Preconception

Brief

Occupational/Environmental

Exposures

In

the

last

decade

more

than

half

of

U.S.

children

were

born

to

working

mothers

and

of

working

men

and

women

were

of

reproductive

age

In

more

than

million

women

age

were

employed

full

time

This

implies

the

need

for

clinicians

to

possess

an

awareness

about

the

impact

of

work

on

the

health

of

their

patients

and

their

future

offspring

Most

chemicals

in

the

workplace

have

not

been

evaluated

for

reproductive

toxicity

and

where

exposure

limits

do

exist

they

were

generally

not

designed

to

mitigate

reproductive

risk

Therefore

many

toxicants

with

unambiguous

reproductive

and

developmental

effects

are

still

in

regular

commercial

or

therapeutic

use

and

thus

present

exposure

potential

to

workers

Examples

of

these

include

heavy

metals

-LRB-

lead

cadmium

-RRB-

organic

solvents

-LRB-

glycol

ethers

percholoroethylene

-RRB-

pesticides

and

herbicides

-LRB-

ethylene

dibromide

-RRB-

and

sterilants

anesthetic

gases

and

anti-cancer

drugs

used

in

healthcare

Surprisingly

many

of

these

reproductive

toxicants

are

well

represented

in

traditional

employment

sectors

of

women

such

as

healthcare

and

cosmetology

Environmental

exposures

also

figure

prominently

in

evaluating

a

woman

's

health

risk

and

that

to

a

pregnancy

Food

and

water

quality

and

pesticide

and

solvent

usage

are

increasingly

topics

raised

by

women

and

men

contemplating

pregnancy

The

microenvironment

of

a

woman

such

as

her

choices

of

hobbies

and

leisure

time

activities

also

come

into

play

Caregivers

must

be

aware

of

their

patients

potential

environmental

and

workplace

exposures

and

weigh

any

risk

of

exposure

in

the

context

of

the

time-dependent

window

of

reproductive

susceptibility

This

will

allow

informed

decision-making

about

the

need

for

changes

in

behavior

diet

hobbies

or

the

need

for

added

protections

on

the

job

or

alternative

duty

assignment

Examples

of

such

environmental

and

occupational

history

elements

will

be

presented

together

with

counseling

strategies

for

the

clinician

Introduction

The

influence

of

environmental

exposures

on

the

general

status

of

health

has

been

increasingly

acknowledged

for

numerous

disease

outcomes

The

connection

between

air

pollution

and

acute

respiratory

disease

for

example

and

more

recently

the

observation

linking

poor

indoor

air

quality

to

increases

in

the

incidence

of

childhood

asthma

has

been

widely

publicized

Such

epidemiologic

observations

are

often

reported

in

the

media

and

when

combined

with

the

growing

public

interest

in

a

clean

and

healthy

environment

have

translated

into

an

increasingly

sophisticated

patient

population

that

expects

healthcare

providers

to

be

conversant

with

the

environmental

contribution

to

disease

risk

Indeed

the

lay

public

often

has

shown

more

interest

in

and

occasionally

knowledge

of

the

relationship

between

health

and

the

environment

than

has

the

medical

community

Medical

educators

and

the

Institute

of

Medicine

-LRB-

IOM

-RRB-

both

have

addressed

this

dilemma

by

promoting

enhanced

medical

student

and

physician

training

in

environmental

medicine

in

order

to

develop

environmentally

literate

physicians

In

this

brief

the

term

environmental

medicine

will

refer

to

diagnosing

and

caring

for

people

exposed

to

chemical

and

physical

hazards

in

their

homes

communities

and

workplaces

through

such

media

as

contaminated

soil

water

and

air

-LSB-

-RSB-

This

definition

was

taken

from

a

IOM

Committee

report

on

Curriculum

Development

in

Environmental

Medicine

The

preconception

clinical

visit

already

includes

environmental

history

queries

regarding

smoking

and

alcohol

use

-LSB-

-RSB-

but

must

be

enlarged

to

address

the

broader

concept

of

environmental

exposures

occurring

in

the

woman

's

home

community

and

workplace

Sources

of

environmental

exposure

Organizing

the

environmental

exposure

history

can

be

facilitated

by

using

the

definition

referred

to

above

and

by

eliciting

information

on

aspects

of

the

patient

's

larger

environment

this

includes

the

specific

community

locale

-LRB-

workplace

and

her

home

environment

-RRB-

and

infrastructure

which

relates

to

air

quality

and

soil

or

water

pollution

all

of

which

the

patient

deals

with

on

a

regular

basis

Community

Surprisingly

a

clinician

would

likely

not

know

if

a

given

patient

in

his/her

practice

was

living

in

an

area

near

by

an

environmentally

polluted

location

such

as

a

National

Priority

Listed

-LRB-

NPL

-RRB-

waste

site

or

a

local

toxic

waste

dump

In

large

metropolitan

areas

many

such

sites

are

present

and

unknown

to

most

citizens

Although

clinically

significant

exposure

to

a

toxic

hazard

present

on

the

site

would

be

unlikely

to

threaten

the

wider

community

a

risk

may

exist

for

some

residents

living

very

close

to

the

site

possibly

allowing

for

soil

or

drinking

water

contamination

In

specific

instances

often

covered

by

the

news

media

patients

would

likely

be

able

to

report

knowledge

of

living

in

the

vicinity

of

such

a

place

Such

a

self-report

response

could

be

elicited

by

simply

asking

the

question

Do

you

live

near

or

have

contact

with

a

waste

site

Probably

the

most

potentially

important

exposure

opportunity

is

via

contaminated

drinking

water

Here

the

biggest

risk

is

usually

from

a

small

private

water

source

such

as

a

well

not

subject

to

municipal

water

treatment

standards

and

testing

According

to

the

EPA

approximately

of

the

U.S.

population

has

a

private

drinking

water

source

Workplace

An

intimate

relationship

exists

between

occupational

and

environmental

health

because

often

the

source

of

environmental

contamination

is

a

former

-LRB-

or

present

day

-RRB-

work

site

The

workplace

is

an

important

special

case

in

environmental

history

taking

because

populations

exposed

at

work

tend

to

be

exposed

at

higher

concentrations

than

the

larger

community

regardless

whether

contamination

is

via

air

water

or

soil

Thus

though

the

working

population

is

smaller

than

the

public

at

large

workers

are

exposed

at

higher

doses

It

is

thus

sometimes

observed

that

workers

are

the

canaries

or

sentinels

exhibiting

first

the

health

effects

which

might

be

expected

in

a

wider

community

exposure

from

an

environmental

pathway

It

is

fair

to

say

that

the

principal

exposure

opportunity

to

an

environmental

reproductive

or

developmental

toxicant

in

patients

will

be

from

their

work

place

Many

toxicants

with

unambiguous

reproductive

and

developmental

effects

are

still

in

regular

commercial

or

therapeutic

use

and

thus

pose

a

continued

potential

risk

in

the

occupational

environment

Several

employment

sectors

where

such

toxicants

commonly

are

found

employ

women

workers

primarily

and

include

laboratory

and

clinical

medicine

printing

and

dry

cleaning

-LSB-

-RSB-

though

this

is

clearly

not

always

the

case

and

the

gender

mix

varies

by

sector

-LRB-

See

Table

-RRB-

Whereas

some

of

these

chemical

toxicants

are

regulated

by

public

health

agencies

the

majority

of

chemicals

considered

for

regulation

are

not

evaluated

for

reproductive

endpoints

-LSB-

-RSB-

but

rather

for

other

toxic

effects

This

gap

in

the

regulatory

safety

net

allows

reproductive

toxicants

to

be

encountered

in

both

environmental

and

work

settings

by

men

and

women.Table

sectors

and

associated

reproductive/develop-mental

toxicantsSectorToxicantExamplesAgriculturePesticides/HerbicidesEthylene

DibromideManufacturingOrganic

SolventsGlycol

ethers

lead

Heavy

MetalsCadmiumDry

CleaningSolventsPerchloroethylenePrintingSolvents/inksPharmaceutical

Compounding/ManufactureHazardous

DrugsAntineoplastics

hormones

immunologic

modifiersHealth

CareBiologicsRubella

CMV

Hepatitis

virusPhysical

AgentsIonizing

Radiation/HeatChemicalsAntineoplastics/Hazardous

Drugs

Anesthetic

Gases

SterilantsPhysical

ExertionLifting/Prolonged

Standing

Shift

WorkNote

Ref

Summarized

from

GAO

Stellman

Home

Here

one

must

consider

the

exposure

opportunities

posed

by

the

woman

's

residential

environment

such

as

those

involved

with

household

tasks

those

related

to

her

pursuit

of

hobbies

and

those

related

to

her

micro-environment

including

diet

Diet

Beyond

the

current

recommendations

related

to

healthy

eating

the

uses

of

non-prescription

herbal

or

alternative

medicines

or

supplements

are

crucial

parts

of

the

medical

history

for

women

planning

a

pregnancy

Also

some

ethnic

home

remedies

must

be

queried

for

as

some

contain

hazardous

contaminants

such

as

lead

and

mercury

-LSB-

-RSB-

There

are

also

several

dietary

cautions

which

should

be

reviewed

with

the

woman

contemplating

pregnancy

One

is

not

new

and

relates

to

the

warning

regarding

exposure

to

food

borne

listeria

infections

Although

listeriosis

may

cause

only

mild

flu-like

symptoms

in

the

pregnant

mother

serious

outcomes

in

the

fetus

may

result

These

include

premature

delivery

stillbirth

or

neonatal

infection

-LSB-

-RSB-

General

advice

relies

upon

avoidance

of

soft

cheese

such

as

feta

Brie

Camembert

blue-veined

and

Mexican-style

cheeses

and

unpasteurized

milk

or

milk

products

-LSB-

-RSB-

Strict

adherence

to

food

preparation

safety

such

as

vigilant

washing

of

raw

fruits

and

vegetables

avoidance

of

undercooked

and

raw

meat

and

careful

separation

of

stored

raw

food

from

uncooked

meats

serves

to

enhance

safety

for

the

entire

family

as

most

food

borne

illness

is

preventable

Consumer

advisory

on

methylmercury

in

fish

In

the

Food

and

Drug

Administration

-LRB-

FDA

-RRB-

and

the

Environmental

Protection

Agency

-LRB-

EPA

-RRB-

issued

a

joint

consumer

advisory

on

methylmercury

in

fish

and

shellfish

The

warning

specifically

targets

women

who

may

become

pregnant

are

pregnant

or

are

nursing

mothers

and

also

includes

young

children

The

document

emphasizes

the

health

benefits

of

fish

in

the

diet

generally

as

a

good

source

of

protein

and

also

warns

of

several

types

of

fish

which

contain

comparatively

higher

concentrations

of

mercury

and

should

be

avoided

These

include

shark

swordfish

King

mackerel

and

Tilefish

Other

types

of

fish

may

be

consumed

in

up

to

two

meals

-LRB-

oz

each

-RRB-

per

week

including

shrimp

canned

light

tuna

-LRB-

but

not

albacore

which

has

more

mercury

-RRB-

salmon

pollock

and

catfish

The

Advisory

also

warns

about

local

fish

advisories

which

are

generally

posted

for

specific

populations

which

supplement

their

diets

with

locally

caught

fish

Appropriate

discussion

of

relevant

advisories

should

take

place

in

the

preconception

visit

and

is

especially

important

for

non-meat

eating

patients

whose

total

dietary

intake

of

fish

may

be

relatively

greater

than

is

the

case

for

meat

consumers

Also

of

note

the

U.S.

