via

nucleotide

sugars

In

accordance

with

this

several

nucleotide

sugar

enzymes

such

as

UDP-glucose

-LRB-

EC

e.g.

-RRB-

and

UDP-glucose

-LRB-

EC

e.g.

-RRB-

are

present

in

haloarchaeal

genomes

Entner-Douderoff

pathway

Variants

of

the

classical

Entner-Douderoff

pathway

are

common

in

the

archaeal

domain

of

life

-LRB-

Danson

and

Hough

-RRB-

Halophilic

archaea

such

as

H.

vallismortis

-LRB-

Altekar

and

Rangaswamy

-RRB-

operate

the

semi-phosphorylated

pathway

where

glucose

is

converted

to

-LRB-

KDG

-RRB-

This

intermediate

is

then

phosphorylated

to

KDPG

and

subsequently

split

into

pyruvate

and

glyceraldehyde

In

Sulfolobus

and

Thermoplasma

a

non-phosphorylated

ED

pathway

has

been

described

-LRB-

in

addition

to

the

semi-phophorylated

pathway

variant

-RRB-

where

KDG

is

cleaved

without

prior

phosphorylation

by

a

novel

bifunctional

KD

-LRB-

P

-RRB-

G

aldolase

-LRB-

same

as

dihydrodipicolinate

synthase

-RRB-

-LRB-

Ahmed

et

al.

-RRB-

Orthologs

for

all

genes

of

the

characterized

Sulfolobus

ED

pathway

cluster

-LRB-

Ahmed

et

al.

Kim

and

Lee

-RRB-

were

found

in

H.

salinarum

H.

marismortui

and

H.

walsbyi

namely

genes

encoding

d-gluconate

dehydratase

-LRB-

EC

gnaD

e.g.

-RRB-

KDG

kinase

-LRB-

EC

kdgK

e.g.

-RRB-

and

KD

-LRB-

P

-RRB-

G

aldolase

-LRB-

kdgA

e.g.

-RRB-

The

ED

pathway

genes

except

kdgK

are

located

in

one

gene

cluster

in

the

H.

salinarum

genome

which

additionally

contains

a

glucose

gene

-LRB-

EC

e.g.

-RRB-

-LRB-

Bonete

et

al.

-RRB-

as

well

as

two

conserved

genes

-LRB-

e.g.

-RRB-

which

are

candidates

for

the

yet

missing

gluconolactonase

-LRB-

EC

-RRB-

The

non-carbohydrate-utilizing

strain

N.

pharaonis

lacks

all

enzyme

genes

for

the

ED

pathway

In

addition

to

their

archaeal

KD

-LRB-

P

-RRB-

G

aldolase

-LRB-

-RRB-

H.

marismortui

-LRB-

-RRB-

H.

walsbyi

-LRB-

-RRB-

and

Haloferax

alicantei

-LRB-

co-located

with

kdgK

-RRB-

also

encode

the

canonical

bacterial-type

KDPG

aldolase

-LRB-

EC

kdgA

-RRB-

which

is

absent

in

Halobacterium

Sulfolobus

and

Thermoplasma

species

This

suggests

operation

of

different

ED

pathway

variants

in

the

two

-LRB-

halo

-RRB-

archaeal

groups

and

probably

a

reduced

semi-phosphorylated

ED

pathway

in

H.

salinarum

which

needs

to

be

investigated

in

future

So

far

experiments

with

glucose-grown

cells

of

H.

salinarum

have

shown

the

conversion

from

glucose

to

gluconate

-LRB-

Sonawat

et

al.

Bhaumik

and

Sonawat

-RRB-

but

have

not

detected

subsequent

reactions

from

gluconate

to

pyruvate

and

glyceraldehyde

when

applying

indirect

enzyme

activity

tests

-LRB-

Rawal

et

al.

-RRB-

Oxidative

pentose

phosphate

pathway

Through

the

oxidative

pentose

phosphate

pathway

phosphorylated

glucose

is

oxidized

to

gluconate

-LRB-

-RRB-

and

then

converted

to

ribulose

-LRB-

-RRB-

by

oxidative

decarboxylation

In

the

archaeal

domain

of

life

the

oxidative

PP

pathway

seems

not

to

exist

because

enzyme

genes

for

this

pathway

are

absent

in

archaeal

genomes

However

haloarchaea

show

glucose

dehydrogenase

activity

-LRB-

EC

-RRB-

in

spite

of

lacking

the

respective

enzyme

gene

-LRB-

Aitken

and

Brown

-RRB-

and

encode

orthologs

of

dehydrogenase

-LRB-

EC

e.g.

-RRB-

Thus

an

operative

albeit

modified

oxidative

PP

pathway

is

indicated

for

haloarchaea

Pentose

metabolism

In

bacteria

pentoses

are

synthesized

via

the

non-oxidative

part

of

the

PP

pathway

where

fructose

-LRB-

-RRB-

and

glyceraldehyde

-LRB-

-RRB-

are

converted

to

ribulose

-LRB-

-RRB-

in

a

complex

five-step

pathway

The

enzyme

gene

set

for

this

pathway

consisting

of

transaldolase

and

-LRB-

EC

-RRB-

transketolase

-LRB-

EC

-RRB-

and

ribulose-phosphate

-LRB-

EC

-RRB-

is

absent

in

most

archaea

except

Methanococcus

jannaschii

and

Thermoplasma

spp.

-LRB-

Soderberg

-RRB-

Individual

genes

encoding

ribose

isomerase

-LRB-

EC

e.g.

-RRB-

for

the

conversion

of

ribose

to

ribulose

are

present

in

archaeal

genomes

Although

Methanocaldococcus

jannaschii

encodes

all

enzyme

genes

for

an

operative

non-oxidative

PP

pathway

relevant

intermediates

-LRB-

i.e.

erythrose

xylose

sedoheptulose

-RRB-

are

absent

in

its

cells

-LRB-

Grochowski

et

al.

-RRB-

Instead

ribulose

is

exclusively

produced

through

the

ribulose

monophosphate

-LRB-

RuMP

-RRB-

pathway

in

M.

jannaschii

This

pathway

is

commonly

employed

for

formaldehyde

fixation

and

detoxification

in

bacteria

but

operates

in

reverse

mode

in

archaea

and

thus

substitutes

for

the

classical

non-oxidative

PP

pathway

-LRB-

Orita

et

al.

Grochowski

et

al.

-RRB-

The

RuMP

pathway

converts

fructose

to

and

then

to

formaldehyde

and

ribulose

involving

-LRB-

-RRB-

and

synthase

-LRB-

-RRB-

These

enzymes

are

encoded

in

all

archaea

except

Thermoplasma

and

haloarchaea

which

presumably

operate

the

non-oxidative

and

a

modified

oxidative

PP

pathway

for

pentose

formation

respectively

-LRB-

Soderberg

-RRB-

Absence

of

the

non-oxidative

branch

of

the

PP

pathway

in

most

archaea

also

results

in

the

absence

of

the

pathway

intermediate

erythrose

-LRB-

-RRB-

which

is

the

precursor

of

aromatic

amino

acids

in

bacteria

While

a

group

of

archaea

-LRB-

e.g.

Pyrococcus

abyssi

-RRB-

encodes

transketolase

required

for

erythrose

synthesis

many

archaea

amongst

them

haloarchaea

lack

transketolase

These

archaea

operate

an

alternative

pathway

for

aromatic

amino

acid

synthesis

starting

from

different

precursors

-LRB-

DKFP

-RRB-

and

l-aspartate

semialdehyde

-LRB-

see

below

-RRB-

-LRB-

White

-RRB-

The

key

enzyme

of

the

reductive

branch

of

the

PP

pathway

for

fixation

is

ribulose-bisphosphate

carboxylase

-LRB-

EC

RuBisCO

-RRB-

RuBisCO

activity

was

previously

detected

in

haloarchaea

such

as

H.

mediterranei

but

not

in

H.

salinarum

-LRB-

Rawal

et

al.

Rajagopalan

and

Altekar

-RRB-

The

acceptor

and

substrate

of

RuBisCO

ribulose

is

not

directly

synthesized

from

ribulose

in

archaea

but

from

the

purine

precursor

-LRB-

PRPP

-RRB-

-LRB-

Finn

and

Tabita

-RRB-

The

required

gene

for

this

conversion

is

present

in

all

haloarchaea

-LRB-

e.g.

-RRB-

while

RuBisCO

-LRB-

-RRB-

is

only

encoded

in

the

N.

pharaonis

genome

Recently

it

has

been

shown

that

purine

and

pentose

metabolism

are

connected

in

archaea

as

archaeal

type

III

RuBisCO

is

involved

in

an

AMP

recycling

pathway

that

is

present

in

N.

pharaonis

-LRB-

see

below

-RRB-

-LRB-

Sato

et

al.

-RRB-

This

novel

pathway

might

be

part

of

a

cyclic

fixation

pathway

in

archaea

consisting

of

-LRB-

i

-RRB-

pentose

formation

-LRB-

PRPP

-RRB-

from

fructose

-LRB-

e.g.

via

the

RuMP

pathway

-RRB-

-LRB-

ii

-RRB-

conversion

of

PRPP

and

adenine

to

AMP

by

adenine

phosphoribosyltransferase

-LRB-

EC

e.g.

-RRB-

-LRB-

iii

-RRB-

AMP

recycling

releasing

adenine

and

-LRB-

and

involving

the

fixation

step

-RRB-

and

-LRB-

iv

-RRB-

gluconeogenesis

for

the

conversion

of

back

to

fructose

-LRB-

Sato

et

al.

-RRB-

Glycerol

metabolism

In

many

hypersaline

habitats

glycerol

is

a

highly

abundant

carbon

source

that

is

produced

by

the

halotolerant

green

algae

Dunaliella

to

protect

itself

from

osmotic

pressure

-LRB-

Borowitzka

et

al.

Phadwal

and

Singh

-RRB-

Haloarchaea

are

able

to

catabolize

the

abundant

glycerol

through

phosphorylation

to

glycerol

and

subsequent

formation

of

dihydroxyacetone

phosphate

-LRB-

DHAP

-RRB-

-LRB-

Rawal

et

al.

