Identification

of

InuR

a

new

Zn

-LRB-

II

-RRB-

transcriptional

activator

involved

in

the

regulation

of

inulinolytic

genes

in

Aspergillus

niger

The

expression

of

inulinolytic

genes

in

Aspergillus

niger

is

co-regulated

and

induced

by

inulin

and

sucrose

We

have

identified

a

positive

acting

transcription

factor

InuR

which

is

required

for

the

induced

expression

of

inulinolytic

genes

InuR

is

a

member

of

the

fungal

specific

class

of

transcription

factors

of

the

Zn

-LRB-

II

-RRB-

type

Involvement

of

InuR

in

inulin

and

sucrose

metabolism

was

suspected

because

of

the

clustering

of

inuR

gene

with

sucB

which

encodes

an

intracellular

invertase

with

transfructosylation

activity

and

a

putative

sugar

transporter

encoding

gene

-LRB-

-RRB-

Deletion

of

the

inuR

gene

resulted

in

a

strain

displaying

a

severe

reduction

in

growth

on

inulin

and

sucrose

medium

Northern

analysis

revealed

that

expression

of

inulinolytic

and

sucrolytic

genes

e.g.

inuE

inuA

sucA

as

well

as

the

putative

sugar

transporter

gene

-LRB-

-RRB-

is

dependent

on

InuR

Genome-wide

expression

analysis

revealed

three

additional

putative

sugar

transporters

encoding

genes

-LRB-

and

-RRB-

which

were

strongly

induced

by

sucrose

in

an

InuR

dependent

way

In

silico

analysis

of

the

promoter

sequences

of

strongly

InuR

regulated

genes

suggests

that

InuR

might

bind

as

dimer

to

two

CGG

triplets

which

are

separated

by

eight

nucleotides

Introduction

Some

plants

store

part

of

their

carbon

as

sucrose

and

as

polymers

of

fructose

-LRB-

fructans

-RRB-

Plant

fructans

often

referred

to

as

inulins

have

predominantly

linkages

between

the

fructose

moieties

and

terminate

with

a

sucrosyl

residue

Inulin

is

present

in

a

range

of

plant

species

In

some

species

such

as

chicory

garlic

Jerusalem

artichoke

and

Dahlia

tubers

about

of

the

plant

fresh

weight

consists

of

inulin

-LRB-

Gupta

and

Kaur

Kaur

and

Gupta

-RRB-

Apart

from

the

use

of

inulin

for

the

production

of

fructose

rich

syrups

inulin

and

fructo-oligosaccharides

-LRB-

FOS

-RRB-

have

attracted

considerable

research

attention

because

of

the

large

number

of

health

benefits

obtained

from

inulin

and

FOS

in

the

diet

of

humans

-LRB-

see

for

a

review

Kaur

and

Gupta

-RRB-

Aspergillus

niger

is

a

saprophytic

fungus

mainly

present

in

the

soil

feeding

preferably

on

organic

matter

such

as

plant

cell

wall

polysaccharides

-LRB-

cellulose

hemi-cellulose

and

pectin

-RRB-

and

on

plant

storage

polysaccharides

-LRB-

starch

and

inulin

-RRB-

A.

niger

is

able

to

produce

various

inulin

degrading

enzymes

including

an

exo-inulinase

an

endo-inulinase

and

an

invertase

-LRB-

Moriyama

et

al.

Ohta

et

al.

Akimoto

et

al.

Berges

et

al.

Boddy

et

al.

L'Hocine

et

al.

-RRB-

The

inulinolytic

enzymes

are

simultaneously

induced

in

the

presence

of

inulin

or

sucrose

indicating

that

the

expression

of

the

genes

encoding

these

enzymes

is

co-regulated

and

controlled

by

a

common

transcription

factor

-LRB-

Yuan

et

al.

-RRB-

Transcriptional

activation

of

catabolic

enzyme

networks

is

often

accomplished

through

transcription

factors

of

the

Zn

-LRB-

II

-RRB-

type

This

type

of

transcription

factor

is

unique

to

fungi

is

the

transcriptional

activator

for

the

expression

of

genes

involved

in

galactose

utilization

and

is

the

best

characterized

Zn

-LRB-

II

-RRB-

transcription

factor

In

general

Zn

-LRB-

II

-RRB-

transcription

factors

possess

a

well

conserved

N-terminal

localized

DNA

binding

motif

-LRB-

-RRB-

followed

by

a

less

well

defined

conserved

domain

known

as

a

fungal

specific

transcription

factor

domain

-LRB-

-RRB-

The

C-terminal

part

of

the

Zn

-LRB-

II

-RRB-

transcription

factor

normally

contains

the

activation

domain

In

the

genome

sequence

of

Saccharomyces

cerevisiae

putative

Zn

-LRB-

II

-RRB-

transcription

factors

have

been

identified

-LRB-

Akache

et

al.

-RRB-

They

are

involved

in

the

transcriptional

control

of

a

wide

variety

of

cellular

processes

-LRB-

Todd

and

Andrianopoulos

Akache

et

al.

-RRB-

including

genes

involved

in

amino

acid

metabolism

-LRB-

ArgRp

Crabeel

et

al.

Kohlhaw

El

Alami

et

al.

-RRB-

sugar

metabolism

-LRB-

Ni

and

Needleman

-RRB-

pyrimidine

biosynthesis

-LRB-

Flynn

and

Reece

-RRB-

In

the

genome

of

filamentous

fungi

even

more

Zn

-LRB-

II

-RRB-

transcription

factors

are

present

In

the

recently

released

genome

sequence

of

A.

niger

CBS

ORFs

were

identified

that

contain

the

Zn

-LRB-

II

-RRB-

motif

and

similar

numbers

are

predicted

from

the

genomes

of

A.

fumigatus

and

A.

nidulans

-LRB-

see

for

comparison

Pel

et

al.

-RRB-

The

role

of

only

a

few

of

the

Zn

-LRB-

II

-RRB-

transcription

factors

in

filamentous

fungi

have

been

studied

in

detail

Filamentous

fungal

Zn

-LRB-

II

-RRB-

proteins

are

involved

in

the

regulation

of

various

processes

which

include

secondary

metabolite

production

-LRB-

e.g.