Department

of

Health

's

Women

's

Infants

and

Children

Program

-LRB-

WIC

-RRB-

sometimes

gives

canned

tuna

as

a

diet

supplement

This

practice

should

be

weighed

in

light

of

the

above

concerns

however

at

least

in

terms

of

the

relative

amount

of

tuna

in

a

weekly

diet

The

complete

consumer

advisory

can

be

found

at

Hobbies

and

home-based

work

The

hobbies

of

concern

generally

would

include

those

involving

similar

types

of

chemical

toxicants

discussed

in

the

occupational

section

above

including

heavy

metals

-LRB-

lead

cadmium

arsenic

-RRB-

and

solvents

-LRB-

paints

-LRB-

other

than

latex

based

-RRB-

furniture

stripper

metal

cleaners

etc.

-RRB-

Hobbies

to

be

discouraged

include

painting

ceramics

stained-glass

window

making

-LRB-

lead

solder

-RRB-

furniture

re-finishing

and

the

like

Leisure

activities

to

be

avoided

include

use

of

saunas

and

hot

tubs

A

number

of

home-based

work

activities

which

resulted

in

contamination

of

the

home

and

family

members

have

been

documented

in

the

literature

Although

uncommon

they

deserve

mention

here

At

one

end

of

the

spectrum

most

involve

the

use

of

metals

either

in

metal

parts

reclamation

-LRB-

lead

batteries

being

melted

down

-RRB-

or

jewelry

making

-LRB-

heating

and

soldering

-RRB-

-LSB-

-RSB-

At

the

other

the

home

environment

may

be

unwittingly

contaminated

by

family

members

who

bring

work

contaminants

home

on

their

shoes

or

dirty

work

clothes

If

the

work

clothes

are

laundered

at

home

there

not

only

is

opportunity

for

exposure

to

the

launderer

but

also

other

household

members

For

this

reason

some

OSHA

standards

require

changing

rooms

so

work

clothes

will

not

secondarily

expose

family

members

in

the

case

of

lead

and

asbestos

work

for

example

-LSB-

-RSB-

Household

exposures

Pesticides

herbicides

and

rodenticides

are

likely

the

most

common

chemical

toxicants

in

the

average

home

Certainly

a

pregnant

woman

or

one

actively

attempting

to

conceive

should

not

apply

any

of

these

-LSB-

-RSB-

Because

the

concept

of

second

hand

exposure

as

described

above

applies

here

also

care

needs

be

taken

so

that

the

chemicals

are

not

tracked

into

or

throughout

the

house

no

matter

who

performs

the

chemical

applications

Of

far

greater

importance

on

a

population

basis

is

second

hand

smoke

in

the

home

and/or

workplace

Numerous

polycyclic

aromatic

hydrocarbons

present

in

cigarette

smoke

are

carcinogenic

and

have

been

shown

to

affect

reproductive

outcomes

in

animal

studies

They

cross

and

affect

the

placenta

and

enter

breast

milk

-LSB-

-RSB-

Laundering

of

contaminated

work

clothes

is

also

a

concern

as

discussed

above

A

hiatus

from

caring

for

the

family

cat

and

litter

box

should

be

mentioned

due

to

the

toxoplasmosis

risk

Fig.

checklist

for

initial

qualitative

evaluation

of

reproductive

hazards

-LRB-

modified

from

Grajewski

-RRB-

Fig.

of

checklist

for

initial

quantitative

evaluation

of

reproductive

hazards

-LRB-

modified

from

Grajewski

-RRB-

Many

non-latex

paints

are

solvent

based

and

contain

small

amounts

of

metals

to

enhance

wear

and

as

preservatives

This

is

especially

true

of

exterior

paint

such

as

that

used

on

porches

or

building

exteriors

or

even

in

the

interiors

of

older

buildings

Rehabbing

older

homes

which

often

involve

paint

stripping

either

with

a

heat

gun

or

a

chemical

stripper

is

particularly

hazardous

Inhalation

is

a

very

efficient

means

of

producing

a

clinically

significant

exposure

to

lead

which

was

commonly

used

years

ago

in

interiors

of

homes

Many

commercial

paint

strippers

contain

methylene

chloride

-LRB-

dichloromethane

-RRB-

which

metabolizes

to

carbon

monoxide

and

is

particularly

toxic

to

the

fetus

Guidance

for

clinician

Certainly

awareness

that

occupational

and

environmental

hazards

encountered

by

patients

may

play

a

clinically

significant

role

in

a

pregnancy

outcome

is

the

first

step

in

effective

patient

management

Therefore

enlarging

the

standard

health

history

form

completed

by

the

patient

on

or

before

the

first

visit

to

obtain

a

more

detailed

environmental

history

is

a

necessary

first

step

and

is

also

often

very

informative

Whereas

a

number

of

preconception

checklists

exist

only

some

of

which

address

fish

consumption

or

residence

near

a

waste

site

most

poorly

capture

or

even

fail

to

query

about

occupation

despite

this

being

the

likely

greatest

environmental

risk

the

patient

faces

A

qualitative

evaluation

of

a

patient

's

potential

exposure

to

reproductive

and

developmental

toxicants

can

be

obtained

with

a

screening

questionnaire

as

seen

in

Fig.

This

involves

examining

occupation

by

industry

sector

of

employment

and

then

by

chemical

physical

and

biological

agents

of

concern

If

a

reproductive

hazard

is

present

in

the

patient

's

work

environment

an

initial

quantitative

assessment

can

be

made

regarding

the

exposure

intensity

-LRB-

Fig.

-RRB-

However

at

this

point

the

clinician

may

want

to

consult

an

occupational

medicine

colleague

to

elicit

a

more

detailed

history

and

assist

in

the

preconception

recommendations

Preconception

management

strategies

should

be

based

on

the

occupational

history

and

include

a

decision

on

the

safety

of

continued

employment

during

the

preconception

period

and

pregnancy

If

the

patient

works

with

bona

fide

reproductive

and

development

toxicants

continued

employment

and

under

what

circumstances

must

be

determined

This

may

involve

identifying

additional

protections

such

as

a

respirator

gloves

or

other

personal

protective

apparel

or

equipment

that

may

make

the

job

safer

for

the

patient

There

are

some

jobs

however

where

it

is

recommended

by

professional

organizations

as

well

as

governmental

safety

and

health

agencies

that

pregnant

persons

or

those

actively

trying

to

conceive

be

provided

with

alternative

duty

-LSB-

-RSB-

A

good

example

here

is

nurses

who

handle

cancer

chemotherapy

-LSB-

-RSB-

However

a

related

issue

involves

crafting

elements

of

an

existing

job

which

the

pregnant

patient

such

as

the

oncology

nurse

may

safely

continue

such

as

patient

education

telephone

triage

and

the

like

This

arrangement

is

termed

alternative

duty

Also

involved

in

preconception

counseling

is

the

potential

impact

on

the

quality

of

breast

milk

if

the

mother

is

planning

to

breastfeed

The

majority

of

organic

solvents

and

pharmaceuticals

absorbed

by

the

mother

in

the

workplace

invariably

make

their

way

into

milk

Thus

both

work

during

pregnancy

and

return

to

work

while

breastfeeding

need

to

be

considered

We

have

thus

far

focused

on

the

working

patient

's

current

job

which

is

generally

considered

the

most

influential

on

pregnancy

outcome

as

most

reproductive

toxicants

are

thought

to

act

via

an

acute

toxicity

mechanism

during

the

three

to

four

months

prior

to

conception

and

pregnancy

-LSB-

-RSB-

However

there

are

also

several

examples

of

remote

past

exposure

that

are

important

to

elicit

though

likely

less

commonly

encountered

An

example

includes

the

patient

who

has

high

bone

lead

stores

from

remote

past

-LRB-

childhood

-RRB-

exposure

that

can

be

mobilized

during

pregnancy

and

expose

the

fetus

-LSB-

-RSB-

Medical

management

at

the

preconception

visit

would

include

a

blood

lead

level

so

this

can

be

tracked

during

pregnancy

Also

maternal

stores

of

fat-soluble

organic

chemicals

such

as

dioxins

and

PCBs

may

also

expose

the

fetus

-LSB-

-RSB-

Positive

responses

to

exposure

to

occupational

or

environmental

agents

by

the

preconception

patient

may

require

consultation

The

Association

of

Occupational

and

Environmental

Clinics

can

supply

referrals

to

clinicians

in

various

locales

nationwide

at

-LRB-

-RRB-

Conclusion

The

preconception

office

clinic

visit

presents

a

strategic

opportunity

to

minimize

the

environmental

and

occupational

sources

of

reproductive

risk

facing

the

preconception

patient

This

requires

the

two-way

exchange

of

information

between

the

patient

and

her

clinician

clarifying

misunderstandings

and

implementing

reasonable

strategies

to

minimize

exposures

from

the

wider

community

in

the

workplace

and

at

home

Taking

an

environmental

and

occupational

history

and

tailoring

recommendations

based

on

that

enlarges

the

likelihood

that

preventable

adverse

pregnancy

outcomes

can

be

avoided

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O


Aspergillus

niger

genome-wide

analysis

reveals

a

large

number

of

novel

alpha-glucan

acting

enzymes

with

unexpected

expression

profiles

The

filamentous

ascomycete

Aspergillus

niger

is

well

known

for

its

ability

to

produce

a

large

variety

of

enzymes

for

the

degradation

of

plant

polysaccharide

material

A

major

carbon

and

energy

source

for

this

soil

fungus

is

starch

which

can

be

degraded

by

the

concerted

action

of

α-amylase

glucoamylase

and

α-glucosidase

enzymes

members

of

the

glycoside

hydrolase

-LRB-

GH

-RRB-

families

and

respectively

In

this

study

we

have

combined

analysis

of

the

genome

sequence

of

A.

niger

CBS

with

microarray

experiments

to

identify

novel

enzymes

from

these

families

and

to

predict

their

physiological

functions

We

have

identified

previously

unknown

family

and

enzymes

in

the

A.

niger

genome

all

of

which

have

orthologues

in

other

aspergilli

Only

two

of

the

newly

identified

enzymes

a

putative

α-glucosidase

-LRB-

AgdB

-RRB-

and

an

α-amylase

-LRB-

AmyC

-RRB-

were

predicted

to

play

a

role

in

starch

degradation

The

expression

of

the

majority

of

the

genes

identified

was

not

induced

by

maltose

as

carbon

source

and

not

dependent

on

the

presence

of

AmyR

the

transcriptional

regulator

for

starch

degrading

enzymes

The

possible

physiological

functions

of

the

other

predicted

family

and

enzymes

including

intracellular

enzymes

and

cell

wall

associated

proteins

in

alternative

α-glucan

modifying

processes

are

discussed

Introduction

Aspergillus

niger

is

a

saprophytic

fungus

well

known

for

its

production

and

secretion

of

a

variety

of

hydrolytic

enzymes

contributing

to

its

ability

to

degrade

plant

polysaccharides

such

as

cellulose

hemicellulose

pectin

starch

and

inulin

-LRB-

De

Vries

and

Visser

Tsukagoshi

et

al.