Nishihara

et

al.

-RRB-

Consistently

haloarchaeal

genomes

encode

glycerol

kinase

-LRB-

EC

e.g.

-RRB-

the

multi-subunit

glycerol

dehydrogenase

-LRB-

EC

e.g.

-RRB-

and

a

potential

ABC

transport

system

-LRB-

e.g.

-RRB-

The

haloalkaliphile

N.

pharaonis

lacks

glycerol

degrading

enzymes

which

might

be

due

to

its

soda

lake

habitat

where

Dunaliella

is

not

a

main

primary

producer

It

should

be

noted

that

glycerol

degradation

and

lipid

formation

occur

through

separate

pathways

in

archaea

meaning

that

the

intermediate

glycerol

derived

from

glycerol

catabolism

is

not

used

for

the

synthesis

of

the

glycerophosphate

backbone

of

archaeal

lipids

-LRB-

see

below

-RRB-

-LRB-

Nishihara

et

al.

-RRB-

In

H.

salinarum

glycerol

can

also

be

converted

to

DHA

by

glycerol

dehydrogenase

-LRB-

Rawal

et

al.

-RRB-

For

this

reaction

a

plasmid-encoded

glycerol

dehydrogenase

-LRB-

EC

-RRB-

is

employed

which

has

been

characterized

and

structurally

elucidated

-LRB-

Offermann

Horn

-RRB-

The

produced

DHA

might

be

phosphorylated

by

DHA

kinase

-LRB-

EC

-RRB-

and

fed

into

the

lower

EM

pathway

However

potential

DHA

kinases

genes

-LRB-

-RRB-

are

only

encoded

by

H.

walsbyi

where

they

probably

depend

on

a

cytosolic

phosphoenolpyruvate-dependent

phosphotransferase

system

-LRB-

-RRB-

-LRB-

Bolhuis

et

al.

-RRB-

Pyruvate

metabolism

and

tricarboxylic

acid

cycle

Pyruvate

metabolism

The

central

metabolite

pyruvate

is

converted

to

acetyl-CoA

by

pyruvate-ferredoxin

oxidoreductase

-LRB-

EC

porAB

-RRB-

-LRB-

Kerscher

and

Oesterhelt

b

-RRB-

and

subsequently

fed

into

the

tricarboxylic

acid

-LRB-

TCA

-RRB-

cycle

NMR

spectroscopy

experiments

for

H.

salinarum

have

shown

that

of

the

flux

is

channelled

into

the

TCA

cycle

via

pyruvate-ferredoxin

oxidoreductase

-LRB-

Ghosh

and

Sonawat

Bhaumik

and

Sonawat

-RRB-

The

remaining

of

the

pyruvate

is

converted

to

the

TCA

intermediate

oxaloacetate

by

pyruvate

carboxylase

-LRB-

EC

-RRB-

in

order

to

fill

up

the

oxaloacetate

pool

of

the

TCA

cycle

when

its

intermediates

are

drawn

off

for

biosynthetic

purposes

-LRB-

Ghosh

and

Sonawat

-RRB-

However

haloarchaea

do

not

encode

archaeal-type

pyruvate

carboxylase

-LRB-

Mukhopadhyay

et

al.

-RRB-

but

biotin

carboxylases

that

are

more

likely

to

be

involved

in

fatty

acid

degradation

-LRB-

e.g.

located

within

a

fatty

acid

degradation

cluster

-RRB-

H.

marismortui

and

H.

walsbyi

encode

a

phosphoenolpyruvate

carboxylase

-LRB-

EC

-RRB-

which

has

been

proposed

to

be

involved

with

a

novel

cytosolic

phosphotransferase

system

-LRB-

-RRB-

-LRB-

Bolhuis

et

al.

-RRB-

During

gluconeogenesis

phosphoenolpyruvate

is

synthesized

from

oxaloacetate

through

malic

enzyme

-LRB-

EC

e.g.

-RRB-

and

pyruvate

water

dikinase

-LRB-

EC

e.g.

-RRB-

in

haloarchaea

The

former

anaplerotic

enzyme

has

been

shown

to

be

active

in

H.

salinarum

while

the

anaplerotic

reactions

catalyzed

by

phosphoenolpyruvate

carboxykinase

-LRB-

EC

-RRB-

and

oxaloacetate

decarboxylase

-LRB-

EC

-RRB-

are

missing

in

haloarchaea

-LRB-

Bhaumik

and

Sonawat

Ghosh

and

Sonawat

-RRB-

Under

anaerobic

conditions

it

has

been

shown

for

H.

salinarum

that

pyruvate

is

primarly

converted

to

alanine

presumably

by

an

aspartate

transaminase

and

to

a

larger

extent

to

lactate

and

acetate

-LRB-

Bhaumik

and

Sonawat

Ghosh

and

Sonawat

-RRB-

In

spite

of

proven

lactate

dehydrogenase

activity

in

H.

salinarum

cell

extracts

-LRB-

Bhaumik

and

Sonawat

-RRB-

no

clear

lactate

dehydrogenase

homolog

has

been

identified

in

haloarchaeal

genomes

In

M.

jannaschii

it

has

been

shown

that

lactate

is

produced

from

lactaldehyde

which

might

be

derived

from

methylglyoxal

-LRB-

Grochowski

et

al.

-RRB-

The

M.

jannaschii

lactaldehyde

dehydrogenase

is

similar

to

several

probable

aldehyde

dehydrogenases

encoded

in

haloarchaea

-LRB-

e.g.

-RRB-

Tricarboxylic

acid

cycle

Although

TCA

cycles

are

highly

variable

and

often

incomplete

within

the

archaeal

domain

of

life

-LRB-

Huynen

et

al.

-RRB-

haloarchaeal

genomes

encode

the

complete

set

of

enzymes

For

H.

salinarum

activity

of

all

these

enzymes

has

been

proven

-LRB-

Aitken

and

Brown

Hubbard

and

Miller

Kerscher

and

Oesterhelt

b

Gradin

et

al.

-RRB-

and

an

operative

TCA

cycle

has

been

shown

by

NMR

spectroscopy

-LRB-

Ghosh

and

Sonawat

-RRB-

In

haloarchaea

pyruvate

and

are

not

converted

by

classical

dehydrogenase

complexes

but

by

oxidoreductases

encoded

by

porAB

and

korAB

genes

respectively

-LRB-

Kerscher

and

Oesterhelt

b

-RRB-

However

haloarchaeal

and

other

archaeal

genomes

further

contain

gene

clusters

encoding

all

components

of

a

dehydrogenase

complex

In

Thermoplasma

acidophilum

it

has

recently

been

shown

that

the

component

of

the

encoded

dehydrogenase

complex

accepts

branched-chain

-LRB-

Heath

et

al.

-RRB-

Most

likely

the

haloarchaeal

dehydrogenase

complex

is

also

involved

in

branched-chain

amino

acid

degradation

When

grown

on

acetate

Haloferax

volcanii

operates

a

glyoxylate

bypass

operon

involving

isocitrate

lyase

-LRB-

EC

-RRB-

and

a

new

type

of

malate

synthase

-LRB-

EC

-RRB-

-LRB-

Serrano

and

Bonete

-RRB-

Homologs

of

both

enzymes

are

present

in

the

N.

pharaonis

-LRB-

-RRB-

and

H.

walsbyi

-LRB-

-RRB-

genomes

while

only

a

probable

malate

synthase

is

encoded

in

H.

marismortui

-LRB-

-RRB-

Halobacterium

lacks

both

glyoxylate

cycle

genes

although

activity

of

both

glyoxylate

cycle

enzymes

has

been

demonstrated

previously

in

this

haloarchaeon

-LRB-

Aitken

and

Brown

-RRB-

Nucleotide

metabolism

De

novo

synthesis

of

nucleotides

The

complete

gene

sets

that

are

required

for

de

novo

synthesis

of

purines

from

ribose

and

for

de

novo

synthesis

of

pyrimidines

from

carbamoylphosphate

and

ribose

are

present

in

haloarchaeal

genomes

Haloarchaea

reveal

an

unusual

domain

fusion

pattern

of

purine

synthesis

enzymes

-LRB-

Fig.

-RRB-

because

they

contain

a

unique

fusion

of

GAR

and

AICAR

transformylases

-LRB-

EC

EC

purN/purH

e.g.

-RRB-

Haloarchaea

do

not

encode

the

novel

AICAR

transformylase

-LRB-

purP

-RRB-

-LRB-

Ownby

et

al.

-RRB-

present

in

most

archaea

but

encode

the

novel

archaeal

variant

of

IMP

cyclohydrolase

-LRB-

purO

e.g.

-RRB-

-LRB-

Graupner

et

al.

-RRB-

The

four

haloarchaeal

strains

show

only

few

differences

in

their

nucleotide

metabolism

namely

in

the

occurrence

of

pyrimidine

kinases

-LRB-

EC

EC

-RRB-

-LRB-

Supplementary

Material

-RRB-

Fig.

rearrangement

of

enzymes

involved

in

the

de

novo

synthesis

of

purines

The

pathway

comprises

steps

from

PRPP

to

IMP

Fusions

of

enzyme

genes

are

indicated

by

linked

boxes

Non-orthologous

enzymes

are

known

for

steps

and

-LRB-

unfilled

boxes

-RRB-

but

further

archaeal

enzymes

for

purine

synthesis

are

unknown

-LRB-

question

marks

-RRB-

Archaeal

type

III

RuBisCO

functions

in

AMP

metabolism

Ribulose-bisphosphate

carboxylase

-LRB-

EC

RuBisCO

-RRB-

is

a

key

enzyme

for

fixation

via

the

Calvin-Benson-Bassham

cycle

in

plants

However

RuBisCO

was

found

to

be

involved

in

bacterial

methionine

cleavage

and

in

the

AMP

metabolism

of

archaea

-LRB-

Sato

et

al.