AflR

Woloshuk

et

al.

-RRB-

pigmentation

-LRB-

e.g.

Tsuji

et

al.

-RRB-

nitrogen

metabolism

-LRB-

NirA

Burger

et

al.

-RRB-

proline

utilization

-LRB-

PrnA

Cazelle

et

al.

Leucine

biosynthesis

-LRB-

Kirkpatrick

and

Schimmel

-RRB-

alcohol

utilization

-LRB-

AlcR

Kulmburg

et

al.

-RRB-

and

sexual

development

-LRB-

RosA

Vienken

et

al.

-RRB-

Well

studied

examples

of

fungal

Zn

-LRB-

II

-RRB-

transcription

factors

required

for

polysaccharide

catabolism

include

AmyR

a

transcriptional

activator

of

starch

degrading

enzymes

-LRB-

Petersen

et

al.

Gomi

et

al.

Tani

et

al.

-RRB-

and

XlnR

a

transcriptional

activator

for

xylanolytic

enzymes

-LRB-

van

Peij

et

al.

Gielkens

et

al.

Hasper

et

al

-RRB-

It

is

well

established

that

the

genes

encoding

many

proteins

required

for

secondary

metabolite

production

are

clustered

in

the

genome

-LRB-

Woloshuk

et

al.

Walton

-RRB-

In

addition

to

several

biosynthetic

enzymes

that

synthesize

the

secondary

metabolites

such

clusters

also

contain

a

Zn

-LRB-

II

-RRB-

transcription

factor

which

is

required

for

the

transcriptional

activation

of

the

genes

within

the

cluster

Gene

clustering

is

not

limited

to

secondary

metabolite

pathways

but

also

found

for

the

proline

utilization

genes

-LRB-

PrnA

Gomez

et

al.

-RRB-

and

for

some

of

the

amylolytic

genes

under

the

control

of

the

regulator

AmyR

Adjacent

to

the

gene

for

the

AmyR

transcription

factor

are

genes

encoding

alpha-glucosidase

-LRB-

AgdA

-RRB-

and

an

alpha-amylase

-LRB-

AmyA

-RRB-

-LRB-

Gomi

et

al.

-RRB-

Also

in

A.

parasiticus

a

sugar

utilization

gene

cluster

has

been

identified

-LRB-

Yu

et

al.

-RRB-

However

it

is

clear

that

clustering

of

genes

with

related

functions

is

not

a

general

rule

since

the

genes

adjacent

to

the

XlnR

transcription

factor

do

not

seem

to

be

involved

in

xylan

degradation

In

a

previous

study

we

showed

that

the

expression

of

the

genes

encoding

inulin

degrading

enzymes

is

co-regulated

and

specifically

induced

in

the

presence

of

inulin

and

sucrose

-LRB-

Yuan

et

al.

-RRB-

The

availability

of

the

genome

sequence

of

A.

niger

allowed

the

rapid

identification

of

Zn

-LRB-

II

-RRB-

transcription

factors

adjacent

to

or

in

close

proximity

to

the

genes

encoding

-LRB-

putative

-RRB-

enzymes

involved

in

inulin

catabolism

We

have

identified

a

Zn

-LRB-

II

-RRB-

cluster

transcription

factor

-LRB-

InuR

-RRB-

which

is

adjacent

to

a

gene

encoding

a

sugar

transporter

and

a

recently

identified

intracellular

invertase

-LRB-

SucB

-RRB-

with

transfructosylation

activity

-LRB-

Goosen

et

al.

-RRB-

In

this

paper

we

show

that

deletion

of

the

inuR

gene

results

in

strongly

impaired

growth

on

inulin

and

sucrose

and

also

abolishes

the

induction

of

the

genes

encoding

the

extracellular

enzymes

involved

in

inulin

and

sucrose

degradation

The

gene

encoding

the

putative

sugar

transporter

located

next

to

inuR

is

co-regulated

with

the

genes

encoding

the

inulinolytic

enzymes

These

results

show

that

InuR

acts

as

a

positive

acting

transcriptional

activator

for

the

induced

expression

of

genes

involved

in

the

breakdown

of

inulin

and

sucrose

and

the

uptake

of

inulin

breakdown

products

Materials

and

methods

Strains

culture

conditions

and

transformation

Aspergillus

niger

strain

used

in

this

study

was

derived

from

the

wild-type

strain

A.

niger

van

Tieghem

-LRB-

CBS

ATCC

-RRB-

-LRB-

Bos

et

al.

-RRB-

The

A.

niger

strain

used

by

DSM

to

sequence

the

genome

is

NRRL

Strain

is

a

pyrG

negative

derivative

of

-LRB-

van

Hartingsveldt

et

al.

-RRB-

and

was

used

to

construct

disruption

strains

A.

niger

strains

were

grown

in

Aspergillus

minimal

medium

-LRB-

MM

-RRB-

-LRB-

Bennet

and

Lasure

-RRB-

or

Aspergillus

complete

medium

-LRB-

CM

-RRB-

consisting

of

MM

medium

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

-RRB-

when

required

For

shake

flask

cultures

A.

niger

strains

were

grown

in

MM

supplemented

with

-LRB-

w/v

-RRB-

carbon

source

and

-LRB-

w/v

-RRB-

casamino

acids

The

conidiospores

were

inoculated

at

a

concentration

of

spores

per

ml

Glucose

and

sucrose

-LRB-

BDH

chemicals

-RRB-

maltodextrin

-LRB-

Avebe

-RRB-

starch

-LRB-

Windmill

Starch

Avebe

-RRB-

xylose

fructose

and

maltose

-LRB-

Sigma-Aldrich

-RRB-

raffinose

-LRB-

Sigma

chemicals

-RRB-

and

inulin

-LRB-

Sensus

Frutafit

Cosun

-RRB-

were

used

as

carbon

sources

For

transfer

experiments

A.