Yuan

et

al.

-RRB-

Starch

is

the

most

abundant

storage

carbohydrate

in

the

plant

kingdom

and

is

present

in

tubers

seeds

and

roots

of

a

variety

of

crop

plants

including

cereals

potatoes

and

manioc

-LRB-

Peters

-RRB-

Starch

is

composed

of

two

different

molecules

-LRB-

-RRB-

amylose

an

unbranched

single

chain

of

α

-LRB-

-RRB-

linked

glucose

residues

and

-LRB-

-RRB-

amylopectin

consisting

of

a

α

-LRB-

-RRB-

linked

glucose

chain

with

α

-LRB-

-RRB-

branches

on

every

glucose

residues

along

the

α

-LRB-

-RRB-

linked

backbone

-LRB-

Robyt

-RRB-

The

degradation

of

starch

is

performed

by

a

variety

of

enzymes

which

are

divided

over

three

Glycoside

Hydrolase

-LRB-

GH

-RRB-

families

based

on

their

sequence

similarity

-LRB-

-RRB-

-LRB-

Coutinho

and

Henrissat

-RRB-

The

first

step

in

starch

degradation

is

the

endo-hydrolysis

of

the

long

polysaccharide

chains

into

shorter

maltooligosaccharides

and

α-limit

dextrins

by

α-amylases

-LRB-

EC

-RRB-

α-Amylases

belong

to

family

a

large

family

containing

various

hydrolysing

and

transglycosylating

enzymes

mostly

acting

on

α

-LRB-

-RRB-

or

α

-LRB-

-RRB-

glycosidic

bonds

Members

of

family

have

a

-LRB-

β/α

-RRB-

barrel

structure

and

can

be

recognized

by

four

highly

conserved

amino

acid

regions

containing

three

catalytic

residues

-LRB-

MacGregor

et

al.

Nakajima

et

al.

-RRB-

After

endo-hydrolysis

subsequent

steps

in

starch

degradation

involve

exo-acting

enzymes

releasing

glucose

This

reaction

is

performed

by

glucoamylase

type

enzymes

of

family

-LRB-

EC

-RRB-

a

relatively

confined

family

with

regard

to

enzyme

specificity

as

all

its

studied

members

hydrolyse

either

α

-LRB-

-RRB-

or

α

-LRB-

-RRB-

bonds

to

release

β-glucose

from

the

non-reducing

end

of

maltooligosaccharides

Most

enzymes

described

thus

far

possess

a

starch-binding

domain

-LRB-

SBD

-RRB-

-LRB-

Sauer

et

al.

-RRB-

a

discrete

C-terminal

region

of

the

protein

that

binds

to

starch

and

facilitates

hydrolysis

-LRB-

Southall

et

al.

-RRB-

Additionally

α

-LRB-

-RRB-

glucosidases

of

family

may

release

α-glucose

from

the

non-reducing

end

of

starch

-LRB-

EC

-RRB-

This

family

also

harbours

other

enzyme

specificities

such

as

α-xylosidase

activity

Several

A.

niger

enzymes

involved

in

starch

degradation

and

their

corresponding

genes

have

been

characterized

and

isolated

A.

niger

glucoamylase

GlaA

-LRB-

family

-RRB-

is

an

important

enzyme

for

the

modification

of

starch

in

the

food

industry

-LRB-

Boel

et

al.

van

Dijck

et

al.

-RRB-

Additionally

one

α-glucosidase

-LRB-

AglA

renamed

AgdA

-RRB-

-LRB-

Nakamura

et

al.

-RRB-

has

been

characterized

previously

as

well

as

three

family

α-amylases

acid

amylase

AamA

and

the

almost

identical

AmyA

and

AmyB

-LRB-

Boel

et

al.

Korman

et

al.

-RRB-

The

transcriptional

regulation

of

the

genes

encoding

starch-degrading

enzymes

has

been

studied

in

several

aspergilli

-LRB-

Nakamura

et

al.

-RRB-

In

general

their

expression

is

high

on

starch

and

induced

by

the

presence

of

-LRB-

iso

-RRB-

maltose

-LRB-

Tsukagoshi

et

al.

Kato

et

al.

-RRB-

The

presence

of

the

inducer

activates

the

Zn

-LRB-

II

-RRB-

transcription

factor

AmyR

which

binds

to

-LRB-

C/A

-RRB-

GG

sequences

in

the

promoter

regions

of

AmyR

target

genes

thereby

activating

their

transcription

-LRB-

Petersen

et

al.

Gomi

et

al.

Tani

et

al.

Ito

et

al.

-RRB-

Recent

studies

have

indicated

that

some

enzymes

in

fungi

may

be

involved

in

the

synthesis

or

modification

of

α-glucan

in

the

fungal

cell

wall

rather

than

in

starch

degradation

The

cell

wall

of

aspergilli

contains

four

major

classes

of

polysaccharides

chitin

α-glucan

β

-LRB-

-RRB-

glucan

and

galactomannan

-LRB-

Fontaine

et

al.

Beauvais

and

Latgé

-RRB-

The

α-glucan

fraction

identified

in

A.

niger

consists

of

two

types

of

molecules

a

linear

polymer

with

alternating

α

-LRB-

-RRB-

-LRB-

-RRB-

glycosidic

bonds

called

nigeran

-LRB-

Barker

and

Carrington

-RRB-

and

pseudonigeran

a

linear

α

-LRB-

-RRB-

glucan

molecule

with

some

-LRB-

-RRB-

α

-LRB-

-RRB-

linkages

-LRB-

Johnston

Horisberger

et

al.

-RRB-

Synthesis

of

α-glucan

is

thought

to

be

carried

out

by

α-glucan

synthase

enzymes

encoded

by

ags

genes

The

first

putative

α-d-glucan

synthase

encoding

gene

-LRB-

-RRB-

was

identified

in

Schizosaccharomyces

pombe

-LRB-

Hochstenbach

et

al.

-RRB-

The

gene

encodes

a

large

three-domain

protein

In

addition

to

the

multi-pass

transmembrane

domain

in

the

C-terminal

part

of

the

protein

two

predicted

catalytic

domains

are

present

The

middle

domain

shows

strong

similarity

to

glycogen

and

starch

synthases

in

Glycosyltransferase

family

-LRB-

GT

-RRB-

and

is

predicted

to

be

involved

in

the

synthesis

of

α-glucan

The

N-terminal

part

of

the

protein

is

similar

to

α-amylases

and

belongs

to

family

This

part

of

the

protein

is

predicted

to

be

localized

extracellularly

and

might

be

involved

in

connecting

two

α

-LRB-

-RRB-

glucan

chains

-LRB-

Grün

et

al.

-RRB-

Apart

from

the

α-glucan

synthases

two

types

of

family

enzymes

were

recently

identified

in

fungi

to

play

a

role

in

fungal

cell

wall

biosynthesis

Marion

et

al.

-LRB-

-RRB-

provided

evidence

for

the

involvement

of

a

putative

α-amylase

-LRB-

-RRB-

in

the

formation

of

α

-LRB-

-RRB-

glucan

in

the

cell

wall

of

Histoplasma

capsulatum

An

knockout

was

unable

to

produce

α

-LRB-

-RRB-

glucan

and

showed

reduced

virulence

In

dimorphic

fungi

like

H.

capsulatum

cell

wall

α-glucan

is

a

known

virulence

factor

-LRB-

Rappleye

et

al.

Rappleye

and

Goldman

-RRB-

The

second

α-amylase-like

enzyme

was

first

studied

in

S.

pombe

-LRB-

Morita

et

al.

-RRB-

Disruption

of

encoding

a

GPI-anchored

protein

resulted

in

hypersensitivity

towards

cell

wall-degrading

enzymes

and

an

aberrant

cell

shape

indicating

that

normal

cell

wall

biosynthesis

was

affected

Disruption

of

a

homologous

gene

-LRB-

agtA

-RRB-

in

A.

niger

also

affected

cell

wall

stability

-LRB-

van

der

Kaaij

et

al.

-RRB-

We

previously

surveyed

the

A.

niger

genome

sequence

to

identify

all

and

family

members

present

in

this

important

industrial

source

for

amylolytic

enzymes

-LRB-

Pel

et

al.

-RRB-

This

resulted

in

identification

of

a

surprisingly

large

number

of

previously

unknown

enzymes

In

this

study

we

have

analysed

their

phylogeny

the

presence

of

specific

protein

features

and

synteny

with

other

Aspergillus

species

which

allowed

the

division

of

the

members

of

each

GH

family

into

several

groups

Additionally

we

studied

the

transcriptional

regulation

of

the

genes

encoding

these

proteins

in

a

wild

type

A.

niger

strain

and

in

a

derived

amyR

deletion

strain

during

their

growth

on

xylose

and

maltose

Only

few

of

the

identified

proteins

were

induced

by

maltose

Expression

of

many

of

the

identified

groups

of

enzymes

including

the

homologues

of

both

S.

pombe

and

H.

capsulatum

was

not

induced

by

maltose

and

was

not

dependent

on

the

presence

of

AmyR

The

possible

involvement

of

these

enzymes

in

cell

wall

α-glucan

synthesis

and

modification

is

discussed

Material

and

methods

Database

mining

of

A.

niger

genome

and

analysis

of

predicted

proteins

The

full

genome

sequence

of

A.

niger

strain

CBS

has

been

deposited

at

the

EMBL

database

with

accession

numbers

-LRB-

Pel

et

al.

-RRB-

and

was

used

for

database

mining

The

nucleotide

accession

numbers

of

A.

niger

genes

as

listed

in

Tables

and

refer

to

this

database

Hidden

Markov

Model

-LRB-

HMM

-RRB-

profiles

were

built

with

the

HMMER

package

-LRB-

Durbin

and

Eddy

-RRB-

-LRB-

-RRB-

based

on

the

amino

acid

sequences

of

known

members

of

and

Proteins

belonging

to

these

families

originating

from

the

different

kingdoms

of

life

were

retrieved

from

the

CAZy

website

at

-LRB-

Coutinho

and

Henrissat

-RRB-

and

the

protein

sequences

were

extracted

from

the

GenBank/GenPept

database

at

and

Swiss-Prot

database

at

The

A.

niger

genome

was

searched

with

the

HMM

profiles

using

the

package

-LRB-

Birney

et

al.