-RRB-

The

latter

pathway

recycles

the

intracellular

pool

of

AMP

produced

by

ADP-dependent

-LRB-

AMP-forming

-RRB-

sugar

kinases

which

are

involved

in

glycolytic

pathways

of

archaea

-LRB-

Kengen

et

al.

Selig

et

al.

-RRB-

N.

pharaonis

is

the

only

haloarchaeal

species

encoding

all

enzyme

genes

for

this

novel

AMP

recycling

pathway

i.e.

AMP

phosphorylase

-LRB-

-RRB-

isomerase

-LRB-

-RRB-

and

RuBisCO

-LRB-

-RRB-

while

H.

salinarum

exhibits

only

the

isomerase

gene

-LRB-

-RRB-

-LRB-

Sato

et

al.

-RRB-

N.

pharaonis

and

some

other

archaea

do

not

possess

ADP-dependent

sugar

kinases

that

would

produce

AMP

though

Instead

AMP

recycling

might

be

part

of

a

cyclic

pathway

for

fixation

as

described

earlier

Lipid

metabolism

Membrane

lipids

of

archaea

consist

of

glycerol

diether

lipids

with

prenyl

side

chains

instead

of

diacylglycerol

esters

Specifically

membranes

of

H.

salinarum

contain

core

lipids

with

two

phytanyl

side

chains

-LRB-

-RRB-

and

to

lesser

extents

also

other

isoprenoid

constituents

such

as

squalenes

-LRB-

-RRB-

phytoenes

-LRB-

-RRB-

menaquinones

-LRB-

-RRB-

and

dolichol

-LRB-

-RRB-

-LRB-

Oesterhelt

Lechner

et

al.

Kushwaha

et

al.

-RRB-

-LRB-

Fig.

-RRB-

Furthermore

H.

salinarum

synthesizes

several

carotenoids

from

prenyl

precursors

preferentially

bacterioruberins

-LRB-

-RRB-

and

photoactive

retinal

-LRB-

-RRB-

-LRB-

Oesterhelt

Oesterhelt

and

Stoeckenius

-RRB-

Retinal

is

incorporated

into

bacteriorhodopsin

and

other

retinal

proteins

which

are

unique

to

haloarchaea

within

the

archaeal

domain

of

life

Although

fatty

acids

are

not

part

of

archaeal

membrane

lipids

small

amounts

of

fatty

acids

-LRB-

-RRB-

have

been

detected

in

membrane

proteins

of

H.

salinarum

-LRB-

Pugh

and

Kates

-RRB-

Other

haloarchaeal

species

likely

possess

similar

membrane

constituents

as

H.

salinarum

because

they

have

the

same

gene

set

for

the

de

novo

synthesis

of

isoprenoids

However

specific

prenyl-based

compounds

might

vary

from

species

to

species

as

in

the

case

of

diether

core

lipids

found

in

haloalkaliphiles

e.g.

N.

pharaonis

-LRB-

and

prenyl

chains

-RRB-

-LRB-

Tindall

et

al.

-RRB-

Fig.

of

isoprenoids

in

halophilic

archaea

The

isoprenoid

precursor

IPP

is

synthesized

via

the

mevalonate

pathway

as

shown

by

labeling

studies

-LRB-

green

reaction

exists

red

reaction

absent

bold

experimental

verification

-RRB-

Various

isoprenoids

detected

in

membranes

of

H.

salinarum

-LRB-

listed

in

boxes

-RRB-

are

synthesized

by

a

series

of

condensation

reactions

with

IPP

which

is

added

in

head

tail

-LRB-

HT

-RRB-

or

head

head

-LRB-

HH

-RRB-

fashion

and

through

desaturase

reactions

-LRB-

-LSB-

-RSB-

-RRB-

Enzyme

gene

sets

for

isoprenoid

synthesis

differ

only

slightly

between

halorarchaea

-LRB-

square

H.

marismortui

circle

H.

walsbyi

diamond

N.

pharaonis

triangle

H.

salinarum

green

gene

exists

red

gene

absent

-RRB-

Bacterial

-LRB-

B

-RRB-

or

archaeal-type

-LRB-

A

-RRB-

enzyme

variants

are

indicated

Superscript

a

indicates

units

are

synthesized

via

the

mevalonate

pathway

starting

from

two

acetyl-CoA

molecules

and

a

still

unknown

arising

from

amino

acid

degradation

-LRB-

Ekiel

et

al.

-RRB-

Prenyl

side

chains

of

membrane

lipids

and

other

isoprenoids

are

derived

via

the

mevalonate

pathway

in

haloarchaea

-LRB-

Ekiel

et

al.

-RRB-

while

the

glycerophosphate

backbone

of

membrane

lipids

is

formed

from

glycerol

This

membrane

precursor

is

derived

from

the

glycolytic

intermediate

DHAP

via

dehydrogenase

-LRB-

EC

e.g.

-RRB-

which

is

ubiquitously

found

in

archaea

-LRB-

Nishihara

et

al.

-RRB-

De

novo

synthesis

of

isoprenoids

-LRB-

mevalonate

pathway

-RRB-

Like

other

archaea

halophiles

synthesize

activated

-LSB-

dimethylallyl

and

isopentenyl

diphosphate

-LRB-

IPP

-RRB-

-RSB-

for

polycondensation

of

prenyl

chains

via

the

mevalonate

pathway

-LRB-

Fig.

-RRB-

A

previous

comparative

analysis

of

the

mevalonate

pathway

-LRB-

Smit

and

Mushegian

-RRB-

identified

gaps

for

three

pathway

steps

in

archaea

namely

the

lack

of

bacterial-type

phosphomevalonate

kinase

-LRB-

EC

-RRB-

diphosphomevalonate

decarboxylase

-LRB-

EC

-RRB-

and

IPP

isomerase

-LRB-

EC

-RRB-

Recently

an

alternative

type

II

IPP

isomerase

has

been

identified

in

archaea

which

belongs

to

the

same

as

lactate

dehydrogenase

-LRB-

Barkley

et

al.

-RRB-

Furthermore

it

has

been

suggested

that

mevalonate

phosphate

is

first

decarboxylated

and

then

phosphorylated

to

synthesize

IPP

while

phosphorylation

of

mevalonate

phosphate

precedes

the

decarboxylation

step

in

bacteria

-LRB-

Grochowski

et

al.

-RRB-

The

archaeal-specific

reactions

involve

a

predicted

phosphomevalonate

decarboxylase

-LRB-

-RRB-

and

a

characterized

isopentenyl

phosphate

kinase

-LRB-

-RRB-

Interestingly

haloarchaea

may

operate

a

chimeric

mevalonate

pathway

-LRB-

Fig.

-RRB-

Like

other

archaea

they

encode

isopentenyl

phosphate

kinase

-LRB-

e.g.

-RRB-

instead

of

a

bacterial

phosphomevalonate

kinase

However

haloarchaea

lack

the

proposed

archaeal

phosphomevalonate

decarboxylase

gene

and

instead

encode

a

bacterial-type

diphospho-mevalonate

decarboxylase

-LRB-

e.g.

-RRB-

Thus

neither

the

classical

bacterial

nor

the

proposed

archaeal

mevalonate

pathway

is

complete

For

the

last

mevalonate

pathway

step

only

N.

pharaonis

and

H.

salinarum

encode

an

archaeal-type

II

IPP

isomerase

-LRB-

e.g.

-RRB-

while

all

four

haloarchaea

possess

a

bacterial-type

IPP

isomerase

-LRB-

e.g.

-RRB-

Future

investigations

are

needed

in

order

to

clarify

whether

archaeal

and

bacterial

enzymes

are

employed

simultaneously

in

haloarchaea

The

acquired

bacterial

enzymes

might

possibly

have

a

higher

substrate

specificity

or

turnover

for

covering

increased

isoprenoid

demands

for

retinal

and

bacterioruberin

biosynthesis

A

functional

mevalonate

pathway

has

been

verified

for

H.

salinarum

by

labeling

studies

-LRB-

Ekiel

et

al.

-RRB-

which

lead

to

the

proposal

of

an

unusual

first

step

Lipid

labeling

patterns

indicated

that

mevalonate

is

not

synthesized

from

three

activated

acetate

precursors

but

rather

from

two

acetyl-CoA

molecules

and

an

unknown

The

latter

is

not

derived

from

acetate

but

from

degraded

amino

acids

such

as

lysine

precursors

derived

via

the

mevalonate

pathway

are

elongated

to

trans

and

cis-polyprenyl

chains

in

head-to-tail

fashion

by

-LRB-

E

-RRB-

and

-LRB-

Z

-RRB-

prenyltransferases

respectively

-LRB-

E

Z

-RRB-

Exact

chain-specificity

of

prenyltransferase

orthologs

needs

to

be

determined

experimentally

but

the

biosynthesis

of

dolichol

and

menaquinones

found

in

H.

salinarum

requires

prenyltransferases

with

long-chain

specifities

Potential

enzymes

for

squalene

and

phytoene

synthesis

through

head-to-head

condensation

are

also

encoded

in

haloarchaeal

genomes

Synthesis

of

carotenoids

and

retinal

For

carotene

biosynthesis

phytoene

is

reduced

to

lycopene

by

phytoene

desaturase

that

is

encoded

in

haloarchaeal

genomes

-LRB-

e.g.

-RRB-

Lycopene

is

the

branching

point

for

the

synthesis

of

β-carotene

-LRB-

-RRB-

and

bacterioruberins

-LRB-

-RRB-

-LRB-

Oesterhelt

-RRB-

The

reactions

that

lead

to

bacterioruberins

have

not

yet

been

elucidated

in

detail

but

lycopene

cyclase

activity

-LRB-

e.g.

-RRB-

converting

lycopene

to

β-carotene

has

been

shown

for

H.

salinarum

-LRB-

Peck

et

al.

-RRB-

The

derived

β-carotene

is

cleaved

by

β-carotene

mono-oxygenase

into

two

retinal

molecules

-LRB-

-RRB-

which

are

incorporated

in

haloarchaeal

rhodopsins

H.

walsbyi

encodes

two

cyanobacterial-like

-LRB-

-RRB-

and

one

plant-like

β-carotene

mono-oxygenase

homologs

-LRB-

-RRB-

and

H.

marismortui

acquired

a

cyanobacterial-like

β-carotene

mono-oxygenase

gene

-LRB-

-RRB-

on

one

of

its

plasmids

-LRB-

Bolhuis

et

al.