niger

strains

were

pre-grown

in

MM

supplemented

with

-LRB-

w/v

-RRB-

xylose

and

-LRB-

w/v

-RRB-

casamino

acids

for

h

at

°C

on

a

rotary

shaker

at

rpm

Then

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

Erlenmeyer

flasks

containing

MM

supplemented

with

-LRB-

w/v

-RRB-

carbon

source

and

incubated

at

°C

for

the

time

indicated

in

the

text

The

mycelium

was

harvested

over

Myracloth

-LRB-

Calbiochem

-RRB-

and

frozen

in

liquid

nitrogen

followed

by

storage

at

°C

prior

to

the

isolation

of

total

RNA

Conidiospores

were

obtained

by

harvesting

spores

from

CM

plates

after

days

of

growth

at

°C

using

a

NaCl

solution

Transformation

of

A.

niger

was

done

as

described

by

Punt

and

van

den

Hondel

-LRB-

-RRB-

using

lysing

enzymes

-LRB-

Sigma

-RRB-

for

protoplastion

The

bacterial

strain

used

for

transformation

and

amplification

of

recombinant

DNA

was

Escherichia

coli

-LRB-

Stratagene

-RRB-

Transformation

of

was

performed

according

to

the

heat

shock

protocol

as

described

by

Inoue

et

al.

-LRB-

-RRB-

Construction

of

the

inuR

pyrG

deletion

strain

The

A.

nigerinuR

gene

was

deleted

by

the

replacement

of

the

complete

open

reading

frame

of

the

inuR

gene

with

the

Aspergillus

oryzaepyrG

gene

The

plasmid

used

to

disrupt

inuR

was

constructed

as

follows

The

DNA

fragments

flanking

the

inuR

ORF

were

amplified

by

PCR

using

genomic

DNA

as

template

Fragments

with

a

length

of

kb

of

flanking

DNA

and

kb

of

flanking

DNA

were

amplified

using

primers

and

and

-LRB-

Supplementary

Table

-RRB-

respectively

Each

primer

was

adapted

with

restriction

sites

as

indicated

for

further

cloning

The

amplified

PCR

fragments

were

digested

with

NotI

and

XhoI

or

XhoI

and

KpnI

respectively

and

cloned

into

pBlue-Script

II

to

obtain

plasmid

and

Subsequently

the

kb

NotI-XhoI

fragment

containing

flanking

of

inuR

from

was

ligated

into

NotI/XhoI

digested

to

give

The

A.

oryzaepyrG

gene

was

isolated

as

a

kb

SalI

BamHI

fragment

obtained

from

plasmid

-LRB-

de

Ruiter-Jacobs

et

al.

-RRB-

and

ligated

into

XhoI/BamHI

digested

to

obtain

pΔinuR

-LRB-

-RRB-

Before

transformation

the

plasmid

pΔinuR

was

linearized

with

NotI

and

transformed

into

A.

nigerpyrG

strain

Uridine

prototrophic

transformants

were

selected

by

incubating

protoplasts

on

agar

plates

containing

MM

without

uridine

Transformants

were

screened

by

PCR

using

primers

and

or

and

-LRB-

Supplementary

Table

-RRB-

Only

transformants

with

a

targeted

deletion

of

the

inuR

gene

should

result

in

the

amplification

of

a

or

kb

PCR

fragment

respectively

PCR

positive

transformants

were

verified

by

Southern

blot

analysis

as

described

by

Sambrook

et

al.

-LRB-

-RRB-

Chromosomal

DNA

from

positive

PCR

transformants

was

isolated

as

described

by

Kolar

et

al.

-LRB-

-RRB-

Ten

μl

of

genomic

DNA

was

digested

for

h

with

U

of

XhoI

or

EcoRI

respectively

The

kb

of

the

flanking

DNA

fragment

of

inuR

was

used

as

probe

for

the

detection

of

inuR

disruptants

A

probe

was

generated

by

digestion

of

the

plasmid

containing

the

inuR

flanking

fragment

with

XhoI

and

BamHI

Fragments

were

purified

from

gel

and

-LSB-

-RSB-

dCTP

labeled

probes

were

synthesized

using

Rediprime

II

DNA

labeling

System

-LRB-

Amersham

Pharmacia

Biotech

-RRB-

according

to

the

instructions

of

the

manufacturer

Complementation

of

inuR

mutant

The

A.

niger

inuR

gene

was

amplified

by

PCR

using

primers

and

-LRB-

Supplementary

Table

-RRB-

and

genomic

DNA

as

a

template

The

primers

corresponded

to

kb

upstream

and

kb

downstream

of

the

A.

niger

inuR

gene

respectively

PCR

was

performed

using

Phusion

High-Fidelity

DNA

Polymerase

-LRB-

Finnzymes

-RRB-

according

to

the

manufacturer

's

instruction

The

amplified

kb

PCR

fragment

was

cloned

into

pGEMT-easy

vector

-LRB-

Promega

-RRB-

to

obtain

the

inuR

complementation

plasmid

was

then

co-transformed

with

harboring

hygromycin

gene

as

selection

marker

-LRB-

Punt

et

al.

-RRB-

into

A.

niger

ΔinuR

strain

-LRB-

see

in

the

text

-RRB-

Hygromycin

resistant

transformants

were

selected

by

incubating

protoplasts

on

agar

plates

containing

MM

with

mg/ml

of

hygromycin

Southern

blot

analysis

was

performed

on

selected

transformants

as

described

by

Sambrook

et

al.