-RRB-

-LRB-

-RRB-

Table

members

of

family

and

identified

in

the

genome

sequence

of

A.

niger

CBS

using

HMM

profiles

Accession

no.GeneFamilyEnzyme

activityFeaturesaAmyR

binding

motifbProposed

biological

α-amylaseSS

Starch

degradation1An12g06930amyAGH13α-AmylaseSS

Starch

degradation2An05g02100amyBGH13α-AmylaseSS

Starch

degradation2An04g06930amyCGH13α-AmylaseSS

degradation3An09g03100agtAGH13α-GlucanotransferaseSS

GPICell

wall

α-glucan

synthesis4An12g02460agtBGH13α-GlucanotransferaseSS

GPI

Cell

wall

α-glucan

α-glucanotransferaseSS

GPICell

wall

α-glucan

α-glucosidase

α-glucosidaseUnknownAn01g13610amyDGH13Putative

α-amylase

wall

α-glucan

α-amylase

wall

α-glucan

debranching

enzyme

Glycogen

branching

enzymeGlycogen

α-glucan

synthaseSSCell

wall

α-glucan

α-glucan

synthaseSS

Cell

wall

α-glucan

α-glucan

synthaseSS

wall

α-glucan

α-glucan

synthaseSSCell

wall

α-glucan

α-glucan

synthaseSSCell

wall

α-glucan

SBD

glucoamylase

878UnknownAn04g06920agdAGH31α-GlucosidaseSS

Starch

α-glucosidaseSS

α-glucosidase

IISSProtein

α-xylosidaseSS

Xyloglucan

α-xylosidase

Xyloglucan

degradationThe

newly

identified

proteins

are

indicated

in

boldaSS

predicted

N-terminal

Signal

Sequence

GPI

predicted

Glycosylphosphatidylinositol

anchor

signal

SBD

predicted

starch-binding

domainbThe

presence

of

consensus

AmyR

binding

motif

-LRB-

-LRB-

A/C

-RRB-

GG

-RRB-

was

analysed

in

the

promoter

region

up

to

kb

upstream

of

the

start

codoncReferences

-LRB-

Boel

et

al.

-RRB-

-LRB-

Korman

et

al.

-RRB-

-LRB-

R.

M.

Van

der

Kaaij

and

X.

L.

Yuan

unpublished

-RRB-

-LRB-

van

der

Kaaij

et

al.

-RRB-

-LRB-

Damveld

et

al.

-RRB-

-LRB-

Boel

et

al.

-RRB-

-LRB-

Nakamura

et

al.

-RRB-

Table

described

family

and

members

from

other

organisms

used

for

the

multiple

sequence

alignments

in

Fig.

no.NameFamilyEnzyme

activityFeaturesaBiological

branching

enzymeGlycogen

metabolismA

debranching

enzymeGlycogen

metabolismS

cerevisiae2P19571BsAmyAGH13α-AmylaseSSStarch

degradationBacillus

sp.3CAA54266BsAglAGH13α-GlucosidaseStarch

degradationBacillus

sp.

GPIα-Glucan

biosynthesisS

GPIα-Glucan

biosynthesisS

GPIα-Glucan

biosynthesisS

GPIα-Glucan

biosynthesisS

biosynthesisH

degradationA

degradationA

nidulans7BAB39856AndAgdBGH31α-GlucosidaseSSStarch

degradationA

IISSProtein

glycosylationT

degradationC

each

A.

niger

protein

identified

a

functionally

or

biochemically

characterized

protein

with

the

highest

similarity

was

used

in

the

phylogenetic

analysis

aSS

predicted

N-terminal

Signal

Sequence

GPI

predicted

Glycosylphosphatidylinositol

anchor

signalbReferences

-LRB-

Sasangka

et

al.

-RRB-

-LRB-

Teste

et

al.

-RRB-

-LRB-

Tsukamoto

et

al.

-RRB-

-LRB-

Nakao

et

al.

-RRB-

-LRB-

Morita

et

al.

-RRB-

-LRB-

Marion

et

al.

-RRB-

-LRB-

Bauer

et

al.

-RRB-

-LRB-

Kato

et

al.

-RRB-

-LRB-

Geysens

et

al.

-RRB-

-LRB-

Geber

et

al.

-RRB-

The

presence

of

signal

peptidase

cleavage

sites

glycosylphosphatidylinositol

-LRB-

GPI

-RRB-

attachment

sites

and

SBD

in

the

obtained

sequences

were

predicted

by

Web-based

tools

at

URL

-LRB-

Bendtsen

et

al.

-RRB-

URL

-LRB-

Eisenhaber

et

al.

-RRB-

and

URL

-LRB-

Marchler-Bauer

and

Bryant

-RRB-

respectively

Multiple

sequence

alignments

of

and

family

members

were

performed

using

DNAMAN

version

-LRB-

Lynnon

BioSoft

Canada

-RRB-

The

alignments

were

based

on

the

full

length

of

the

predicted

proteins

except

in

case

of

predicted

α-glucan

synthases

for

which

only

the

N-terminal

part

encoding

the

family

domain

was

used

for

the

alignment

The

phylogenetic

relationship

was

calculated

by

using

Optimal

Alignment

-LRB-

Thompson

et

al.

-RRB-

with

gap

opening

penalty

and

gap

extension

penalty

of

and

respectively

A

bootstrapped

test

of

phylogeny

was

performed

by

the

Neighbour-Joining

method

using

replicates

Wherever

possible

one

protein

with

described

activity

was

included

for

each

of

the

groups

identified

based

on

phylogenetic

analysis

Strains

and

transformations

The

wild

type

A.

niger

strain

used

in

this

study

is

a

derivative

of

A.

niger

van

Tieghem

-LRB-

CBS

ATCC

-RRB-

-LRB-

Bos

et

al.

-RRB-

Strain

is

a

pyrG

negative

derivative

of

-LRB-

van

Hartingsveldt

et

al.

-RRB-

and

was

used

to

construct

the

amyR

disruption

strain

A.

niger

strains

were

grown

in

Aspergillus

minimal

medium

-LRB-

MM

-RRB-

-LRB-

Bennett

and

Lasure

-RRB-

or

Aspergillus

complete

medium

-LRB-

CM

-RRB-

consisting

of

MM

with

the

addition

of

-LRB-

w/v

-RRB-

yeast

extract

and

-LRB-

w/v

-RRB-

casamino

acids

Growth

medium

was

supplemented

with

mM

uridine

-LRB-

Serva

Germany

-RRB-

when

required

Transformation

of

A.

niger

was

performed

as

described

earlier

-LRB-

Punt

and

van

den

Hondel

-RRB-

using

lysing

enzymes

-LRB-

Sigma

USA

-RRB-

for

protoplastation

The

bacterial

strain

used

for

transformation

and

amplification

of

recombinant

DNA

was

Escherichia

coli

-LRB-

Stratagene

USA

-RRB-

Transformation

of

was

performed

according

to

the

heat

shock

protocol

-LRB-

Inoue

et

al.

-RRB-

Disruption

of

the

maltose

utilization

activator

amyR

in

A.

niger

Plasmid

containing

the

A.

nigeramyR

gene

as

a

kb

NsiI

fragment

in

was

kindly

provided

by

P.

van

Kuyk

-LRB-

Wageningen

University

the

Netherlands

-RRB-

and

was

used

to

disrupt

the

amyR

gene

The

construction

of

the

amyR

deletion

cassette

was

performed

as

follows

The

BamHI

EcoRI

fragment

and

NsiI

SalI

fragment

flanking

the

amyR

ORF

at

the

and

region

respectively

were

isolated

from

The

isolated

NsiI

SalI

fragment

was

cloned

into

to

obtain

plasmid

Subsequently

a

BamHI

SalI

fragment

carrying

the

A.

oryzaepyrG

gene

obtained

from

plasmid

-LRB-

de

Ruiter-Jacobs

et

al.

-RRB-

was

inserted

into

which

resulted

in

plasmid

The

BamHI

EcoRI

fragment

isolated

from

was

ligated

into

resulting

in

the

amyR

deletion

plasmid

-LRB-

pΔamyR

-RRB-

Prior

to

transformation

to

pΔamyR

was

linearized

with

EcoRI

Uridine

prototrophic

transformants

were

selected

by

their

ability

to

grow

on

MM

without

uridine

After

two

rounds

of

purification

transformants

were

tested

for

their

ability

to

grow

on

starch

Approximately

of

the

pyrG

transformants

showed

defective

growth

on

MM

agar

plates

containing

starch

as

sole

carbon

source

Six

independent

putative

amyR

deletion

strains

-LRB-

-RRB-

with

identical

phenotypes

were

obtained

Southern

blot

analysis

confirmed

proper

deletion

and

a

single

integration

of

the

amyR

disruption

cassette

at

the

amyR

locus

Strain

was

used

for

further

analysis

and

we

will

refer

to

this

strain

as

the

ΔamyR

strain

in

the

remaining

of

this

paper

Culture

conditions

RNA

preparation

microarray

experiments

and

data

analysis

RNA

extracted

from

the

A.

niger

ΔamyR

strain

and

its

parental

strain

-LRB-

-RRB-

grown

on

different

carbon

sources

were

used

for

microarray

experiments

using

custom-made

Affymetrix

GeneChip

Microarrays

kindly

provided

by

DSM

Food

Specialties

-LRB-

Delft

The

Netherlands

-RRB-

All

experiments

for

each

growth

condition

-LRB-

culturing

the

mycelia

RNA

extractions

and

microarray

hybridizations

-RRB-

were

performed

twice

as

independent

biological

experiments

Aspergillus

niger

spores

-LRB-

spores

ml

-RRB-

were

inoculated

in

ml

MM

supplemented

with

-LRB-

w/v

-RRB-

xylose

-LRB-

Sigma

-RRB-

and

-LRB-

w/v

-RRB-

casamino

acids

and

grown

for

h

at

°C

on

a

rotary

shaker

at

rpm

The

mycelium

was

harvested

by

suction

over

a

nylon

membrane

and

washed

with

MM

without

carbon

source

Aliquots

of

g

wet

weight

of

mycelium

were

transferred

to

ml

Erlenmeyer

flasks

containing

ml

MM

supplemented

with

-LRB-

w/v

-RRB-

carbon

source

-LSB-

maltose

-LRB-

Sigma

-RRB-

or

xylose

-RSB-

and

incubated

at

°C

for

a

further

or

h

The

pH

of

all

cultures

grown

for

h

was

equal

to

the

pH

at

the

time

of

transfer

-LRB-

pH

-RRB-

Cultures

grown

for

h

were

buffered

at

pH

by

the

addition

of

mM

of

citric

acid/sodium

citrate

to

allow

comparison

between

the

and

the

ΔamyR

strain

at

this

time

point

The

mycelium

was

harvested

over

Miracloth

filter

frozen

in

liquid

nitrogen

and

stored

at

°C

Total

RNA

was

isolated

from

mycelia

using

TRIzol

reagent

-LRB-

Invitrogen

-RRB-

and

RNA

quality

was

verified

by

analyzing

aliquots

with

glyoxal/DMSO

gel

electrophoresis

and

Agilent

Bioanalyzer

Lab

on

chip

system

-LRB-

Agilent

Technologies

USA

-RRB-

Processing

labeling

and

hybridization

of

cRNA

to

A.

niger

Affymetrix

GeneChips

were

performed

according

to

the

corresponding

Affymetrix

protocols

for

Eukaryotic

Target

Preparation

and

Eukaryotic

Target

hybridization

For

probe

array

washing

and

staining

the

protocol

Antibody

Amplification

for

Eukaryotic

Targets

was

followed

Hybridized

probe

array

slides

were

scanned

with

Agilent

technologies

Gene

Array

Scanner

at

a

μm

resolution

and

a

wavelength

of

nm

Affymetrix

Microarray

Suite

software

was

used

to

calculate

the

signal

and

P-values

and

to

set

the

algorithm

's

absolute

call

flag

which

indicates

the

reliability

of

the

data

points

according

to

P

-LRB-

present

-RRB-

M

-LRB-

marginal

-RRB-

and

A

-LRB-

absent

-RRB-

The

data

on

each

chip

were

globally

scaled

to

an

arbitrary

target

gene

intensity

of

The

complete

microarray

data

were

deposited

into

ArrayExpress

with

an

accession

at

The

pre-scaled

data

from

each

hybridization

experiment

was

then

normalized

for

statistical

analysis

using

Genespring

software

-LRB-

Silicon

Genetics

USA

-RRB-

The

per

chip

normalization

was

performed

to

ensure

that

the

overall

characteristic

of

the

expression

distribution

such

as

median

should

be

the

same

for

all

the

chips

For

the

genome-wide

analysis

we

focused

on

maltose-induced

genes

and

therefore

a

pre-filtering

of

data

was

performed

to

select

for

genes

whose

detection

calls

are

present

in

both

maltose

duplicate

samples

in

the

wild-type

strain

-LRB-

-RRB-

The

selected

dataset

was

used

to

perform

a

one-way

ANOVA

analysis

under

the

test

type

of

parametric

test

do

n't

assume

variances

equal

Fold

changes

in

expression

between

two

different

conditions

were

then

computed

for

genes

with

P

based

on

one-way

ANOVA

analysis

Results

Identification

of

glycoside

hydrolase

family

and

genes

in

the

A.

niger

CBS

genome

sequence

α-Amylases

glucoamylases

and

α-glucosidases

members

of

families

and

respectively

are

the

three

main

types

of

enzymes

involved

in

breakdown

of

starch

by

aspergilli

-LRB-

Tsukagoshi

et

al.