-RRB-

H.

salinarum

and

N.

pharaonis

lack

β-carotene

mono-oxygenase

homologs

and

must

therefore

possess

a

still

unknown

non-orthologous

enzyme

for

the

oxidative

cleavage

of

β-carotene

Previously

brp

and

blh

have

been

shown

to

play

a

role

in

regulation

or

synthesis

of

retinal

-LRB-

Peck

et

al.

-RRB-

Fatty

acid

metabolism

Even-numbered

fatty

acids

like

palmitic

and

stearic

acid

are

part

of

membrane

proteins

but

not

of

membrane

lipids

in

archaea

-LRB-

Pugh

and

Kates

-RRB-

For

example

palmitic

acid

is

associated

with

halorhodopsin

as

a

free

fatty

acid

in

H.

salinarum

-LRB-

Kolbe

et

al.

-RRB-

The

origin

of

these

fatty

acids

is

unclear

because

archaea

do

not

encode

components

of

a

fatty

acid

synthase

complex

In

methanogenic

archaea

biosynthesis

of

fatty-acid-like

compounds

from

by

repeated

chain

elongation

has

been

reported

-LRB-

White

-RRB-

For

the

degradation

of

activated

fatty

acids

via

the

ß-oxidation

pathway

gene

candidates

are

present

in

haloarchaea

and

most

other

archaea

However

since

chain-length

specifity

of

these

enzymes

is

currently

unknown

fatty

acid

degradation

might

still

be

limited

to

short

chain

lengths

-LRB-

e.g.

for

derivatives

of

branched-chain

amino

acids

-RRB-

A

series

of

genes

were

assigned

for

Natronomonas

-LRB-

genes

-RRB-

and

Haloarcula

-LRB-

genes

-RRB-

for

the

repeated

four-step

reaction

sequence

of

the

ß-oxidation

pathway

indicating

a

versatile

fatty-acid

metabolism

in

these

two

species

In

contrast

few

ß-oxidation

genes

are

present

in

H.

salinarum

-LRB-

genes

-RRB-

and

H.

walsbyi

-LRB-

genes

-RRB-

In

accordance

to

these

findings

growth

experiments

showed

that

N.

pharaonis

is

able

to

grow

on

fatty

acids

of

various

lengths

as

carbon

source

-LRB-

especially

-RRB-

while

fatty

acids

seem

not

to

be

utilized

by

H.

salinarum

as

growth

is

reduced

or

diminished

by

long-chain

-LRB-

-RRB-

and

medium-chain

fatty

acids

-LRB-

-RRB-

respectively

-LRB-

Konigsmaier

-RRB-

Haloarchaea

likely

degrade

odd-numbered

fatty

acids

because

they

possess

probable

propionyl-CoA

carboxylase

-LRB-

EC

e.g.

-RRB-

methylmalonyl-CoA

epimerase

-LRB-

EC

e.g.

-RRB-

as

well

as

methylmalonyl-CoA

mutase

-LRB-

EC

e.g.

-RRB-

Natronomonas

further

encodes

enzymes

that

are

probably

involved

in

propionate

catabolism

-LRB-

-RRB-

Amino

acid

synthesis

While

certain

haloarchaea

such

as

Haloarcula

hispanica

-LRB-

Hochuli

et

al.

-RRB-

do

not

require

any

amino

acids

for

growth

H.

salinarum

is

grown

in

synthetic

media

with

to

amino

acids

-LRB-

Oesterhelt

and

Krippahl

Grey

and

Fitt

-RRB-

Through

metabolic

pathway

reconstruction

and

comparison

it

can

be

proposed

that

H.

salinarum

has

indeed

reduced

capabilities

to

synthesize

amino

acids

Specifically

H.

salinarum

lacks

gene

clusters

for

valine

leucine

isoleucine

lysine

and

arginine

-LRB-

Supplementary

Material

-RRB-

Furthermore

because

of

reduced

folate

biosynthesis

methionine

biosynthesis

via

folate-dependent

methionine

synthase

-LRB-

EC

-RRB-

might

be

affected

in

H.

salinarum

The

predicted

set

of

essential

amino

acids

fits

well

to

the

set

of

amino

acids

that

can

be

sensed

by

H.

salinarum

-LRB-

Oren

-RRB-

except

for

lysine

which

is

not

an

attractant

signal

and

for

cysteine

which

is

sensed

by

BasT

-LRB-

Kokoeva

et

al.

-RRB-

in

spite

of

the

fact

that

all

enzyme

genes

for

cysteine

biosynthesis

are

present

in

its

genome

The

synthesis

of

several

amino

acids

-LRB-

glutamate

glutamine

proline

aspartate

asparagine

alanine

serine

phenylalanine

tryptophan

histidine

-RRB-

by

H.

salinarum

has

been

verified

by

NMR

labeling

studies

-LRB-

Ekiel

et

al.

Bhaumik

and

Sonawat

Ghosh

and

Sonawat

Engelhard

et

al.

-RRB-

-LRB-

Fig.

-RRB-

For

H.

marismortui

N.

pharaonis

and

H.

walsbyi

complete

independence

from

supplemented

amino

acids

can

be

concluded

from

comparative

analysis

-LRB-

Supplementary

Material

-RRB-

De

novo

synthesis

of

all

amino

acids

has

already

been

proven

for

N.

pharaonis

by

the

development

of

a

synthetic

medium

without

amino

acid

supplements

-LRB-

Falb

et

al.

Oberwinkler

-RRB-

The

three

haloarchaea

that

do

not

require

amino

acids

are

also

able

to

utilize

several

nitrogen

sources

because

they

posess

gene

sets

required

for

urea

conversion

as

well

as

for

nitrate

and

ammonia

assimilation

-LRB-

Falb

et

al.

-RRB-

-LRB-

Supplementary

Material

-RRB-

Amino

acid

biosynthesis

pathways

of

the

three

haloarchaea

show

only

few

differences

i.e.

for

proline

lysine

serine

and

glycine

synthesis

-LRB-

see

below

-RRB-

Glutamate

family

-LRB-

glutamate

glutamine

proline

arginine

-RRB-

The

biosynthesis

of

glutamate

from

the

TCA

cycle

intermediate

is

a

major

metabolic

conversion

in

haloarchaea

as

shown

by

NMR

labeling

studies

in

Halobacterium

Labeled

pyruvate

alanine

acetate

and

glycerol

were

mainly

found

to

be

incorporated

into

glutamate

and

a

considerable

part

of

the

flux

through

the

TCA

cycle

was

shown

to

be

channelled

to

glutamate

-LRB-

Ghosh

and

Sonawat

Ekiel

et

al.

-RRB-

Three

paralogous

glutamate

dehydrogenase

genes

were

found

in

Natronomonas

-LRB-

-RRB-

Halobacterium

-LRB-

-RRB-

and

Haloarcula

-LRB-

-RRB-

but

only

one

gene

in

Haloquadratum

-LRB-

-RRB-

The

activity

of

two

Halobacterium

glutamate

dehydrogenases

with

NADP

-LRB-

-RRB-

and

NAD

-LRB-

-RRB-

cofactor

specificity

has

been

investigated

in

detail

-LRB-

Bonete

et

al.

Perez-Pomares

et

al.

Hayden

et

al.

-RRB-

In

N.

pharaonis

H.

walsbyi

and

H.

marismortui

glutamate

is

also

derived

by

glutamate

synthase

-LRB-

e.g.

-RRB-

from

and

glutamine

a

reaction

that

is

part

of

a

proposed

ammonia

assimilation

pathway

-LRB-

e.g.

-RRB-

-LRB-

Falb

et

al.

-RRB-

It

should

be

noted

that

haloarchaeal

cells

amass

glutamate

in

high

concentrations

as

a

compatible

osmolyte

-LRB-

Kokoeva

et

al.

Desmarais

et

al.

-RRB-

Glutamine

is

derived

from

glutamate

by

glutamate-ammonia

ligase

-LRB-

EC

e.g.

-RRB-

which

is

present

in

all

haloarchaea

Glutamate

might

further

be

converted

to

l-glutamate

and

subsequently

to

proline

The

required

enzymes

encoded

by

the

proCBA

gene

cluster

are

only

found

in

the

genomes

of

Natronomonas

-LRB-

-RRB-

and

Haloquadratum

-LRB-

-RRB-

but

are

absent

in

Halobacterium

and

Haloarcula

Biosynthesis

of

proline

by

H.

salinarum

has

been

shown

previously

-LRB-

Ghosh

and

Sonawat

-RRB-

indicating

an

alternative

pathway

in

the

latter

two

haloarchaea

Proline

might

be

synthesized

via

dehydrogenase

-LRB-

EC

-RRB-

and

proline

dehydrogenase

-LRB-

EC

putA

-RRB-

This

notion

is

supported

by

the

fact

that

the

phylogenetic

profiles

of

putA

is

complementary

to

that

of

the

proABC

cluster

i.e.

putA

is

only

encoded

in

H.

salinarum

-LRB-

-RRB-

and

H.

marismortui

-LRB-

-RRB-

but

is

missing

in

N.

pharaonis

and

H.

walsbyi

Proline

might

also

be

derived

by

the

cyclisation

of

ornithine

as

shown

for

M.

jannaschii

-LRB-

Graupner

and

White

-RRB-

Homologs

of

ornithine

cyclodeaminase

-LRB-

EC

-RRB-

are

encoded

in

all

haloarchaea

-LRB-

e.g.

-RRB-

Glutamate

is

also

a

precursor

of

ornithine

which

is

converted

to

arginine

by

urea

cycle

enzymes

The

genomes

of

Natronomonas

Haloquadratum

and

Haloarcula

possess

argXCBDEF

clusters

-LRB-

e.g.