-LRB-

-RRB-

Construction

of

the

ΔinuR

ΔamyR

double

deletion

strain

To

make

a

pyrG-strain

of

the

ΔinuR

strain

-LRB-

-RRB-

this

strain

was

incubated

on

MM

plates

containing

mg/ml

of

FOA

and

mM

uracil

FOA

resistant

uracil

requiring

mutants

were

transformed

with

harboring

the

A.

nigerpyrG

or

with

pPyrE

harboring

the

A.

niger

pyrE

gene

on

a

kb

SstII

subclone

respectively

One

of

the

selected

FOA

resistant

mutants

was

complemented

with

the

pyrG

gene

indicating

that

the

uracil

auxotrophy

was

caused

by

a

mutation

in

the

pyrG

gene

The

strain

was

then

used

to

construct

a

ΔinuR

ΔamyR

double

disruption

strain

by

transforming

plasmid

pΔamyR

which

also

contains

the

pyrG

selection

marker

-LRB-

Yuan

et

al.

submitted

-RRB-

Uridine

prototrophic

transformants

were

selected

by

incubating

protoplasts

on

agar

plates

containing

MM

without

uridine

Transformants

that

showed

normal

growth

on

glucose

but

not

on

starch

were

selected

One

possible

ΔinuRΔamyR

double

mutant

-LRB-

-RRB-

was

verified

by

Southern

analysis

and

used

for

further

analysis

Northern

blot

analysis

Total

RNA

isolation

Northern

analysis

and

synthesis

of

DNA

probes

was

performed

as

described

in

Yuan

et

al.

-LRB-

-RRB-

The

primers

used

to

generate

probe

are

shown

in

Supplementary

Table

Microtiter

plate

growth

assay

Growth

of

A.

niger

strains

were

determined

using

a

BioAssay

Reader

-LRB-

Perkin

Elmer

Life

Sciences

-RRB-

Spores

-LRB-

-RRB-

were

inoculated

in

each

well

of

a

microtiter

plate

-LRB-

Nalge

Nunc

International

USA

-RRB-

and

incubated

at

°C

for

h.

Each

well

contained

μl

of

MM

containing

-LRB-

w/v

-RRB-

of

one

of

the

various

carbon

sources

each

supplemented

with

-LRB-

w/v

-RRB-

casamino

acids

to

stimulate

spore

germination

Six

replicates

of

each

condition

were

made

Growth

was

monitored

by

measuring

turbidity

-LRB-

-RRB-

at

intervals

Microarray

experiments

and

data

analysis

RNA

extracted

from

the

A.

niger

ΔinuR

strain

and

wild-type

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-

For

microarray

experiments

mycelia

were

isolated

from

grown

cultures

by

transfer

experiment

-LRB-

see

above

-RRB-

and

each

growth

condition

was

performed

in

duplicate

as

independent

biological

experiments

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

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

pre-scaled

chip

data

from

each

hybridization

experiment

were

normalized

using

Genespring

software

-LRB-

Silicon

Genetics

-RRB-

Normalization

with

default

parameters

in

Genespring

software

-LRB-

Per

Chip

Normalize

to

percentile

Per

Gene

Normalize

to

median

-RRB-

was

used

For

this

study

we

focused

on

sucrose

induced

genes

Therefore

pre-filtering

of

data

was

performed

to

select

for

genes

whose

detection

calls

are

present

in

both

sucrose

duplicate

samples

of

wild-type

strain

The

selected

data

set

was

further

performed

for

one-way

ANOVA

analysis

Fold

changes

in

expression

levels

between

two

different

conditions

were

then

computed

for

genes

with

P

based

on

One-way

ANOVA

analysis

and

changes

by

more

than

twofold

were

considered

significant

and

are

reported

here

Microarray

data

were

deposited

into

ArrayExpress

with

an

accession

number

at

In

silico

analysis

of

regulatory

elements

of

co-regulated

genes

The

kb

promoter

sequences

of

the

seven

sucrose

induced

InuR

dependant

genes

-LRB-

InuE

-RRB-

-LRB-

InuA

-RRB-

-LRB-

SucA

-RRB-

and

were

obtained

from

the

strain

The

kb

promoter

regions

were

analyzed

on

both

strands

using

DNA

Analyzer

module

of

Phylosopher

-LRB-

Genedata

A.G.

Basel

Switzerland

-RRB-

using

the

Gibbs

Sampling

Strategy

for

Multiple

Alignment

-LRB-

Lawrence

et

al.

-RRB-

Results

Identification

of

potential

transcriptional

regulators

involved

in

inulin

catabolism

A.

niger

is

able

to

produce

various

inulinolytic

enzymes

involved

in

the

modification

or

degradation

of

inulin

and

sucrose

The

transcriptional

regulation

of

the

genes

encoding

the

inulinolytic

enzymes

of

A.

niger

including

an

exo-inulinase

-LRB-

InuE

-RRB-

an

endo-inulinase

-LRB-

InuA

-RRB-

and

an

invertase

-LRB-

SucA

-RRB-

has

been

studied

previously

-LRB-

Moriyama

et

al.

Yuan

et

al.

-RRB-

In

addition

to

the

extracellur

enzymes

two

additional

potential

intracellular

inulinolytic

enzymes

-LRB-

SucB

and

SucC

-RRB-

were

predicted

from

the

A.

niger

genome

sequence

-LRB-

Yuan

et

al.

-RRB-

These

proteins

contain

all

the

conserved

domains

of

the

family

to

which

inulinolytic

enzymes

belong

Finally

a

potential

ORF

was

found

in

the

genome

which

showed

homology

to

the

group

of

exo-inulinases

Because

the

predicted

ORF

lacked

several

of

the

conserved

domains

and

contained

several

frame

shift

mutations

this

gene

-LRB-

inuQ

-RRB-

was

considered

to

be

a

pseudogene

-LRB-

Yuan

et

al.

-RRB-

The

expression

of

the

genes

encoding

the

extracellular

inulinolytic

enzymes

is

co-regulated

and

induced

on

inulin

and

sucrose

suggesting

that

these

genes

are

under

control

of

a

single

transcription

factor

-LRB-

Yuan

et

al.

-RRB-

Some

transcriptional

activators

involved

in

sugar

catabolism

are

clustered

in

the

genome

with

their

target

genes

-LRB-

Gomi

et

al.

Yu

et

al.

-RRB-

To

identify

possible

candidate

transcription

factors

involved

in

the

regulation

of

inulinolytic

genes

we

searched

the

A.

niger

genome

sequence

for

transcription

factors

located

close

to

the

genes

encoding

the

inulinolytic

enzymes

We

found

that

inuA

-LRB-

-RRB-

and

inuQ

-LRB-

-RRB-

are

located

next

to

each

other

and

that

also

a

Zn

-LRB-

II

-RRB-

transcription

factor

encoding

gene

-LRB-

-RRB-

was

located

adjacent

to

inuQ

The

orientation

of

the

three

ORFs

is

in

the

same

direction

indicating

that

they

do

not

share

a

common

promoter

region

-LRB-

Fig.