-RRB-

To

identify

all

genes

encoding

enzymes

that

might

play

a

role

in

starch

utilization

or

other

α-glucan

modifying

processes

in

A.

niger

the

genome

of

A.

niger

CBS

was

searched

with

HMM

profiles

based

on

known

enzymes

from

families

and

This

resulted

in

the

retrieval

of

a

total

of

protein

sequences

including

previously

unknown

proteins

-LRB-

Pel

et

al.

-RRB-

as

listed

in

Table

The

predicted

proteins

were

annotated

based

on

their

similarity

to

known

enzymes

Gene

names

were

assigned

based

on

this

annotation

and

do

not

necessarily

match

the

activity

of

the

enzymes

as

this

is

often

unknown

Two

approaches

were

combined

to

predict

putative

functions

in

cellular

processes

for

this

surprisingly

large

number

of

newly

identified

proteins

First

phylogenetic

trees

were

constructed

using

the

and

family

members

identified

in

the

A.

niger

genome

as

well

as

functionally

characterized

proteins

from

other

organisms

with

similarity

to

the

identified

A.

niger

proteins

-LRB-

Fig.

-RRB-

Second

using

DNA

microarrays

the

expression

of

all

the

A.

niger

genes

encoding

and

enzymes

was

examined

in

both

the

A.

niger

wild

type

strain

and

the

ΔamyR

strain

derived

after

growth

on

different

carbon

sources

-LRB-

Fig.

Table

-RRB-

Both

the

and

the

ΔamyR

strains

were

pregrown

in

xylose

for

h

and

mycelia

were

transferred

to

either

xylose

or

maltose

media

and

grown

further

for

or

h.

Expression

levels

were

determined

based

on

geometric

mean

data

of

biological

duplicate

samples

We

will

discuss

each

enzyme

family

in

detail

and

combine

the

findings

in

A.

niger

with

the

predicted

proteins

present

in

the

genomes

of

A.

fumigatus

-LRB-

Nierman

et

al.

-RRB-

A.

nidulans

-LRB-

Galagan

et

al.

-RRB-

and

A.

oryzae

-LRB-

Machida

et

al.

-RRB-

Fig.

phylogenetic

tree

of

A.

niger

-LRB-

a

-RRB-

and

-LRB-

b

-RRB-

enzymes

and

several

closest

homologues

from

other

species

Newly

identified

proteins

in

the

genome

of

A.

niger

are

shown

in

bold

A

description

of

each

protein

is

listed

in

Tables

and

Bootstrap

values

are

indicated

on

the

node

of

each

branch

The

tree

was

created

with

DNAMAN

using

gap

and

extension

penalties

of

and

respectively

The

scale

bar

corresponds

to

a

genetic

distance

of

substitution

per

positionFig

profiles

of

A.

niger

family

and

enzymes

Accession

numbers

of

the

gene

names

are

given

in

Tables

and

Strain

and

time

points

after

transfer

from

the

preculture

are

indicated

on

the

right-hand

side

The

numeric

values

and

Present/Absent

calls

from

the

expression

data

are

provided

as

Supplementary

Tables

Identification

and

transcriptional

regulation

of

family

members

The

HMMer

search

for

family

enzymes

in

the

A.

niger

genome

resulted

in

the

identification

of

protein

sequences

of

which

had

not

been

identified

previously

-LRB-

Table

-RRB-

Table

displays

the

four

conserved

regions

typical

for

family

proteins

as

identified

in

these

enzymes

A

phylogenetic

tree

was

produced

combining

the

A.

niger

family

enzymes

with

several

functionally

characterized

family

proteins

from

other

organisms

-LRB-

Fig.

-RRB-

The

combination

of

this

phylogenetic

analysis

with

a

functional

annotation

of

the

proteins

revealed

six

recognizable

subgroups

Table

of

the

four

conserved

regions

of

all

family

enzymes

identified

in

A.

niger

as

well

as

in

four

Aah

proteins

from

S.

pombe

and

from

H.

capsulatumThe

seven

residues

generally

conserved

in

family

are

indicated

in

bold

and

the

three

catalytic

residues

are

additionally

underlined

The

group

to

which

the

proteins

are

assigned

as

described

in

this

paper

is

indicated

Group

I

consists

of

four

extracellular

α-amylases

Three

of

these

are

the

previously

characterized

extracellular

α-amylases

acid-amylase

-LRB-

AamA

-RRB-

and

α-amylases

AmyA

and

AmyB

-LRB-

Korman

et

al.

-RRB-

One

new

extracellular

α-amylase

was

identified

and

named

AmyC

This

protein

displays

high

similarity

with

the

known

A.

niger

α-amylases

-LRB-

identity

to

AmyA

and

AmyB

identity

to

AamA

-RRB-

The

amyC

gene

is

located

in

a

gene

cluster

also

containing

an

α-glucosidase

gene

-LRB-

agdA

-RRB-

and

the

amyR

gene

encoding

the

AmyR

transcription

factor

Expression

of

aamA

in

A.

niger

was

not

detectable

in

xylose

media

but

was

strongly

induced

in

the

presence

of

maltose

-LRB-

Fig.

-RRB-

The

expression

of

aamA

was

reduced

to

a

non-detectable

level

in

the

ΔamyR

strain

Expression

of

amyA

and

amyB

was

not

detected

in

A.

niger

in

any

of

the

conditions

tested

-LRB-

Fig.

-RRB-

-LRB-

see

Discussion

-RRB-

The

expression

of

the

newly

identified

amyC

gene

was

relatively

low

compared

to

the

aamA

gene

At

h

after

transfer

from

the

preculture

the

expression

level

of

amyC

was

independent

of

the

carbon

source

while

after

h

the

expression

level

was

reduced

on

xylose

compared

to

maltose

Additionally

the

expression

on

maltose

was

reduced

to

in

the

ΔamyR

strain

-LRB-

Table

-RRB-

The

presence

of

three

putative

AmyR-binding

elements

in

the

promoter

region

of

amyC

further

suggests

that

its

expression

is

controlled

by

AmyR

-LRB-

Table

-RRB-

The

A.

niger

acid

amylase

-LRB-

encoded

by

aamA

-RRB-

in

the

CBS

strain

does

not

contain

a

predicted

SBD

whereas

its

homologues

in

A.

nidulans

A.

fumigatus

and

other

aspergilli

contain

a

full-length

SBD

-LRB-

Table

-RRB-

In

addition

the

presence

of

a

functional

SBD

in

the

AamA

protein

of

A.

niger

was

suggested

because

of

the

purification

of

AamA

via

its

SBD

from

culture

fluid

of

a

ΔglaA

strain

and

subsequent

demonstration

of

the

SBD

with

specific

antibodies

-LRB-

M.

F.

Coeffet-Le

Gal

and

D.

Archer

personal

communication

University

of

Nottingham

UK

-RRB-

We

therefore

PCR

amplified

the

aamA

gene

and

its

flanking

regions

using

genomic

DNA

as

a

template

determined

its

DNA

sequence

and

compared

it

to

the

aamA

gene

and

its

flanking

region

from

CBS

The

comparison

revealed

that

the

aamA

gene

of

A.

niger

strain

does

include

an

SBD

and

that

the

strain

harbours

a

deletion

of

nucleotides

right

after

the

part

encoding

the

-LRB-

β/α

-RRB-

barrel

causing

a

frame

shift

and

the

introduction

of

a

stop

codon

resulting

in

a

truncated

protein

-LRB-

Fig

-RRB-

After

the

deletion

the

DNA

sequence

continues

by

encoding

part

of

a

predicted

SBD

but

this

part

is

not

translated

-LRB-

Fig

-RRB-

The

SBD

in

AamA

found

in

the

A.

niger

strain

is

also

predicted

to

be

present

in

A.

niger

strain

ATCC

-LRB-

Baker

-RRB-

Group

II

contains

three

putative

GPI-anchored

enzymes

recently

identified

as

α-glucanotransferases

and

named

AgtA

AgtB

and

AgtC

respectively

-LRB-

van

der

Kaaij

et

al.

-RRB-

This

subgroup

of

proteins

is

characterized

by

the

presence

of

two

hydrophobic

signal

sequences

The

N-terminal

signal

sequence

is

predicted

to

serve

for

translocation

to

the

endoplasmic

reticulum

-LRB-

ER

-RRB-

whereas

the

C-terminal

sequence

is

predicted

to

be

replaced

by

a

preassembled

glycosylphosphatidylinositol

-LRB-

GPI

-RRB-

anchor

in

the

ER

-LRB-

Orlean

-RRB-

The

three

enzymes

cluster

together

with

the

α-amylases

in

the

phylogenetic

tree

-LRB-

Fig.

-RRB-

but

can

be

distinguished

from

the

α-amylases

by

their

catalytic

domains

which

are

clearly

different

from

the

consensus

sequence

for

the

α-amylase

family

In

all

three

proteins

one

or

two

highly

conserved

histidines

in

conserved

regions

I

and

IV

are

replaced

by

other

hydrophilic

residues

-LRB-

Table

-RRB-

Family

members

without

these

histidine

residues

which

are

part

of

the

active

site

are

very

rare

-LRB-

Uitdehaag

et

al.

-RRB-

Mutation

of

these

residues

generally

results

in

reduced

activity

or

altered

reaction

specificity

of

the

enzymes

-LRB-

Chang

et

al.

Leemhuis

et

al.

-RRB-

Interestingly

both

the

conserved

His-residues

in

Regions

I

and

IV

are

also

missing

in

all

-LRB-

putative

-RRB-

α-glucan

synthases

-LRB-

Table

-RRB-

Other

aspergillus

genomes

harbour

two

or

three

Agt

homologues

-LRB-

Fig

Table

-RRB-

all

sharing

the

aberrant

conserved

regions

and

predicted

GPI-anchoring

In

the

A.

niger

genome

both

agtB

and

agtC

are

located

next

to

genes

encoding

putative

α-glucan

synthases

and

this

arrangement

of

genes

is

conserved

in

other

aspergilli

The

agtA

gene

is

constitutively

expressed

in

both

the

wild

type

strain

and

the

ΔamyR

strain

under

all

growth

conditions

examined

-LRB-

Fig.