-RRB-

containing

all

required

enzymes

-LRB-

except

the

argA

gene

-RRB-

for

the

de

novo

synthesis

of

ornithine

as

well

as

a

probable

transcription

regulator

ArgX

The

complete

gene

set

for

arginine

biosynthesis

is

absent

in

Halobacterium

Instead

halobacterial

arginine

requirements

are

covered

by

the

uptake

of

external

arginine

via

a

verified

arginine-ornithine

antiporter

-LRB-

-RRB-

-LRB-

J.

Tittor

unpublished

results

-RRB-

encoded

next

to

the

arginine

deiminase

pathway

cluster

on

plasmid

Aspartate

family

-LRB-

aspartate

alanine

asparagine

threonine

methionine

lysine

-RRB-

Aspartate

is

derived

from

the

TCA

cycle

intermediate

oxaloacetate

by

aspartate

transaminase

-LRB-

EC

-RRB-

of

which

several

paralogs

are

present

in

haloarchaeal

genomes

-LRB-

e.g.

and

-RRB-

These

seem

to

be

also

involved

in

pyruvate

transamination

to

alanine

since

experiments

showed

that

the

transaminase

which

is

involved

in

alanine

synthesis

in

Halobacterium

uses

aspartate

instead

of

glutamate

as

amino

group

donor

-LRB-

Bhaumik

and

Sonawat

-RRB-

In

agreement

with

this

no

clear

orthologs

of

glutamate-pyruvate

transaminase

-LRB-

EC

-RRB-

have

been

found

in

haloarchaeal

genomes

Labeling

studies

showed

that

aspartate

is

converted

to

asparagine

inH

salinarum

-LRB-

Engelhard

et

al.

-RRB-

and

consistently

all

haloarchaeal

genomes

encode

asparagine

synthase

-LRB-

EC

e.g.

-RRB-

In

a

pathway

analogous

to

proline

synthesis

from

glutamate

aspartate

is

likely

to

be

converted

to

l-aspartate

semialdehyde

This

compound

is

not

only

a

precursor

of

threonine

methionine

and

lysine

in

archaea

but

is

also

required

for

aromatic

amino

acid

synthesis

via

a

modified

shikimate

pathway

-LRB-

see

below

-RRB-

Enzyme

genes

for

threonine

biosynthesis

from

l-aspartate

semialdehyde

via

l-homoserine

and

O-phospho-l-homoserine

are

present

in

all

haloarchaea

-LRB-

e.g.

-RRB-

In

M.

jannaschii

O-phospho-l-homoserine

is

not

only

converted

to

threonine

but

also

to

homocysteine

-LRB-

White

-RRB-

Additionally

haloarchaea

might

synthesize

homocysteine

from

homoserine

via

O-acetyl-l-homoserine

-LRB-

but

not

via

O-succinyl-l-homoserine

-RRB-

This

pathway

requires

clustered

metX

and

metY

genes

-LRB-

e.g.

-RRB-

in

case

of

direct

sulfhydrylation

of

O-acetyl-l-homoserine

Alternatively

O-acetyl-l-homoserine

could

be

converted

to

cystathionine

and

subsequently

to

homocysteine

by

thiol-lyase

synthase

-LRB-

e.g.

-RRB-

In

the

final

step

methionine

is

likely

derived

from

homocysteine

by

cobalamine-independent

methionine

synthase

-LRB-

EC

metE

-RRB-

of

which

two

to

three

paralogs

are

found

in

haloarchaea

-LRB-

-RRB-

except

Halobacterium

-LRB-

-RRB-

Since

methionine

synthase

depends

on

the

availability

of

folate

reduced

folate

synthesis

in

H.

salinarum

might

also

affect

its

synthesis

of

methionine

The

diaminopimelate

-LRB-

DAP

-RRB-

pathway

for

the

synthesis

of

lysine

has

already

been

proven

for

H.

hispanica

-LRB-

Hochuli

et

al.

-RRB-

Enzymes

for

the

DAP

pathway

are

present

in

Natronomonas

Haloquadratum

and

Haloarcula

i.e.

the

dapABD

gene

cluster

-LRB-

e.g.

dapC

is

synonym

with

argD

-RRB-

as

well

as

lysA

-LRB-

e.g.

-RRB-

and

argG

genes

-LRB-

e.g.

-RRB-

Gaps

within

the

DAP

pathway

-LRB-

dapE

dapF

-RRB-

occuring

in

Haloarcula

and

Natronomonas

are

likely

to

be

bridged

by

still

unknown

non-orthologous

enzymes

Halobacterium

is

not

capable

of

lysine

biosynthesis

because

it

does

not

encode

any

of

the

dap

genes

Serine

family

-LRB-

serine

glycine

cysteine

-RRB-

Serine

biosynthesis

might

occur

via

two

different

pathways

Within

the

phosphorylated

pathway

glycerate

a

glycolytic

intermediate

is

oxidized

to

which

is

further

converted

to

phosphoserine

and

to

serine

by

transaminase

and

phosphatase

activity

Alternatively

non-phosphorylated

glycerate

is

directly

oxidized

to

hydroxypyruvate

and

then

transaminated

All

four

haloarchaea

possess

genes

for

phosphoglycerate

dehydrogenase

-LRB-

EC

serA

e.g.

-RRB-

and

phosphoserine

phosphatase

-LRB-

EC

serB

e.g.

-RRB-

of

the

phosphorylated

synthesis

pathway

However

the

enzyme

gene

for

the

intermediate

pathway

step

phosphoserine

transaminase

-LRB-

EC

serC

-RRB-

is

missing

Nevertheless

studies

in

Methanococcales

revealed

that

broad-specificity

class

V

aspartate

transaminases

-LRB-

e.g.

-RRB-

catalyze

this

reaction

-LRB-

Helgadottir

et

al.

-RRB-

Apart

from

the

phosphorylated

serine

synthesis

pathways

Natronomonas

Haloquadratum

and

Haloarcula

might

also

operate

the

non-phosphorylated

pathway

because

these

species

encode

a

probable

hydroxypyruvate

reductase

-LRB-

EC

e.g.

-RRB-

for

the

initial

step

from

glycerate

to

hydroxypyruvate

One

of

the

class

V

aspartate

transaminases

might

then

catalyze

the

transaminase

reaction

from

hydroxypyruvate

to

serine

Glycine

is

potentially

derived

from

serine

by

glycine

hydroxymethyltransferase

-LRB-

EC

e.g.

-RRB-

in

all

haloarchaea

Halobacterium

might

additionally

synthesize

glycine

from

threonine

employing

threonine

aldolase

-LRB-

EC

-RRB-

which

is

unique

to

the

archaeal

domain

of

life

H.

salinarum

would

thus

be

adapted

to

its

reduced

folate

synthesis

-LRB-

see

below

-RRB-

as

the

folate-dependent

glycine

hydroxymethyltransferase

is

circumvented

All

haloarchaea

except

H.

walsbyi

encode

components

of

the

glycine

cleavage

system

-LRB-

EC

-RRB-

which

is

encoded

in

a

single

gene

cluster

in

Halobacterium

-LRB-

-RRB-

Future

investigations

might

elucidate

whether

metabolic

fluxes

differ

for

the

serine

metabolism

of

Halobacterium

and

the

other

haloarchaea

Similar

to

the

conversion

of

homoserine

to

homocysteine

via

O-acetyl-l-homoserine

serine

is

likely

to

be

converted

in

all

halobacteria

to

O-acetyl-serine

-LRB-

cysE

e.g.

-RRB-

and

then

to

cysteine

by

the

incorporation

of

sulfide

-LRB-

cysK

e.g.

-RRB-

An

alternative

cysteine

biosynthesis

pathway

employed

by

M.

jannaschii

which

lacks

cysE

and

cysK

genes

starts

from

homocysteine

and

phosphoserine

and

leads

via

cystathionine

to

cysteine

-LRB-

White

-RRB-

This

pathway

requires

thiol-lyases

and

might

be

also

operative

in

haloarchaea

Recently

also

a

tRNA-dependent

synthesis

pathway

has

been

reported

for

M.

jannaschii

that

leads

from

phosphoserine

an

intermediate

of

the

phosphorylated

serine

pathway

to

Cys-tRNA

-LRB-

Sauerwald

et

al.

-RRB-

Similar

tRNA-dependent

pathways

have

been

suggested

for

glutamine

and

asparagine

synthesis

from

glutamate

and

aspartate

-LRB-

Di

Giulio

-RRB-

For

cysteine

biosynthesis

sulfide

needs

to

be

assimilated

from

sulfate

via

adenylylsulfate

and

sulfite

Although

archaea

encode

a

gene

for

the

small

subunit

-LRB-

cysD

-RRB-

of

the

sulfate

adenylyltransferase

-LRB-

EC

e.g.

-RRB-

they

lack

the

GTPase

subunit

-LRB-

cysN

-RRB-

Genes

for

the

two

subsequent

sulfate

assimilation

steps

-LRB-

cysC

cysH

-RRB-

are

present

in

some

archaea

-LRB-

e.g.

Aeropyrum

pernix

-RRB-

but

are

not

present

in

any

of

the

haloarchaeal

genomes

Alternatively

thiosulfate

might

be

converted

to

sulfite

by

thiosulfate

sulfurtransferase

-LSB-

EC

e.g.

-LRB-

N-terminus

-RRB-

-RSB-

and

subsequently

to

sulfide

by

one

of

the

potential

sulfite

reductases

-LSB-

e.g.

-LRB-

C-terminus

-RRB-

not

in

H.

salinarum

-RSB-

Biosynthesis

of

branched

chain

amino

acids

-LRB-

valine

leucine

isoleucine

-RRB-

All

enzyme

genes

for

the

de

novo

synthesis

of

valine

leucine

and

isoleucine

are

present

in

Natronomonas

Haloquadratum

and

Haloarcula

but

not

in

Halobacterium

Threonine-ammonia

lyase

-LRB-

EC

e.g.