-RRB-

Fig.

representation

of

the

clustering

of

putative

transcription

factors

with

inulinolytic

genes

in

the

genome

of

A.

niger

and

and

inuR

encode

putative

transcription

factors

The

genes

related

to

inulin

degradation

include

inuA

-LRB-

endo-inulinase

-RRB-

inuQ

-LRB-

pseudogene

-RRB-

sucA

-LRB-

invertase

-RRB-

sucB

-LRB-

homologous

to

sucA

-RRB-

and

-LRB-

putative

sugar

transporter

-RRB-

encodes

a

hypothetical

protein

without

known

function

The

arrows

indicate

the

transcriptional

orientations

of

the

genes

In

the

proximity

of

sucA

-LRB-

-RRB-

a

second

transcription

factor

encoding

gene

was

found

This

transcription

factor

contains

a

zinc

finger

DNA

binding

motif

Between

this

gene

and

sucA

an

additional

ORF

-LRB-

-RRB-

is

present

This

amino-acid

protein

encodes

a

hypothetical

protein

without

any

conserved

motifs

A

BlastP

search

revealed

that

this

protein

has

no

close

orthologs

in

other

fungal

genomes

The

transcription

factor

encoded

by

shows

strong

sequence

similarity

to

the

S.

cerevisiae

-LRB-

e-value

-RRB-

an

essential

transcription

factor

that

contributes

to

normal

cell

proliferation

-LRB-

Gangwani

et

al.

-RRB-

Clustered

with

sucB

-LRB-

-RRB-

a

third

putative

transcription

factor

encoding

gene

-LRB-

-RRB-

was

found

which

also

belonged

to

the

Zn

-LRB-

II

-RRB-

transcription

factor

family

Between

sucB

and

an

additional

ORF

-LRB-

-RRB-

is

located

-LRB-

Fig.

-RRB-

The

protein

encoded

by

this

gene

has

all

the

characteristics

of

a

sugar

transporter

protein

In

the

direct

proximity

of

the

two

remaining

genes

encoding

inulinolytic

enzymes

-LRB-

InuE

and

SucC

-RRB-

no

potential

transcription

factors

were

found

The

three

transcription

factors

identified

via

clustering

were

considered

to

be

good

candidates

to

be

involved

in

the

transcriptional

regulation

of

the

inulinolytic

system

of

A.

niger

In

order

to

study

their

possible

role

in

inulin

utilization

deletion

strains

of

these

transcription

factors

were

constructed

Deletion

of

did

not

have

any

measurable

effect

on

growth

on

inulin

or

sucrose

in

comparison

with

the

wild-type

strain

which

indicates

that

this

gene

is

not

required

for

inulin

utilization

Growth

of

the

deletion

strain

on

xylose

glucose

fructose

starch

and

maltose

was

also

identical

to

the

growth

of

the

wild-type

strain

-LRB-

data

not

shown

-RRB-

A

deletion

strain

of

was

never

obtained

although

over

transformants

were

screened

A

possible

explanation

might

be

that

is

an

essential

gene

As

indicated

above

is

most

homologous

to

the

essential

transcription

factor

of

S.

cerevisiae

Since

the

homology

with

does

not

indicate

involvement

of

this

transcription

factor

in

inulin

utilization

no

further

attempts

were

made

to

obtain

the

deletion

strain

As

shown

in

detail

below

deletion

of

resulted

in

a

strain

which

showed

a

severe

growth

defect

on

inulin

and

on

sucrose

For

the

remainder

of

the

paper

we

will

refer

to

as

inuR

InuR

homologs

were

also

identified

in

the

genomes

of

other

Aspergilli

The

alignment

of

the

InuR

proteins

is

given

in

Fig.

The

strong

conservation

of

the

transcription

factor

among

Aspergilli

suggest

that

the

regulation

of

inulinolytic

gene

expression

is

mediated

in

the

various

Aspergilli

by

the

InuR

proteins

present

in

the

different

fungi

Fig.

of

InuRp

and

its

orthologs

in

Aspergilli

The

amino

acid

sequences

of

the

binuclear

DNA-binding

motif

-LRB-

-RRB-

and

the

Fungal

specific

transcription

factor

domain

-LRB-

-RRB-

are

underlined

The

conserved

six

cystein

residues

are

indicated

by

stars

AoInuRp

AgInuRp

AfInuRp

and

AdInuRp

are

InuR

protein

orthologs

from

A.

oryzae

A.

niger

A.

fumigatus

and

A.

nidulans

respectively

InuR

encodes

a

Zn

-LRB-

II

-RRB-

transcription

factor

that

is

required

for

growth

on

inulin

Based

on

the

analysis

of

the

genomic

sequence

the

predicted

open

reading

frame

of

the

inuR

encoding

gene

is

bp

long

and

interrupted

by

introns

with

sizes

of

and

bp

respectively

The

predicted

InuR

protein

sequence

is

comprised

of

amino

acid

residues

which

would

result

in

a

protein

with

a

calculated

molecular

mass

of

kDa

Analysis

of

the

predicted

InuR

protein

indicated

that

InuR

contains

two

conserved

domains

One

is

the

Zn

-LRB-

II

-RRB-

-LRB-

-RRB-

binuclear

cluster

which

represents

the

DNA-binding

domain

at

the

end

-LRB-

residues

-RRB-

The

domain

is

very

well

conserved

-LRB-

-RRB-

and

binds

two

Zn

atoms

which

coordinate

folding

of

the

domain

A

BlastP

search

revealed

orthologous

transcription

factors

in

the

genome

of

A.

nidulans

A.

oryzae

and

A.

fumigatus

-LRB-

see

above

Fig.