-RRB-

Expression

of

agtB

was

only

detected

h

after

transfer

regardless

of

the

carbon

source

and

independent

of

AmyR

while

expression

of

agtC

was

not

detected

Group

III

consists

of

two

putative

intracellular

α-glucosidases

named

AgdC

-LRB-

-RRB-

and

AgdD

-LRB-

-RRB-

The

protein

sequences

contained

all

residues

commonly

conserved

in

the

α-amylase

superfamily

-LRB-

Table

-RRB-

The

predicted

intracellular

proteins

lack

clear

similarity

to

any

previously

characterized

fungal

protein

although

similar

enzymes

are

predicted

in

A.

oryzae

A.

nidulans

and

A.

fumigatus

-LRB-

Fig

-RRB-

Their

most

related

functionally

characterized

homologue

is

an

α-glucosidase

from

Bacillus

sp.

-LRB-

Nakao

et

al.

-RRB-

Expression

of

agdC

was

low

and

not

induced

on

maltose

and

expression

of

agdD

was

not

observed

-LRB-

Fig.

-RRB-

Group

IV

contains

two

putative

intracellular

α-amylases

named

AmyD

-LRB-

-RRB-

and

AmyE

-LRB-

-RRB-

which

share

identity

AmyD

was

recently

characterized

as

an

α-amylase

with

low-hydrolyzing

activity

on

starch

and

related

substrates

-LRB-

Van

der

Kaaij

et

al.

-RRB-

AmyD

and

AmyE

are

similar

to

a

recently

identified

protein

from

H.

capsulatum

which

was

shown

to

be

involved

in

cell

wall

α-glucan

synthesis

The

latter

protein

has

not

been

characterized

biochemically

Functionally

characterized

enzymes

with

similarity

to

this

cluster

therefore

included

only

bacterial

enzymes

of

which

maltohexaose-forming

α-amylase

of

alkalophilic

Bacillus

sp.

-LRB-

Tsukamoto

et

al.

-RRB-

had

the

highest

similarity

No

significant

expression

of

amyD

or

amyE

was

detected

in

our

experiments

-LRB-

Fig.

-RRB-

Predicted

enzymes

highly

similar

to

A.

niger

AmyD

and

AmyE

were

also

present

in

other

Aspergillus

species

-LRB-

Fig

Table

-RRB-

Like

the

A.

niger

amyE

gene

the

orthologues

in

A.

nidulans

-LRB-

-RRB-

and

in

A.

oryzae

-LRB-

-RRB-

are

clustered

in

the

genome

with

genes

encoding

α-glucanotransferases

and

α-glucan

synthases

-LRB-

Table

-RRB-

The

two

A.

niger

proteins

in

Group

V

could

be

reliably

annotated

as

enzymes

involved

in

glycogen

metabolism

glycogen

branching

enzyme

-LRB-

GbeA

-RRB-

and

glycogen

debranching

enzyme

-LRB-

GdbA

-RRB-

A

homologue

for

each

of

these

enzymes

is

present

in

the

other

Aspergillus

genome

sequences

Transcriptional

analysis

in

A.

niger

showed

that

both

genes

were

expressed

both

on

xylose

and

maltose

and

that

their

expression

was

unaffected

in

the

ΔamyR

strain

-LRB-

Fig.

-RRB-

Group

VI

contains

the

five

predicted

α-glucan

synthase

genes

-LRB-

Damveld

et

al.

-RRB-

The

derived

proteins

are

highly

similar

-LRB-

-RRB-

to

each

other

and

all

contain

the

two

catalytic

domains

-LRB-

and

-RRB-

characteristic

for

these

proteins

Both

agsB

and

agsC

are

clustered

in

the

genome

with

genes

encoding

the

aforementioned

α-glucanotransferases

agtC

and

agtB

respectively

In

both

cases

the

direction

of

transcription

of

the

pair

of

genes

is

such

that

they

can

be

transcribed

from

their

intergenic

region

Expression

analysis

in

our

microarray

data

collection

which

includes

data

from

various

time

points

on

several

carbon

sources

-LRB-

glucose

maltose

xylose

inulin

sucrose

-RRB-

indicate

that

agsC

and

agtB

are

co-expressed

at

a

later

growth

stage

independent

of

the

carbon

source

-LRB-

data

not

shown

-RRB-

Expression

of

both

agsB

and

its

neighbouring

gene

agtC

was

not

detected

in

any

of

the

growth

conditions

The

expression

levels

of

agsA

and

agsD

were

very

low

or

not

detectable

AgsE

was

highly

expressed

in

all

experiments

independent

of

AmyR

-LRB-

Fig.

-RRB-

As

expected

several

proteins

with

high

similarity

to

the

A.

niger

α-glucan

synthases

are

predicted

from

the

other

Aspergillus

genomes

although

the

number

of

homologues

present

is

highest

in

A.

niger

-LRB-

Fig

Table

-RRB-

Identification

and

transcriptional

regulation

of

family

members

An

HMMer

search

for

family

members

in

the

A.

niger

genome

returned

two

predicted

proteins

the

previously

described

glucoamylase

GlaA

-LRB-

Boel

et

al.

-RRB-

and

an

unknown

predicted

protein

named

GlaB

-LRB-

-RRB-

The

GlaB

protein

lacks

both

an

N-terminal

signal

sequence

and

an

SBD

and

displays

a

low

similarity

of

with

GlaA

These

two

types

of

family

enzymes

are

also

recognized

in

other

aspergilli

studied

-LRB-

Fig

-RRB-

All

predicted

proteins

similar

to

GlaB

lack

both

a

signal

for

secretion

and

an

SBD

features

typically

present

in

the

fungal

enzymes

described

to

date

A.

niger

glaA

was

expressed

on

xylose

and

strongly

induced

on

maltose

as

described

previously

-LRB-

Fowler

et

al.

-RRB-

The

induction

of

glaA

was

AmyR

dependent

Expression

of

glaB

was

not

detected

in

any

of

the

conditions

tested

-LRB-

Fig.

-RRB-

Identification

and

transcriptional

regulation

of

family

members

HMMer

searches

in

the

genome

of

A.

niger

revealed

the

presence

of

seven

family

members

of

which

only

one

was

previously

identified

as

an

α-glucosidase

-LRB-

aglA

-RRB-

-LRB-

Nakamura

et

al.

-RRB-

We

propose

to

name

all

-LRB-

putative

-RRB-

α-glucosidases

in

A.

niger

Agd

enzymes

similar

to

the

nomenclature

in

A.

nidulans

and

to

rename

AglA

as

AgdA

to

prevent

confusion

with

α-galactosidases

-LRB-

den

Herder

et

al.

-RRB-

The

phylogenetic

tree

-LRB-

Fig.

-RRB-

shows

the

presence

of

-LRB-

at

least

-RRB-

four

subgroups

within

family

In

the

group

of

the

α-glucosidases

AgdA

clusters

with

AgdB

a

predicted

extracellular

protein

AgdB

has

some

similarity

to

two

A.

nidulans

enzymes

-LRB-

identity

-RRB-

with

both

α

and

β-glucosidase

activity

-LRB-

Bauer

et

al.

-RRB-

and

α-glucosidase

B

-LRB-

AndAgdB

identity

-RRB-

with

strong

transglycosylation

activity

-LRB-

Kato

et

al.

-RRB-

However

of

the

true

orthologues

of

AgdB

has

been

characterized

-LRB-

Fig

-RRB-

Transcript

analysis

revealed

that

agdB

was

regulated

similar

to

agdA

as

the

strong

induction

of

both

genes

in

the

presence

of

maltose

was

dependent

on

the

presence

of

amyR

However

where

most

AmyR-regulated

genes

-LRB-

aamA

glaA

and

agdA

-RRB-

reached

their

highest

level

of

induction

after

h

growth

on

maltose

the

expression

level

of

agdB

was

decreased

after

h

compared

to

h

-LRB-

Fig.

-RRB-

From

the

remaining

five

family

members

AgdE

-LRB-

-RRB-

shows

similarity

to

Trichoderma

reesei

glucosidase

II

-LRB-

TrAguII

-RRB-

-LRB-

Geysens

et

al.

-RRB-

This

type

of

α-glucosidases

is

located

in

the

ER

where

it

is

involved

in

the

trimming

of

α

-LRB-

-RRB-

linked

glucose

residues

from

N-glycan

core

structure

which

may

be

attached

to

proteins

designated

to

be

secreted

-LRB-

Geysens

et

al.

-RRB-

AgdE

contains

a

predicted

signal

sequence

which

might

serve

to

direct

the

protein

to

the

ER

The

agdE

gene

was

constitutively

expressed

in

all

conditions

tested

-LRB-

Fig.

-RRB-

consistent

with

its

predicted

function

as

an

α-glucosidase

II

Two

additional

family

members

AgdF

-LRB-

-RRB-

and

AgdG

-LRB-

-RRB-

lack

similarity

to

any

functionally

described

proteins

Expression

of

both

genes

was

not

detected

in

any

of

the

tested

growth

conditions

The

two

final

family

members

named

AxlA

and

AxlB

are

highly

similar

to

from

A.

nidulans

which

was

recently

characterized

as

an

α-xylosidase

-LRB-

Bauer

et

al.

-RRB-

Gene

axlA

was

highly

expressed

in

the

presence

of

xylose

whereas

no

expression

was

detected

in

maltose

grown

cultures

-LRB-

Fig.

-RRB-

The

high

expression

of

axlA

on

xylose

further

supports

its

putative

function

as

a

xylanolytic

enzyme

The

gene

encoding

the

putatively

intracellular

AxlB

was

expressed

at

a

very

low

level

independent

of

the

carbon

source

-LRB-

Fig.

-RRB-

A

BLASTP

search

in

the

genomes

of

A.

fumigatus

A.

nidulans

and

A.

oryzae

for

family

enzymes

resulted

in

a

similar

collection

of

enzymes

as

identified

in

A.

niger

-LRB-

Fig

-RRB-

Several

clusters

of

orthologous

proteins

are

distinguishable

but

the

assignment

of

an

enzymatic

activity

to

these

clusters

is

not

yet

possible

due

to

a

lack

of

well

studied

homologues

for

these

enzymes

thus

requiring

biochemical

studies

in

our

future

work

Genome-wide

analysis

of

AmyR

dependent

maltose

induced

genes

using

microarrays

The

expression

analysis

of

genes

encoding

family

and

enzymes

in

the

A.

niger

genome

revealed

that

the

expression

of

only

a

limited

number

of

genes

was

induced

by

maltose

in

an

AmyR

dependent

way

In

fact

only

four

genes

-LRB-

aamA

glaA

agdA

and

agdB

-RRB-

showed

the

predicted

expression

pattern

for

genes

encoding

enzymes

involved

in

the

breakdown

of

starch

-LRB-

Fig.