-RRB-

which

is

required

to

synthesize

the

isoleucine

precursor

is

encoded

in

all

haloarchaea

The

threonine

pathway

leading

to

isoleucine

is

employed

by

H.

hispanica

-LRB-

flux

-RRB-

but

it

is

simultaneuosly

operated

together

with

the

pyruvate

pathway

leading

via

citramalate

to

isoleucine

-LRB-

flux

-RRB-

-LRB-

Hochuli

et

al.

-RRB-

However

no

sequences

are

yet

available

in

public

repositories

for

enzymes

of

this

pathway

e.g.

citramalate

lyase

-LRB-

EC

-RRB-

A

third

potential

pathway

from

glutamate

to

isoleucine

via

methylaspartate

-LRB-

mesaconate

pathway

-RRB-

is

not

employed

by

H.

hispanica

-LRB-

Hochuli

et

al.

-RRB-

Mesaconate

pathway

genes

-LRB-

mamABC

-RRB-

were

found

in

the

genomes

of

H.

salinarum

and

H.

marismortui

however

-LRB-

see

below

-RRB-

In

Halobacterium

may

further

be

derived

from

methionine

by

methionine

gamma-lyase

-LRB-

EC

-RRB-

a

reaction

that

releases

volatile

methanthiol

from

the

cells

-LRB-

Nordmann

et

al.

-RRB-

The

fact

that

Halobacterium

encodes

enzyme

genes

for

synthesis

but

lacks

other

isoleucine

synthesis

enzymes

implies

that

is

a

precursor

for

further

metabolic

pathways

in

haloarchaea

Biosynthesis

of

aromatic

amino

acids

-LRB-

phenylalanine

tyrosine

tryptophan

histidine

-RRB-

The

complete

gene

set

for

the

classical

shikimate

pathway

where

is

derived

from

erythrose

-LRB-

-RRB-

and

phosphoenolpyruvate

-LRB-

-RRB-

is

only

present

in

Thermoplasma

spp.

and

some

other

archaea

-LRB-

Soderberg

-RRB-

In

methanogenic

archaea

is

synthesized

via

a

novel

pathway

starting

from

l-aspartate

semialdehyde

-LRB-

-RRB-

and

-LRB-

DKFP

-RRB-

-LRB-

White

-RRB-

The

DKFP

pathway

is

also

employed

by

haloarchaea

as

shown

by

the

incorporation

of

labeled

aspartate

into

the

of

tryptophan

in

H.

salinarum

-LRB-

Engelhard

et

al.

-RRB-

Furthermore

the

two

novel

DKFP

pathway

enzymes

for

alternative

synthesis

2-amino-3,7-dideoxy-d-threo-hept-6-ulosonate

synthase

-LRB-

together

with

DhnA-type

fructose

bisphosphate

aldolase

e.g.

-RRB-

and

synthase

type

II

-LRB-

e.g.

-RRB-

-LRB-

Porat

et

al

-RRB-

are

encoded

in

haloarchaeal

genomes

The

precursor

of

the

modified

shikimate

pathway

DKFP

presumably

derives

from

methylglyoxal

rather

than

from

nucleoside

diphosphate

sugars

in

archaea

-LRB-

White

and

Xu

-RRB-

Methylglyoxal

is

not

created

by

methylglyoxal

synthase

-LRB-

EC

-RRB-

which

is

absent

in

archaea

but

from

glyceraldehyde

using

triosephosphate

isomerase

-LRB-

EC

e.g.

-RRB-

Another

glycolytic

enzyme

a

multifunctional

class

I

fructose-biphosphate

aldolase

-LRB-

e.g.

-RRB-

catalyzes

the

subsequent

transaldolase

reaction

of

methylglyoxal

with

fructose

-LRB-

or

fructose

-RRB-

to

DKFP

The

remaining

biosynthesis

steps

from

to

chorismate

follow

the

classical

shikimate

pathway

in

haloarchaea

Enzyme

genes

for

biosynthesis

of

tryptophan

tyrosine

and

phenylalanine

from

chorismate

are

also

present

in

haloarchaeal

genomes

Consistent

with

this

de

novo

synthesis

of

phenylalanine

and

tryptophan

has

been

proven

for

H.

salinarum

-LRB-

Ekiel

et

al.

Engelhard

et

al.

-RRB-

A

second

synthesis

pathway

for

aromatic

amino

acids

in

archaea

has

recently

been

described

for

M.

maripaludis

-LRB-

Porat

et

al.

-RRB-

where

aromatic

amino

acids

are

synthesized

through

the

incorporation

of

exogenous

aryl

acids

catalyzed

by

a

ferredoxin-dependent

indolepyruvate

oxidoreductase

-LRB-

ior

-RRB-

This

pathway

is

presumably

not

present

in

halophilic

archaea

because

their

only

two

ferredoxin-dependent

oxidoreductases

have

been

already

assigned

otherwise

-LRB-

porAB

korAB

-RRB-

-LRB-

Kerscher

and

Osterhelt

b

-RRB-

Histidine

biosynthesis

genes

are

partly

clustered

within

haloarchaeal

genomes

All

enzymes

for

histidine

biosynthesis

starting

from

ribose

are

encoded

except

for

histidinol-phosphatase

-LRB-

EC

-RRB-

An

unknown

alternative

enzyme

is

indicated

for

the

missing

pathway

step

especially

since

histidine

biosynthesis

has

already

been

proven

experimentally

in

H.

salinarum

-LRB-

Ekiel

et

al.

-RRB-

Amino

acid

degradation

Glutamate

and

aspartate

degradation

Glutamate

and

aspartate

are

major

carbon

substrates

for

halophilic

archaea

that

are

fed

into

the

TCA

cycle

and

subsequently

into

the

respiratory

chain

for

ATP

production

Several

transaminases

are

encoded

in

haloarchaea

which

likely

catalyze

the

conversion

of

aspartate

glutamate

and

other

amino

acids

to

TCA

cycle

intermediates

In

H.

salinarum

and

H.

marismortui

glutamate

might

further

be

degraded

to

mesaconate

by

methylaspartate

mutase

-LRB-

EC

-RRB-

and

methylaspartate

ammonia-lyase

-LRB-

EC

-RRB-

Genes

for

the

mesaconate

pathway

-LRB-

mamABC

-RRB-

are

only

encoded

in

the

two

haloarchaea

and

very

few

bacteria

i.e.

two

E.

coli

strains

as

well

as

a

Clostridium

and

Treponema

species

In

thermophilic

anaerobic

bacteria

conversion

of

mesaconate

to

citramalate

and

subsequently

to

pyruvate

and

acetate

has

been

proven

-LRB-

Plugge

et

al.

-RRB-

However

the

respective

enzymes

for

the

established

reactions

are

not

yet

known

Mesaconate

is

also

a

potential

precursor

of

isoleucine

in

haloarchaea

although

not

for

the

haloarchaeon

H.

hispanica

-LRB-

Hochuli

et

al.

-RRB-

Arginine

metabolism

Urea

cycle

genes

for

the

conversion

of

ornithine

to

arginine

were

found

in

all

halophilic

strains

but

arginase

-LRB-

EC

-RRB-

converting

arginine

back

to

ornithine

is

only

present

in

H.

marismortui

Instead

H.

salinarum

metabolizes

arginine

to

ornithine

with

concomitant

ATP

production

via

the

arginine

deiminase

pathway

-LRB-

Hartmann

et

al.

-RRB-

This

pathway

is

rare

amongst

prokaryotes

and

H.

salinarum

is

the

only

archaeon

that

has

acquired

an

arginine

deiminase

gene

cluster

-LRB-

arcRACB

plasmid

-RRB-

-LRB-

Ruepp

and

Soppa

-RRB-

encoding

all

required

arginine

deiminase

pathway

enzymes

as

well

as

a

probable

transcription

regulator

ArcR

The

gene

for

an

arginine-ornithine

antiporter

-LRB-

-RRB-

-LRB-

J.

Tittor

unpublished

results

-RRB-

is

also

located

next

to

the

arginine

deiminase

gene

cluster

Archaea

also

utilize

arginine

for

the

biosynthesis

of

polyamines

such

as

putrescine

spermidine

or

spermine

-LRB-

Graham

et

al.

-RRB-

which

seem

to

be

required

in

archaea

for

nucleosome

maintenance

in

high-temperature

environments

-LRB-

Higashibata

et

al.

-RRB-

Orthologs

of

a

novel

arginine

decarboxylase

-LRB-

EC

e.g.

-RRB-

-LRB-

Graham

et

al.

-RRB-

and

agmatinase

-LRB-

EC

e.g.

-RRB-

for

the

conversion

of

arginine

to

putrescine

are

present

in

haloarchaeal

genomes

For

pathway

steps

leading

from

putrescine

to

spermidine

no

enzyme

genes

are

encoded

in

halophilic

archaea

Branched-chain

amino

acid

degradation

For

amino

acids

with

a

more

complex

carbon

scaffold

i.e.

branched-chain

and

aromatic

amino

acids

separate

biosynthesis

and

degradation

pathways

regulated

independently

from

each

other

have

evolved

Catabolism

of

valine

leucine

and

isoleucine

first

involves

a

transaminase

-LRB-

e.g.

-RRB-

for

the

conversion

of

branched-chain

amino

acids

to

the

respective

The

following

decarboxylation

is

likely

catalyzed

by

a

previously

unassigned

dehydrogenase

multienzyme

complex

-LRB-

e.g.

-RRB-

whose

component

has

recently

been

shown

to

accept

branched-chain

in

T.

acidophilum

-LRB-

Heath

et

al.

-RRB-

The

derived

activated

fatty

acids

are

likely

to

be

further

degraded

to

acetyl-CoA

and

propionyl-CoA

Aromatic

amino

acid

degradation

Histidine

is

likely

to

be

degraded

to

glutamate

via

the

urocanate

pathway

by

H.

salinarum

and

H.

marismortui

encoding

hutUGIH

gene

clusters

-LRB-

e.g.

-RRB-

The

two

haloarchaea

-LRB-

and

A.

pernix

-RRB-

also

acquired

the

only

archaeal

tryptophanases

-LRB-

EC

-RRB-

and

H.

salinarum

the

only

archaeal

kynureninase

-LRB-

EC

-RRB-

for

potential

indole

or

anthranilate

formation

from

tryptophan

respectively

Further

haloarchaea

-LRB-

except

H.

walsbyi

-RRB-

encode

orthologs

-LRB-

e.g.