-RRB-

The

most

similar

transcription

factors

to

InuR

which

have

been

functionally

characterized

apart

from

the

proposed

orthologs

in

A.

oryzae

A.

fumigatus

and

A.

nidulans

are

the

AmyR

transcription

factors

from

the

different

Aspergillus

species

Subsequent

phylogenetic

analysis

indicated

that

the

subgroup

of

InuR

transcription

factors

is

most

closely

related

to

the

group

of

AmyR

transcription

factors

-LRB-

data

not

shown

-RRB-

Besides

the

Zn

-LRB-

II

-RRB-

motif

a

second

conserved

domain

known

as

the

middle

homology

region

-LRB-

MHR

-RRB-

or

domain

is

present

in

Zn

-LRB-

II

-RRB-

transcription

factors

The

region

is

thought

to

assist

the

Zn

-LRB-

II

-RRB-

cluster

in

DNA

target

discrimination

-LRB-

Schjerling

and

Holmberg

-RRB-

In

the

A.

niger

InuR

protein

the

domain

compromises

aa

and

the

domain

is

also

present

in

the

InuR

proteins

of

the

other

Aspergilli

Construction

and

growth

properties

of

the

inuR

disruptant

in

A.

niger

An

inuR

disruptant

mutant

was

constructed

as

described

in

the

Materials

and

methods

Putative

disruption

strains

were

identified

by

PCR

screening

and

further

verified

by

Southern

blot

analysis

Strain

was

selected

to

further

characterize

the

InuR

function

and

we

will

refer

to

this

strain

as

the

ΔinuR

strain

The

effect

of

the

disruption

of

the

inuR

gene

on

the

utilization

of

various

carbon

sources

was

analyzed

on

agar

plates

Growth

and

conidiation

of

the

ΔinuR

strain

was

strongly

reduced

on

plates

containing

inulin

in

comparison

to

the

wild-type

strain

-LRB-

Fig.

-RRB-

As

shown

growth

of

the

ΔinuR

strain

on

inulin

was

not

completely

abolished

After

days

of

growth

a

thin

colony

with

a

reduced

degree

of

conidiation

was

formed

Some

effect

on

growth

and

conidiation

was

also

observed

for

the

ΔinuR

strain

growing

on

plates

containing

sucrose

and

to

a

lesser

extent

on

raffinose

The

differences

in

growth

on

sucrose

and

raffinose

were

most

clear

after

days

of

growth

-LRB-

Fig.

-RRB-

Both

the

colony

size

and

the

degree

of

conidiation

of

the

ΔinuR

strain

and

the

wild-type

strain

after

days

of

growth

were

very

similar

-LRB-

data

not

shown

-RRB-

The

ΔinuR

strain

showed

a

similar

growth

phenotype

to

the

wild-type

on

MM

plates

containing

fructose

glucose

maltose

starch

xylose

sorbitol

or

glycerol

as

sole

carbon

source

-LRB-

Fig.

and

data

not

shown

-RRB-

Fig.

of

A.

niger

strains

on

the

utilization

of

different

carbon

sources

The

wild-type

-LRB-

-RRB-

and

inuR

disruptant

-LRB-

ΔinuR

-RRB-

amyR

disruptant

-LRB-

ΔamyR

-RRB-

and

double

mutant

-LRB-

ΔamyRΔinuR

-RRB-

strains

were

grown

on

MM

plates

containing

different

carbon

sources

at

°C

for

days

Growth

of

the

ΔinuR

strain

on

different

carbon

sources

was

also

determined

by

analyzing

growth

in

a

microtiter

plate

well

containing

MM

with

various

carbon

sources

Equal

numbers

of

spores

of

the

wild-type

strain

-LRB-

-RRB-

and

ΔinuR

strain

were

inoculated

and

plates

were

incubated

at

°C

For

each

condition

at

least

four

replicates

were

made

and

the

average

value

of

the

replicates

and

its

standard

deviation

value

were

determined

as

shown

in

Fig.

The

ΔinuR

showed

a

clear

growth

defect

on

inulin

and

sucrose

A

small

but

reproducible

growth

difference

in

growth

on

raffinose

on

agar

plates

was

also

observed

in

the

microtiter

plate

analysis

Initially

the

growth

curves

of

the

wild-type

and

the

ΔinuR

strain

on

raffinose

were

identical

but

after

h

growth

of

the

ΔinuR

strain

seem

to

be

retarded

-LRB-

Fig.

-RRB-

The

ΔinuR

strain

grew

equally

well

as

the

wild-type

strain

on

MM

with

maltodextrin

maltose

glucose

xylose

or

fructose

as

sole

carbon

source

-LRB-

Fig.

and

data

not

shown

-RRB-

In

conclusion

the

growth

analysis

of

the

ΔinuR

strain

indicates

that

the

Zn

-LRB-

II

-RRB-

transcription

factor

is

required

for

the

efficient

utilization

of

inulin

and

and

to

a

lesser

extent

raffinose

and

it

may

therefore

act

as

a

positive

transcriptional

activator

of

the

genes

encoding

the

inulinolytic

enzymes

To

further

substantiate

that

the

defective

growth

of

the

ΔinuR

strain

on

inulin

was

caused

by

the

disruption

of

the

inuR

gene

a

plasmid

containing

the

inuR

gene

with

kb

upstream

and

kb

downstream

region

was

constructed

and

co-transformed

into

the

ΔinuR

strain

with

-LRB-

Punt

et

al.

-RRB-

Hygromycin

resistant

transformants

were

analyzed

for

growth

on

inulin

and

subjected

to

Southern

blot

analysis

Transformants

that

showed

restored

growth

on

inulin

all

harbor

the

inuR

gene

while

transformants

showing

defective

growth

on

inulin

did

not

contain

the

inuR

gene

-LRB-

data

not

shown

-RRB-

The

restoration

of

the

wild-type

growth

on

inulin

upon

retransformation

of

the

ΔinuR

strain

with

a

functional

copy

of

the

inuR

gene

indicated

that

the

inability

to

grow

on

inulin

was

indeed

a

consequence

of

inuR

disruption

Fig.

plate

assay

of

growth

of

inuR

disruptant

on

different

carbon

sources

A.

niger

wild-type

-LRB-

open

circles

-RRB-

strain

and

inuR

disruptant

-LRB-

ΔinuR

solid

circles

-RRB-

were

inoculated

in

microtiter

plate

wells

and

each

well

was

inoculated

with

spores

in

μl

of

MM

containing

carbon

source

and

casamino

acids

The

growth

was

monitored

by

measuring

culture

turbidity

-LRB-

-RRB-

and

read

every

h.