-RRB-

To

identify

additional

genes

with

a

possible

role

in

starch

metabolism

the

expression

of

all

predicted

open

reading

frames

on

the

Affymetrix

microarray

chips

was

analysed

Comparison

of

the

transcriptome

of

the

wild

type

strain

-LRB-

-RRB-

grown

for

h

after

transfer

on

either

xylose

or

maltose

identified

genes

that

were

significantly

induced

by

maltose

-LRB-

One-way

ANOVA

analysis

P

and

change

in

expression

level

-RRB-

A

set

of

genes

was

expressed

higher

in

compared

to

the

ΔamyR

strain

-LRB-

One-way

ANOVA

analysis

P

-RRB-

after

the

transfer

of

these

strains

from

xylose

to

maltose

Combining

the

two

gene

sets

resulted

in

a

collection

of

six

genes

that

were

induced

on

maltose

in

an

AmyR-dependent

manner

-LRB-

Fig.

Tables

-RRB-

Five

of

these

were

assigned

to

the

category

of

carbohydrate

transport

and

metabolism

according

to

FunCat

-LRB-

Ruepp

et

al.

-RRB-

including

the

three

genes

encoding

known

extracellular

starch

degrading

enzymes

-LRB-

aamA

glaA

and

agdA

-RRB-

and

a

putative

α-glucosidase

-LRB-

agdB

-RRB-

-LRB-

Table

-RRB-

Gene

encoding

a

protein

with

high

similarity

-LRB-

-RRB-

to

a

Candida

intermedia

glucose/xylose

symporter

-LRB-

Leandro

et

al.

-RRB-

was

induced

by

maltose

this

induction

was

not

observed

in

the

ΔamyR

strain

The

sixth

induced

gene

-LRB-

-RRB-

encodes

a

protein

highly

similar

-LRB-

-RRB-

to

Kluyveromyces

lactis

phosphoenolpyruvate

carboxykinase

-LRB-

Kitamoto

et

al.

-RRB-

which

functions

in

gluconeogenesis

by

catalyzing

the

conversion

of

oxaloacetate

into

phosphoenolpyruvate

It

should

be

noted

that

the

pH

of

the

growth

medium

remained

during

the

h

growth

period

after

transfer

Fig.

of

microarray

analysis

for

maltose

induced

and

AmyR

dependent

genes

Venn-diagram

showing

the

number

of

genes

induced

on

maltose

compared

to

xylose

in

A.

niger

and

the

number

of

genes

induced

in

compared

to

the

ΔamyR

deletion

strain

The

number

of

genes

both

induced

by

maltose

and

dependent

on

AmyR

is

indicated

in

bold

The

maltose

induced

and

AmyR

dependent

genes

which

are

present

in

both

and

h

after

transfer

from

a

preculture

are

shown

in

the

boxTable

of

six

maltose

induced

and

AmyR

dependent

genes

in

A.

niger

at

h

-LRB-

pH

-RRB-

after

transfer

from

precultureORF

xyloseCallΔamyR

maltoseCallFold

inductionP-valuePutative

functionMaltose

vs

vs

ΔamyRCarbohydrate

transport

and

Acid

alpha-amylase

Extracellular

alpha-glucosidase

Extracellular

alpha-glucosidase

Glucoamylase

GlaA

Putative

monosaccharide

Putative

phosphoenolpyruvate

carboxykinase

aGene

name

in

bold

indicates

that

the

gene

is

induced

on

maltose

in

an

AmyR

dependant

way

after

both

and

hbThe

expression

level

was

based

on

the

geometric

mean

value

of

the

duplicate

samples

and

the

deviation

values

between

the

duplicate

samples

are

indicated

The

P-value

was

based

on

ANOVA

analysiscP

M

or

A

represent

detection

calls

for

present

marginal

or

absent

respectively

Genes

were

divided

over

different

functional

groups

according

to

FunCat

-LRB-

Ruepp

et

al.

-RRB-

Table

of

the

maltose

induced

and

AmyR

dependent

genes

in

A.

niger

at

h

-LRB-

-RRB-

after

transfer

from

precultureORF

xyloseCallΔamyR

maltoseCallFold

inductionP-valuePutative

functionMaltose

vs

vs

ΔamyRCarbohydrate

transport

and

Acid

alpha-amylase

Extracellular

alpha-glucosidase

Extracellular

alpha-glucosidase

P

Putative

hexose

transport

protein

P

Putative

monosaccharide

Glucoamylase

GlaA

Extracellular

alpha-amylase

Putative

hexose

Sugar/H

symporter

Putative

isoamyl

alcohol

oxidase

Putative

phosphoglycerate

mutaseAmino

acid

transport

and

P

Similarity

to

tyrosinase

protein

Cell

rescue

defence

and

virulence

P

Similarity

to

integral

membrane

protein

Similarity

to

integral

membrane

protein

P

Similarity

to

integral

membrane

proteinProtein

M

Similarity

to

UDP-galactose

P

Unknown

P

Unknowna

b

cSee

footnotes

of

Table

A

similar

comparative

analysis

was

performed

for

samples

taken

for

cultures

that

had

grown

for

h

after

the

transfer

To

prevent

differences

in

pH

between

the

and

the

ΔamyR

strain

after

h

of

growth

the

pH

of

the

medium

was

buffered

to

using

mM

of

citric

acid/sodium

citrate

to

allow

comparison

between

the

and

the

ΔamyR

strain

at

this

time

point

as

the

medium

of

the

ΔamyR

strain

did

not

acidify

as

quickly

as

the

medium

of

the

strain

A

total

of

genes

were

significantly

induced

by

maltose

in

comparison

with

xylose

and

genes

were

higher

expressed

in

compared

with

the

ΔamyR

strain

-LRB-

one-way

ANOVA

analysis

P

-RRB-

By

combining

the

two

sets

we

identified

genes

which

were

induced

by

maltose

and

whose

induction

was

AmyR

dependent

-LRB-

Fig.

Table

-RRB-

Nine

of

these

genes

encode

proteins

involved

in

carbohydrate

transport

and

metabolism

from

which

five

were

also

identified

as

differentially

expressed

after

h

-LRB-

aamA

glaA

agdA

agdB

and

-RRB-

Additionally

identified

genes

included

amyC

encoding

an

extracellular

α-amylase

three

genes

encoding

putative

sugar

transporters

and

several

other

genes

belonging

to

various

functional

categories

-LRB-

Tables

-RRB-

At

this

stage

we

can

not

exclude

that

additional

overlapping

genes

have

escaped

our

attention

because

of

the

different

pH

values

of

the

medium

after

transfer

which

also

could

affect

gene

expression

The

results

from

the

microarray

experiments

were

validated

using

Northern

blot

analysis

for

a

selected

number

of

amylolytic

genes

These

genes

include

glaA

aamA

agdB

and

amyC

As

shown

in

supplementary

Fig.

the

results

of

the

Northern

hybridizations

are

in

good

agreement

with

the

microarray

data

-LRB-

Supplementary

Table

-RRB-

The

Northern

analysis

also

revealed

an

additional

mRNA

of

a

larger

size

for

the

amyC

gene

h

after

transfer

The

detection

of

two

different-sized

mRNAs

suggests

two

different

mRNA

start

sites

or

different

polyadenylation

sites

but

the

exact

sequence

of

the

different

mRNAs

and

possible

consequences

for

the

gene

model

have

not

been

addressed

so

far

Interestingly

the

transcription

factor

gene

amyR

was

also

induced

and

h

after

transfer

to

maltose

compared

to

the

transfer

to

xylose

-LRB-

P-value

P-value

respectively

-RRB-

indicating

transcriptional

regulation

of

the

amyR

gene

itself

As

the

AmyR

transcription

factor

is

per

se

missing

in

the

deletion

strain

it

is

not

appropriate

to

include

the

amyR

gene

in

the

group

of

maltose

induced

AmyR

dependent

genes

However

the

promoter

region

of

the

amyR

gene

contains

two

AmyR

binding

motifs

allowing

for

the

possibility

that

AmyR

can

induce

its

own

expression

Discussion

The

full

inventory

of

glycoside

hydrolases

belonging

to

families

and

in

A.

niger

strain

CBS

has

recently

been

described

-LRB-

Pel

et

al.

-RRB-

Members

of

these

protein

families

in

aspergilli

including

A.

niger

have

been

studied

extensively

mainly

because

of

their

industrial

relevance

Nevertheless

several

groups

of

new

alpha-glucan

acting

enzymes

that

had

not

been

identified

previously

were

identified

These

novel

enzymes

are

conserved

among

several

Aspergillus

species

as

well

as

other

Ascomycetes

indicating

that

they

may

play

an

important

role

in

fungal

metabolism

In

the

present

study

we

have

combined

transcriptional

analysis

and

more

detailed

phylogenetic

analysis

to

further

understand

their

function

The

family

in

A.

niger

contains

six

separate

groups

of

amylase-type

enzymes

-LRB-

Fig.

-RRB-

of

which

three

groups

had

not

been

described

thus

far

The

best-described

group

comprises

the

extracellular

α-amylases

and

is

now

extended

with

AmyC

in

addition

to

the

three

known

α-amylases

Overexpression

of

the

amyC

gene

in

A.

niger

resulted

in

increased

levels

of

endo-α-amylase

activity

in

the

medium

indicating

that

this

gene

indeed

encodes

an

extracellular

α-amylase

-LRB-

R.

M.

Van

der

Kaaij

and

X.

L.

Yuan

unpublished

results

-RRB-

The

relatively

low

expression

of

amyC

compared

to

other

starch

degrading

enzymes

may

explain

why

this

protein

has

not

been

identified

previously

The

localization

of

the

amyC

gene

in

the

genome

is

noteworthy

as

it

is

part

of

a

small

cluster

of

amylolytic

genes

-LRB-

with

agdA

-RRB-

and

their

transcriptional

regulator

gene

amyR

A

similar

organization

is

observed

in

the

genomes

of

other

aspergilli

such

as

A.

nidulans

and

A.

oryzae

-LRB-

Gomi

et

al.

-RRB-

In

A.

fumigatus

the

same

cluster

is

extended

with

a

glucoamylase

-LRB-

similar

to

GlaA

-RRB-

Gene

clusters

of

transcriptionally

co-regulated

genes

in

filamentous

fungi

are

often

involved

in

the

same

process

e.g.

secondary

metabolite

production

-LRB-

Woloshuk

et

al.

-RRB-

or

catabolism

of

amino

acids

-LRB-

Hull

et

al.

-RRB-

A

possible

function

of

AmyC

might

be

to

act

as

a

scouting

enzyme

for

the

presence

of

starch

resulting

in

the

subsequent

activation

of

AmyR

by

starch-derived

molecules

-LRB-

maltose

or

isomaltose

-RRB-

Alternatively

AmyC

might

be

regulated

by

another

system

additional

to

AmyR

e.g.

pH-regulated

expression

or

its

expression

might

be

upregulated

locally

e.g.

in

hyphal

tips

The

lack

of

detectable

expression

of

amyA

and

amyB

in

A.

niger

is

puzzling

Both

genes

were

expressed

in

A.

niger

strain

CBS

in

a

fed-batch

fermentation

using

glucose

as

a

carbon

source

-LRB-

Pel

et

al.

-RRB-

but

batch

cultivation

of

A.

niger

on

glucose

did

not

result

in

detectable

expression

in

microarray

studies

-LRB-

E.

Martens

H.

Kools

P.