-RRB-

of

the

recently

described

l-tyrosine

decarboxylase

-LRB-

EC

-RRB-

-LRB-

Kezmarsky

et

al.

-RRB-

which

is

required

for

synthesis

of

methanofuran

in

M.

jannaschii

Complete

aromatic

amino

acid

degradation

pathways

could

not

be

reconstructed

but

probable

aromatic

compound

dioxygenases

-LRB-

e.g.

-RRB-

are

encoded

in

all

haloarchaeal

genomes

Cofactor

metabolism

Bacteria

and

archaea

are

commonly

able

to

synthesize

cofactors

de

novo

However

the

relevant

biosynthesis

pathways

are

often

not

completely

understood

i.e.

early

pathway

steps

leading

to

biotin

and

thiamine

In

fact

many

genes

-LRB-

e.g.

thiI

moaA

-RRB-

which

have

been

associated

with

coenzyme

biosynthesis

have

not

yet

been

assigned

to

specific

metabolic

reactions

Upon

metabolic

reconstruction

archaea

reveal

many

gaps

where

enzyme

genes

are

replaced

by

still

unknown

non-orthologous

genes

Although

some

novel

enzymes

have

been

elucidated

in

recent

years

e.g.

novel

classes

of

GTP

cyclohydrolases

-LRB-

e.g.

-RRB-

-LRB-

Graham

et

al.

El

Yacoubi

et

al.

Grochowski

et

al.

-RRB-

several

pathway

gaps

in

archaeal

coenzyme

metabolism

remain

to

be

filled

Taking

these

limitations

into

account

de

novo

synthesis

pathways

for

common

cofactors

are

likely

in

haloarchaea

The

compared

haloarchaeal

genomes

show

surprisingly

different

gene

sets

for

cofactor

synthesis

pathways

and

might

have

differing

synthetic

capabilities

for

coenzymes

-LRB-

Supplementary

Material

-RRB-

N.

pharaonis

has

the

most

complete

set

of

cofactor

synthesis

genes

and

has

been

shown

to

grow

independently

of

cofactors

-LRB-

Falb

et

al.

-RRB-

It

is

capable

of

synthesizing

menaquinone

coenzyme

A

tetrahydrofolate

-LRB-

THF

-RRB-

molybdopterin

hemes

cobamide

flavins

nicotinamide

derivates

-LRB-

NAD

NADP

-RRB-

biotin

thiamine

and

pyridoxal

The

latter

is

presumably

synthesized

by

novel

pyridoxal

synthesis

enzymes

-LRB-

pdxS/pdxT

e.g.

-RRB-

like

in

B.

subtilis

-LRB-

Raschle

et

al.

-RRB-

While

most

enzymes

for

biotin

synthesis

are

encoded

in

N.

pharaonis

-LRB-

bioA

absent

-RRB-

and

H.

marismortui

-LRB-

bioA

and

bioD

absent

-RRB-

H.

salinarum

and

H.

walsbyi

lack

the

complete

biotin

synthesis

gene

set

Thiamine

biosynthesis

pathways

might

also

differ

amongst

haloarchaea

because

a

couple

of

probable

thiamine

biosynthesis

genes

found

in

N.

pharaonis

are

partly

missing

in

other

haloarchaea

i.e.

thiM

-LRB-

-RRB-

thiE

-LRB-

-RRB-

and

tenA

-LRB-

-RRB-

For

the

latter

two

enzymes

genes

there

are

non-orthologous

variants

in

haloarchaea

-LRB-

including

N.

pharaonis

-RRB-

tenA

is

analogous

to

thiC

-LRB-

-RRB-

and

thiN

-LRB-

-LRB-

fused

to

thiD

-RRB-

-RRB-

to

thiE

-LRB-

Morett

et

al.

-RRB-

Finally

gene

sets

for

folate

metabolism

differ

greatly

between

various

haloarchaeal

strains

-LRB-

see

below

-RRB-

leading

to

the

reduced

folate

synthesis

capability

in

H.

salinarum

-LRB-

Levin

et

al.

-RRB-

Consistent

with

these

findings

biotin

thiamine

and

folate

are

supplied

to

the

synthetic

medium

of

Halobacterium

-LRB-

Oesterhelt

and

Krippahl

-RRB-

The

utilization

of

several

cofactors

has

been

confirmed

in

haloarchaea

such

as

the

use

of

nicotinamide

derivates

by

glutamate

dehydrogenases

-LRB-

NAD

NADP

-RRB-

-LRB-

Hayden

et

al.

-RRB-

the

association

of

coenzyme

A

and

ferredoxin

to

pyruvate

and

ferredoxin

oxidoreductases

-LRB-

-RRB-

-LRB-

Kerscher

and

Oesterhelt

b

-RRB-

and

the

incorporation

of

flavin

in

dodecin

-LRB-

-RRB-

-LRB-

Bieger

et

al.

-RRB-

Respiratory

chains

of

haloarchaea

further

involve

menaquinone

and

certain

hemes

-LRB-

Oesterhelt

Sreeramulu

et

al.

Scharf

et

al.

Mattar

and

Engelhard

Falb

et

al.

-RRB-

Several

copies

of

proteinaceous

cofactors

e.g.

ferredoxin

thioredoxin

halocyanin

-LRB-

Mattar

et

al.

-RRB-

and

Fe-S

proteins

were

also

found

in

haloarchaeal

genomes

These

are

likely

used

in

various

redox

reactions

Heme

and

cobamide

synthesis

Menaquinone

-LRB-

men

-RRB-

and

heme

-LRB-

hem

-RRB-

biosynthesis

gene

clusters

are

present

in

all

haloarchaeal

genomes

All

heme

synthesis

genes

for

steps

leading

from

glutamate

to

uroporphyrinogen

III

have

been

found

but

genes

for

successive

modifications

of

the

porphyrin

system

to

protoheme

-LRB-

e.g.

by

hemE

-RRB-

are

absent

as

in

most

other

archaea

-LRB-

Supplementary

Material

-RRB-

Thus

an

alternative

heme

biosynthesis

pathway

via

as

shown

for

M.

barkeri

seems

likely

-LRB-

Buchenau

et

al.

-RRB-

A

novel

archaeal

variant

of

heme

A

synthase

-LRB-

-RRB-

-LRB-

Lewin

and

Hederstedt

-RRB-

for

the

conversion

of

heme

O

to

heme

A

is

present

in

haloarchaea

-LRB-

e.g.

-RRB-

Halobacterium

assimilates

corrinoid

precursors

and

cobamides

through

a

high-affinity

transport

system

but

is

also

able

to

synthesize

cobamide

-LRB-

coenzyme

-RRB-

de

novo

-LRB-

Woodson

et

al.

-RRB-

Cobamide

synthesis

starting

from

uroporphyrinogen

III

has

mainly

been

studied

in

bacteria

e.g.

in

Pseudomonas

denitrificans

-LRB-

cob

genes

-RRB-

which

uses

an

aerobic

pathway

and

in

Salmonella

typhimurium

-LRB-

cbi/cob

genes

-RRB-

and

Bacillus

megaterium

which

employ

an

anaerobic

pathway

-LRB-

Rodionov

et

al.

-RRB-

The

two

pathway

variants

differ

in

their

cobalt

integration

step

occurring

early

in

the

anaerobic

-LRB-

S.

typhimurium

cbiK

B.

megaterium

cbiX

-RRB-

but

late

within

the

aerobic

pathway

-LRB-

P.

denitrificans

cobN

-RRB-

Haloarchaea

encode

both

cobalt

chelatases

the

oxygen-dependent

-LRB-

cobN

e.g.

-RRB-

and

the

oxygen-independent

type

-LRB-

cbiX

e.g.

-RRB-

-LRB-

Supplementary

Material

-RRB-

Experimental

data

showed

however

that

cobamides

are

synthesized

under

aerobic

conditions

in

Halobacterium

-LRB-

Woodson

et

al.

-RRB-

Haloarchaeal

genomes

encode

most

known

cob

genes

but

homologs

of

P.

denitrificanscobG

cobF

cobK

-LRB-

S.

typhimirium

cobJ

-RRB-

and

cobP

-LRB-

S.

typhimirium

cobU

-RRB-

are

absent

This

indicates

alternative

enzymes

and

reactions

in

archaeal

cobalamin

synthesis

For

example

the

bifunctional

bacterial

cobP

enzyme

gene

-LRB-

EC

-RRB-

is

replaced

by

two

novel

genes

cbiZ

-LRB-

e.g.

-RRB-

and

cobY

-LRB-

e.g.

-RRB-

in

archaea

-LRB-

Woodson

et

al.

Woodson

and

Escalante-Semerena

-RRB-

The

novel

archaeal

variant

of

phosphate

phosphatase

-LRB-

EC

cobZ

-RRB-

-LRB-

Zayas

et

al.

-RRB-

for

the

synthesis

of

the

cobamide

α-ligand

is

not

present

in

haloarchaea

however

Their

genomes

-LRB-

except

H.

salinarum

-RRB-

contain

the

bacterial-type

phosphatase

gene

-LRB-

cobC

in

Salmonella

e.g.

-RRB-

encoded

next

to

the

hemCXD

cluster

-LRB-

-RRB-

in

Natronomonas

The

cobamide

α-ligand

is

derived

by

cleavage

of

the

ribityl

tail

from

flavin

mononucleotide

cofactor

by

BluB

-LRB-

-RRB-

-LRB-

Campbell

et

al.

-RRB-

of

which

distant

homologs

are

present

in

haloarchaea

-LRB-

e.g.

-RRB-

Folate

synthesis

Folic

acid

and

its

derivates

are

synthesized

from

p-aminobenzoate

-LRB-

PAB

-RRB-

and

a

pteridine

intermediate

-LRB-

Fig.