Each

condition

was

performed

in

six

replicates

The

standard

deviation

value

is

indicated

InuR

is

required

for

the

induction

of

inulinolytic

genes

To

further

establish

the

role

of

InuR

as

a

positive

acting

transcriptional

regulator

for

inulinolytic

gene

expression

Northern

blot

analysis

was

performed

to

determine

the

mRNA

level

of

the

various

inulinolytic

enzyme

encoding

genes

both

in

the

ΔinuR

strain

and

the

WT

strain

We

previously

showed

that

the

expression

of

the

genes

encoding

the

exo-inulinase

-LRB-

inuE

-RRB-

endo-inulinase

-LRB-

inuA

-RRB-

invertase

-LRB-

sucA

-RRB-

was

detected

only

in

the

presence

of

inulin

and

sucrose

and

not

on

other

carbon

sources

tested

such

as

xylose

glucose

maltose

or

starch

Expression

of

the

sucB

gene

was

low

on

all

the

carbon

sources

tested

and

slightly

induced

in

the

presence

of

inulin

and

sucrose

-LRB-

Yuan

et

al.

-RRB-

In

order

to

clarify

whether

the

expression

of

these

inulinolytic

genes

was

controlled

by

InuR

both

the

wild-type

strain

and

the

ΔinuR

strain

were

pre-grown

for

h

in

MM

containing

xylose

as

a

carbon

source

After

the

pre-growth

equal

amounts

of

mycelium

was

transferred

to

fresh

MM

containing

-LRB-

w/v

-RRB-

inulin

or

-LRB-

w/v

-RRB-

sucrose

as

a

carbon

source

At

different

time

points

after

the

transfer

RNA

was

extracted

from

mycelia

and

subjected

to

Northern

blot

analysis

-LRB-

Fig.

-RRB-

The

transfer

to

inulin

and

sucrose

resulted

in

an

induction

of

the

inulinolytic

enzymes

in

the

wild-type

strain

The

induction

of

genes

is

much

faster

on

sucrose

than

on

inulin

and

is

explained

by

a

much

more

rapid

degradation

and

uptake

of

the

carbon

source

as

observed

previously

-LRB-

Yuan

et

al.

-RRB-

In

the

ΔinuR

strain

no

induction

of

expression

was

found

for

the

inuE

and

inuA

genes

after

transfer

to

inulin

and

a

very

low

level

of

sucA

mRNA

was

detected

after

the

transfer

to

inulin

The

transfer

to

sucrose

did

not

result

in

a

detectable

induction

of

any

of

the

three

inulinolytic

genes

in

the

ΔinuR

strain

A

slight

induction

of

expression

of

sucB

in

the

presence

of

inulin

and

sucrose

seems

also

dependent

on

InuR

-LRB-

Fig.

-RRB-

The

expression

of

the

gene

encoding

a

putative

sugar

transporter

in

relation

to

inulin

and

sucrose

was

also

examined

As

discussed

above

is

clustered

and

located

between

the

inuR

and

the

sucB

genes

-LRB-

Fig.

-RRB-

Expression

of

is

strongly

induced

in

response

to

the

presence

of

inulin

and

sucrose

and

dependant

on

the

InuR

transcription

factor

-LRB-

Fig.

-RRB-

No

or

very

low

expression

levels

of

were

detected

when

grown

on

maltose

glucose

fructose

or

xylose

or

after

a

transfer

to

maltose

indicating

that

expression

is

specific

for

growth

on

inulin

and

sucrose

-LRB-

data

not

shown

-RRB-

The

inuR

gene

was

constantly

expressed

at

a

relatively

low

level

on

the

various

carbon

sources

tested

-LRB-

inulin

sucrose

maltose

fructose

or

glucose

data

not

shown

-RRB-

suggesting

that

the

activation

of

the

InuR

transcription

factor

is

regulated

post-transcriptionally

Fig.

blot

analysis

of

inulinolytic

genes

in

wild-type

-LRB-

-RRB-

and

the

ΔinuR

strain

from

mycelia

transferred

from

xylose

-LRB-

preculture

-RRB-

to

inulin

or

sucrose

Total

RNA

was

isolated

from

mycelia

growing

in

inulin

or

sucrose

at

different

time

points

-LRB-

and

h

-RRB-

as

indicated

after

the

transfer

Genome

wide

transcriptional

profiling

of

the

InuR

regulon

in

A.

niger

The

experiments

described

above

suggest

that

InuR

is

a

transcriptional

activator

of

inulinolytic

genes

and

required

for

the

efficient

utilization

of

inulin

and

sucrose

In

addition

to

regulating

the

inulinolytic

genes

it

is

possible

that

InuR

also

regulates

other

genes

in

the

genome

through

direct

or

indirect

processes

in

the

pathway

of

inulin

or

sucrose

utilization

To

identify

additional

genes

with

a

possible

role

in

inulin

or

sucrose

metabolism

we

performed

genome

wide

expression

analysis

using

A.

niger

Affymetrix

GeneChip

arrays

The

A.

niger

wild-type

strain

and

the

ΔinuR

strain

were

pre-grown

for

h

in

-LRB-

w/v

-RRB-

xylose

and

transferred

to

fresh

medium

containing

-LRB-

w/v

-RRB-

sucrose

as

carbon

source

and

grown

for

h

on

sucrose

As

a

control

the

mycelium

from

the

strain

was

also

transferred

to

fresh

-LRB-

w/v

-RRB-

xylose

medium

For

each

culture

condition

a

duplicate

experiment

was

carried

out

RNA

was

isolated

from

each

of

the

samples

and

subjected

to

microarray

analysis

-LRB-

see

Materials

and

methods

-RRB-

To

identify

genes

that

were

induced

by

sucrose

we

first

compared

the

expression

profile

of

A.