Schaap

personal

communication

-RRB-

These

results

indicate

a

difference

between

both

strains

with

regard

to

either

the

presence

of

amyA/B

in

A.

niger

strain

or

in

the

transcriptional

regulation

of

amyA/B

In

the

recently

released

genome

sequence

of

A.

niger

strain

ATCC

from

the

Joint

Genome

Institute

-LRB-

Baker

-RRB-

we

could

not

identify

orthologues

of

the

AmyA/AmyB

proteins

suggesting

that

these

genes

might

be

actually

absent

from

the

strain

The

absence

or

lack

of

expression

of

amyA/amyB

is

consistent

with

the

observation

that

the

A.

niger

strain

-LRB-

background

-RRB-

with

gene

disruptions

for

both

aamA

and

glaA

grows

very

poorly

on

starch

-LRB-

Weenink

et

al.

-RRB-

confirming

that

AmyA

and

AmyB

do

not

contribute

significantly

to

the

amylolytic

potential

of

The

microarray

analysis

revealed

that

in

addition

to

the

amyA/B

genes

five

more

amylolytic

genes

-LRB-

agtC

amyD

agdD

agdG

and

glaB

-RRB-

were

not

detected

-LRB-

Absent

calls

-RRB-

in

any

of

the

conditions

tested

However

orthologous

genes

were

identified

in

the

genome

of

A.

niger

strain

suggesting

that

these

genes

are

not

absent

from

the

genome

but

rather

not

expressed

under

the

tested

conditions

In

this

study

we

have

identified

three

new

groups

within

the

family

that

had

not

been

described

before

in

aspergilli

α-amylase-like

proteins

predicted

to

be

GPI-anchored

enzymes

-LRB-

group

II

-RRB-

-LRB-

subfamily

as

defined

by

Stam

et

al.

-RRB-

intracellular

α-glucosidases

-LRB-

group

III

-RRB-

-LRB-

unknown

subfamily

-RRB-

and

intracellular

α-amylases

-LRB-

group

IV

-RRB-

-LRB-

subfamily

-RRB-

Detailed

biochemical

studies

of

two

GPI-anchored

proteins

revealed

that

they

are

α-glucanotransferases

acting

mainly

on

maltooligosaccharides

-LRB-

van

der

Kaaij

et

al.

-RRB-

The

expression

of

these

genes

was

not

regulated

in

response

to

maltose

metabolism

or

AmyR

The

Agt

enzymes

are

homologues

of

a

group

of

four

GPI-anchored

amylase-like

proteins

recently

identified

in

S.

pombe

-LRB-

-RRB-

which

have

similarly

aberrant

conserved

regions

-LRB-

Table

-RRB-

A

deletion

of

one

of

these

homologues

resulted

in

aberrant

morphology

of

the

cell

and

an

increased

sensitivity

towards

cell

wall

degrading

enzymes

-LRB-

Morita

et

al.

-RRB-

A

similar

phenotype

was

observed

for

an

A.

niger

knockout

of

agtA

-LRB-

van

der

Kaaij

et

al.

-RRB-

Such

a

phenotype

can

be

explained

if

α-glucan

synthesis

is

affected

The

clustering

of

agt

genes

with

genes

encoding

α-glucan

synthases

in

all

four

Aspergillus

species

studied

and

the

expression

pattern

of

the

A.

niger

agt

genes

both

support

the

hypothesis

that

Agt

enzymes

play

a

role

in

α-glucan

synthesis

Aspergillus

niger

contains

two

putative

intracellular

α-amylases

AmyD

and

AmyE

that

belong

to

the

recently

identified

subfamily

-LRB-

Stam

et

al.

-RRB-

A

homologous

gene

in

H.

capsulatum

was

shown

to

be

essential

for

the

formation

of

α

-LRB-

-RRB-

glucan

in

the

cell

wall

-LRB-

Marion

et

al.

-RRB-

The

possibility

of

a

role

for

subfamily

enzymes

in

the

cell

wall

of

Aspergilli

is

strengthened

by

their

location

in

the

genome

A.

niger

A.

nidulans

and

A.

oryzae

all

contain

a

cluster

of

genes

containing

a

homologue

of

as

well

as

ags

and

agt

genes

-LRB-

Table

-RRB-

Both

the

GPI-anchored

α-glucanotransferases

and

the

family

α-amylase-like

proteins

are

only

present

in

fungi

with

cell

wall

α-glucan

and

not

in

the

true

yeasts

which

lack

this

type

of

cell

wall

glucans

However

a

study

with

heterologously

expressed

AmyD

did

not

confirm

a

role

for

this

enzyme

in

α

-LRB-

-RRB-

glucan

formation

as

the

enzyme

only

showed

a

-LRB-

low

-RRB-

hydrolyzing

activity

on

substrates

with

α

-LRB-

-RRB-

glycosidic

bonds

in

vitro

-LRB-

van

der

Kaaij

et

al.

-RRB-

The

expression

levels

of

amyD

and

amyE

were

below

detection

level

and

thus

could

not

confirm

nor

refute

the

possible

involvement

of

these

proteins

in

cell

wall

α-glucan

synthesis

in

A.

niger

At

this

moment

no

function

can

be

assigned

to

the

predicted

intracellular

α-glucosidases

AgdC

and

AgdD

and

their

homologues

identified

in

other

fungi

The

enzymes

show

similarity

to

a

Bacillus

α-glucosidase

as

well

as

to

a

maltase

from

C.

albicans

and

might

therefore

be

involved

in

the

intracellular

hydrolysis

of

maltose

Alternatively

these

enzymes

could

play

a

role

in

trehalose

metabolism

as

they

show

some

similarity

to

bacterial

hydrolases

Trehalose

is

known

to

be

a

common

reserve

carbohydrate

in

fungi

including

A.

niger

-LRB-

Wolschek

and

Kubicek

Arguelles

-RRB-

A

surprising

finding

from

the

genome

mining

of

A.

niger

was

the

high

number

of

GH

enzymes

with

a

predicted

intracellular

location

This

group

included

not

only

the

starch-acting

enzymes

described

here

but

also

other

predicted

intracellular

glycoside

hydrolases

such

as

β-glucosidase

chitinase

β-mannosidase

β-xylosidase

rhamnosidase

and

invertase

present

in

various

Aspergillus

genomes

-LRB-

Pel

et

al.

Goosen

et

al.

-RRB-

Most

of

these

proteins

were

predicted

to

be

exo-acting

i.e.

releasing

terminal

mono

or

disaccharide

residues

from

the

non-reducing

end

of

the

substrate

In

A.

niger

the

genes

encoding

these

proteins

are

expressed

at

a

relatively

low

level

compared

to

the

extracellular

starch

degrading

enzymes

sometimes

even

below

the

detection

limit

In

a

recent

publication

expression

of

intracellular

chitinases

in

A.

nidulans

was

found

to

be

induced

during

the

autolysis

phase

of

the

culture

-LRB-

Yamazaki

et

al.

-RRB-

A

role

for

these

and

other

predicted

intracellular

proteins

during

the

autolysis

phase

would

explain

their

low

expression

levels

observed

in

A.

niger

during

vegetative

growth

as

well

as

their

lack

of

secretion

signals

The

genome-wide

expression

analysis

identified

only

five

genes

that

were

induced

by

maltose

in

an

AmyR

dependent

way

and

h

after

the

transfer

to

maltose

Among

them

are

genes

encoding

known

starch

degrading

enzymes

-LRB-

aamA

glaA

-RRB-

two

-LRB-

putative

-RRB-

α-glucosidases

-LRB-

agdA

agdB

-RRB-

and

a

putative

sugar

transporter

protein

-LRB-

-RRB-

As

described

in

the

Results

section

the

samples

taken

after

and

h

after

transfer

were

grown

at

different

pH

values

-LRB-

pH

and

respectively

-RRB-

which

might

influence

our

results

and

lead

to

an

underestimation

of

the

number

of

genes

that

are

induced

by

maltose

in

an

AmyR

dependant

way

at

both

and

h

as

additional

pH-controlled

gene

expression

might

also

occur

To

fully

explore

this

both

strains

should

be

cultivated

at

controlled

conditions

at

identical

pH.

The

specificity

of

the

putative

sugar

transporter

encoded

by

the

gene

has

not

been

examined

but

its

expression

profile

suggests

that

it

might

function

as

a

glucose

or

a

maltose

transporter

Three

additional

sugar

transporters

were

expressed

higher

on

maltose

than

on

xylose

after

h

-LRB-

Table

-RRB-

One

of

them

has

been

functionally

characterized

as

a

high-affinity

glucose

transporter

-LRB-

Vankuyk

et

al.

-RRB-

which

is

in-line

with

its

expression

profile

The

two

additional

sugar

transporters

encoding

genes

-LRB-

and

-RRB-

showed

an

interesting

expression

pattern

with

induced

expression

on

maltose

but

also

relatively

high

expression

levels

on

xylose

-LRB-

Table

-RRB-

suggesting

that

these

transporters

might

have

a

broad

sugar

transporting

activity

In

the

ΔamyR

strain

both

their

expression

levels

were

strongly

reduced

also

relative

to

the

xylose

conditions

indicating

that

the

deletion

of

AmyR

might

have

had

an

indirect

effect

A

possible

explanation

is

that

disruption

of

the

AmyR

transcription

factor

resulted

in

low

levels

of

extracellular

enzymes

converting

maltose

to

glucose

and

consequently

stress

due

to

low

glucose

levels

The

low

availability

of

glucose

might

be

a

signal

to

downregulate

mstC

and

if

these

genes

encode

low-affinity

sugar

transporters

To

further

address

these

observations

additional

array

analysis

should

be

performed

using

the

ΔamyR

strain

grown

on

xylose

Interestingly

the

amyR

gene

itself

was

also

induced

by

the

presence

of

maltose

suggesting

that

its

regulation

takes

place

at

least

partly

at

the

transcriptional

level

Furthermore

conserved

AmyR

binding

motifs

are

present

in

the

promoters

of

all

co-regulated

genes

including

the

amyR

promoter

region

with

the

exception

of

aamA

-LRB-

Table

-RRB-

Possibly

the

aamA

promoter

contains

a

currently

unknown

AmyR

binding

motif

Alternatively

an

AmyR

binding

site

might

be

present

in

the

promoter

region

of

aamA

in

the

background

The

results

presented

in

this

paper

suggest

that

A.

niger

can

metabolize

maltose

by

inducing

the

expression

of

a

limited

amount

of

enzymes

and

sugar

transporters

Other

members

of

the

and

protein

families

might

function

in

starch

metabolism

in

different

conditions

-LRB-

solid

state

growth

or

influenced

by

pH

nitrogen

metabolism

etc.

-RRB-

or

they

may

be

involved

in

other

processes

such

as

the

synthesis

of

cell

wall

α-glucan

or

the

glycosylation

of

proteins

To

determine

the

exact

function

of

these

enzymes

will

require

further

biochemical

characterization

in

combination

with

the

detailed

analysis

of

gene

deletion

mutants

Electronic

supplementary

material

Below

is

the

link

to

the

electronic

supplementary

material

Supplementary

material

-LRB-

DOC

kb

-RRB-

aspergillus niger
glucoamylase
amyr
starch
maltose
cell wall

alpha-amylase
alpha-glucosidase
alpha-glucan
starch-binding domain