Supplementary

Material

-RRB-

The

latter

is

derived

from

GTP

by

a

series

of

reactions

involving

folE

folB

and

folK

genes

all

of

them

are

missing

in

the

archaeal

domain

of

life

except

for

H.

marismortui

This

species

has

acquired

an

extensive

folate

metabolism

gene

cluster

located

on

one

of

its

plasmid

which

contains

canonical

GTP

cyclohydrolase

I

-LRB-

EC

folE

-RRB-

Recently

a

novel

archaeal

GTP

cyclohydrolase

-LRB-

e.g.

-RRB-

producing

a

cyclic

phosphate

intermediate

has

been

reported

in

archaea

-LRB-

El

Yacoubi

et

al.

Grochowski

et

al.

-RRB-

but

novel

enzyme

genes

replacing

folB

and

folK

remain

to

be

elucidated

PAB

is

usually

derived

from

chorismate

by

aminodeoxychorismate

synthase

-LRB-

EC

pabAB

-RRB-

and

aminodeoxychorismate

lyase

-LRB-

EC

pabC

-RRB-

which

are

clustered

in

haloarchaeal

genomes

-LRB-

e.g.

-RRB-

These

enzymes

have

previously

been

annotated

as

copies

of

anthranilate

synthase

-LRB-

same

-RRB-

and

branched-chain

amino

acid

aminotransferase

-LRB-

same

-RRB-

respectively

The

pabA

gene

-LRB-

-RRB-

of

H.

salinarum

strain

-LRB-

but

not

of

strain

-RRB-

is

disrupted

by

an

insertion

element

though

so

that

de

novo

synthesis

of

folate

is

likely

omitted

in

this

strain

Proposed

haloarchaeal

pab

genes

have

not

yet

been

characterized

and

it

should

be

noted

that

labeling

studies

for

M.

maripaludis

suggest

the

synthesis

of

PAB

from

and

not

from

chorismate

in

this

archaeon

-LRB-

Porat

et

al.

-RRB-

Fig.

biosynthesis

and

metabolism

in

halophilic

archaea

Sets

of

enzyme

genes

which

are

involved

in

folate

metabolism

differ

considerably

between

haloarchaeal

species

-LRB-

square

H.

marismortui

circle

H.

walsbyi

diamond

N.

pharaonis

triangle

H.

salinarum

hexagon

H.

volcanii

green

gene

exists

red

gene

absent

-RRB-

For

details

see

Supplementary

Material

For

folate

synthesis

the

pteridine

intermediate

and

PAB

are

converted

to

and

subsequently

to

by

the

gene

products

of

folP

and

folC

-LRB-

Fig.

-RRB-

Dihydrofolate

is

then

reduced

to

by

dihydrofolate

reductase

-LRB-

EC

folA

-RRB-

which

is

encoded

in

all

haloarchaea

-LRB-

e.g.

-RRB-

except

H.

salinarum

Tetrahydrofolate

synthesis

in

H.

salinarum

occurs

via

a

novel

alternative

pathway

instead

where

is

reduced

to

by

a

flavin-binding

Prd

linker

domain

of

the

FolC-Prd-FolP

fusion

protein

-LRB-

e.g.

-RRB-

-LRB-

Levin

et

al.

-RRB-

Tetrahydropteroate

is

subsequently

converted

to

tetrahydrofolate

by

the

FolC

domain

The

canonical

dihydrofolate

reductase

FolA

is

not

only

involved

in

de

novo

synthesis

of

folate

but

is

also

required

for

the

regeneration

of

dihydrofolate

produced

by

thymidylate

synthase

-LRB-

EC

thyA

-RRB-

This

enzyme

is

replaced

by

an

alternative

folate-independent

enzyme

encoded

by

thyX

-LRB-

-RRB-

in

case

a

chromosomal

folA

gene

is

absent

like

in

H.

salinarum

and

H.

marismortui

-LRB-

Supplementary

Material

-RRB-

Tetrahydrofolate

is

likely

metabolized

to

formyl

and

methyl-THF

in

haloarchaea

However

only

H.

walsbyi

and

the

H.

marismortui

plasmid

encode

all

required

enzyme

genes

involved

in

folate

coenzyme

metabolism

-LRB-

e.g.

-RRB-

Conclusions

Comparative

analysis

of

enzyme

gene

sets

of

four

halophilic

archaea

reveals

analogous

metabolic

routes

for

the

biosynthesis

of

nucleotides

and

prenyl-based

lipids

Biosynthesis

pathways

for

all

proteinogenic

amino

acids

are

present

for

H.

marismortui

H.

walsbyi

and

N.

pharaonis

while

H.

salinarum

has

no

biosynthetic

capabilities

for

five

amino

acids

The

loss

of

amino

acid

synthesis

genes

and

the

acquisition

of

a

catabolic

gene

clusters

for

arginine

and

glutamate

fermentation

in

Halobacterium

might

have

been

driven

by

the

constant

availability

of

external

amino

acids

under

extreme

salt

conditions

where

few

other

halophiles

compete

for

nutrients

Several

pathway

variations

were

observed

for

folate-dependent

enzymes

such

as

the

occurrence

of

different

types

of

thymidylate

synthases

or

the

circumvention

of

glycine

hydroxymethyltransferase

by

threonine

aldolase

These

differences

might

be

due

to

varying

gene

sets

for

folate

metabolism

in

particular

the

replacement

of

the

canonical

dihydrofolate

reductase

by

dihydropteroate

reductase

in

H.

salinarum

-LRB-

Levin

et

al.

-RRB-

While

some

haloarchaea

may

require

thiamine

and

biotin

supplements

all

halophilic

archaea

possess

the

required

enzyme

genes

for

the

de

novo

synthesis

of

cobamide

hemes

flavin

and

nicotinamide

nucleotides

folate

coenzyme

A

menaquinone

molybdopterin

pyridoxal

and

several

proteinaceous

cofactors

like

halocyanin

The

compared

haloarchaeal

species

can

best

be

distinguished

by

their

catabolic

and

central

intermediary

pathways

While

H.

marismortui

has

a

versatile

sugar

and

nitrogen

metabolism

H.

walsbyi

encodes

a

unique

sugar

phosphotransferase

system

and

H.

salinarum

is

characterized

by

a

variable

energy

metabolism

Its

plasmids

acquired

arginine

deiminase

and

cytochrome

d

oxidase

gene

clusters

-LRB-

-RRB-

as

well

as

the

gene

for

glycerol

dehydrogenase

N.

pharaonis

is

not

able

to

catabolize

glycerol

-LRB-

that

is

not

common

in

its

alkaline

environment

-RRB-

and

is

further

incapable

of

sugar

degradation

However

the

haloalkaliphilic

strain

encodes

an

archaeal-type

RuBisCO

that

is

probably

involved

in

a

novel

carbon

dioxide-fixation

pathway

-LRB-

Sato

et

al.

-RRB-

It

appears

that

halophilic

archaea

have

adopted

several

strategies

in

order

to

adapt

to

the

nutritional

conditions

of

hypersaline

environments

e.g.

by

gain/loss

of

metabolic

pathways

acquirement

of

plasmid-encoded

enzymes

and

utilization

of

secretion

enzymes

-LRB-

Supplementary

Material

-RRB-

Interestingly

haloarchaeal

genomes

often

encode

bacterial-type

enzymes

instead

of

-LRB-

or

alongside

-RRB-

non-orthologous

archaeal

variants

e.g.

bacterial-type

phosphate

phosphatase

-LRB-

cobC

-RRB-

folate

reductase

-LRB-

folA

-RRB-

and

β-carotene

monooxygenases

Within

the

mevalonate

pathway

two

bacterial-type

enzymes

-LRB-

IPP

isomerase

diphosphomevalonate

decarboxylase

-RRB-

exist

which

might

have

been

acquired

to

cover

high

isoprenoid

demands

of

haloarchaea

Future

studies

might

elucidate

whether

halophilic

archaea

employ

different

enzyme

sets

for

the

synthesis

of

their

membrane

lipids

and

carotenes

the

latter

of

which

are

unique

to

the

haloarchaeal

branch

of

the

archaeal

domain

The

compiled

data

on

haloarchaeal

metabolism

present

a

valuable

resource

for

future

system

biology

approaches

-LRB-

Gonzalez

et

al.

-RRB-

because

current

knowledge

of

enzyme

variants

domain

rearrangements

and

enzyme

substrate

specifities

has

been

included

throughout

Further

many

of

the

predicted

reactions

and

enzymes

have

been

linked

to

available

experimental

data

from

enzyme

activity

tests

and

labeling

studies

conducted

in

the

model

species

H.

salinarum

Some

of

these

experimental

results

however

disagree

with

genomic

findings

e.g.

for

enzymes

of

the

glyoxylate

cycle

-LRB-

Aitken

and

Brown

-RRB-

and

the

Entner-Douderoff

pathway

-LRB-

Rawal

et

al.

-RRB-

-LRB-

Fig.

-RRB-

These

inconsistencies

might

be

due

to

still

unknown

non-orthologous

enzymes

or

may

result

from

investigating

different

strains

of

Halobacterium

which

have

been

renamed

several

times

within

the

past

decades

Although

many

pathway

gaps

in

archaeal

metabolic

networks

have

been

closed

in

recent

years

novel

enzyme

and

pathway

variants

still

remain

to

be

discovered

within

the

archaeal

domain

of

life

In

haloarchaea

pathways

for

ribose

synthesis

and

for

retinal

formation

need

to

be

investigated

A

future

in-depth

analysis

of

abundant

non-orthologous

gene

displacements

in

halophilic

and

other

archaea

might

further

give

interesting

insights

into

the

evolution

of

metabolic

pathways

Electronic

supplementary

material

Below

is

the

link

to

the

electronic

supplementary

material

Supplementary

Material

-LRB-

DOC

MB

-RRB-

Supplementary

Material

-LRB-

DOC

KB

-RRB-

Supplementary

Material

-LRB-

DOC

KB

-RRB-

Supplementary

Material

-LRB-

DOC

KB

-RRB-

metabolism
archaea
haloarchaea
halobacterium salinarum
metabolic pathways
enzymes