niger

wild-type

strain

grown

in

sucrose

to

that

of

A.

niger

wild-type

strain

grown

in

xylose

h

after

transfer

The

expression

of

genes

was

more

than

twofold

increased

by

sucrose

-LRB-

One-way

ANOVA

analysis

P

-RRB-

-LRB-

Supplementary

Table

-RRB-

To

identify

genes

that

were

induced

on

sucrose

in

an

InuR

dependant

way

the

expression

profile

of

the

A.

niger

wild-type

strain

grown

in

sucrose

was

compared

to

that

of

the

ΔinuR

strain

after

the

h

transfer

to

sucrose

A

total

of

genes

were

identified

where

expression

was

significantly

increased

on

sucrose

in

the

strain

but

not

in

the

ΔinuR

strain

-LRB-

One-way

ANOVA

analysis

P

and

change

-RRB-

-LRB-

Supplementary

Table

-RRB-

Finally

by

combining

these

data

sets

genes

were

identified

for

which

expression

was

induced

by

sucrose

in

an

InuR

dependent

manner

-LRB-

Table

-RRB-

Table

of

sucrose

induced

and

InuR

dependent

genes

in

A.

niger

at

h

after

transfer

from

precultureORF

sucrose

xylose

amyR

sucrose

Fold

inductionP

valueFunctionSucrose

versus

vs

ΔinuRCarbohydrate

transport

and

metabolism

aP

A

Invertase

P

A

A

Putative

fructose

facilitator

P

P

M

P

M

P

A

P

A

P

Putative

monosaccharide

transporter

P

P

P

Putative

monosaccharide

transporter

Amino

acid

transport

and

metabolism

P

P

P

Putative

asparagine

synthase

P

p

P

Putative

neutral

amino

acid

permease

Lipid

transport

and

metabolism

P

P

P

A

Putative

ethanolamine

kinase

P

P

A

A

Putative

glycerophosphoinositol

transporter

Phosphate

transport

and

metabolism

P

P

A

Putative

phosphate-repressible

phosphate

permease

Nucleotide

transport

and

metabolism

P

P

P

Putative

phosphoribosylformylglycinamidine

synthase

Other

transport

mechanism

P

P

P

A

Putative

multidrug

transporter

P

P

P

Putative

ABC

multidrug

transporter

P

P

P

Putative

ABC

transporterTranscription

P

A

M

A

Putative

DNA-directed

RNA

polymerase

P

A

P

A

Putative

Ran-GTPase-activating

proteinProtein

synthesis

P

P

P

Putative

RNA-binding

protein

P

P

P

Putative

translation

initiation

factor

P

P

P

Putative

ribosome

biogenesis

protein

P

P

P

Putative

asparaginyl-tRNA

synthetaseProtein

fate

P

P

P

Putative

importin

unit

P

P

P

A

Unknown

protein

P

P

P

Unknown

protein

P

A

P

Unknown

protein

P

P

P

Unknown

proteinaThe

expression

level

was

based

on

the

geometric

mean

value

of

the

duplicate

samples

and

the

deviation

values

between

the

duplicate

samples

are

indicated

P

M

or

A

representing

detection

calls

for

present

marginal

or

absent

respectively

The

P-value

was

based

on

One-way

ANOVA

analysis

Genes

were

divided

over

different

functional

groups

according

to

FunCat

-LRB-

Ruepp

et

al.

-RRB-

The

predicted

protein

sequences

of

these

genes

were

assigned

to

various

functional

categories

-LRB-

Ruepp

et

al.

Mewes

et

al.

-RRB-

and

shown

in

Table

Interestingly

genes

with

the

highest

fold

changes

-LRB-

change

-RRB-

almost

all

fall

into

the

category

of

carbohydrate

transport

and

metabolism

Among

them

are

the

genes

encoding

the

extracellular

inulinolytic

enzymes

sucA

inuE

and

inuA

as

well

as

the

above

mentioned

putative

sugar

transporter

gene

-LRB-

-RRB-

supporting

the

previous

Northern

analysis

An

additional

gene

that

showed

a

very

high

fold-change

and

belongs

to

the

category

of

carbohydrate

transport

and

metabolism

is

encodes

a

putative

sugar

transporter

which

is

over

induced

by

sucrose

in

a

wild-type

strain

when

compared

to

xylose

and

the

induction

was

greatly

reduced

by

when

inuR

gene

was

deleted

BlastP

search

revealed

that

the

transporter

displayed

the

highest

identity

to

a

fructose

transporter

protein

-LRB-

-RRB-

from

Zygosaccharomyces

bailii

-LRB-

Pina

et

al.

-RRB-

An

additional

putative

sugar

transporter

gene

-LRB-

-RRB-

was

induced

on

sucrose

in

an

InuR

dependant

way

In

contrast

to

the

other

two

transporters

-LRB-

and

-RRB-

this

transporter

is

relatively

highly

expressed

on

both

xylose

and

in

the

ΔinuR

mutant

resulting

in

relative

small

fold-changes

An

additional

gene

-LRB-

-RRB-

was

strongly

induced

on

sucrose

-LRB-

about

-RRB-

in

an

InuR

dependant

way

The

protein

encoded

by

is

annotated

as

a

multidrug

resistance

-LRB-

MDR

-RRB-

protein

because

of

its

highest

identity

to

other

MDR

proteins

of

the

Major

Facilitator

Superfamily

of

transporters

Since

the

substrates

of

MDR

proteins

are

hard

to

predict

on

the

basis

of

the

amino

acid

sequence

the

annotation

should

be

interpreted

with

care

and

does

not

exclude

the

possibility

that

this

protein

has

a

role

in

sugar

transport

Apart

from

the

transporter

which

might

function

as

a

fructose

transporter

based

on

the

strong

homology

towards

-LRB-

Pina

et

al.

-RRB-

a

useful

prediction

of

substrates

of

the

other

putative

transporters

can

not

be

made

In

the

list

of

induced

genes

-LRB-

Table

-RRB-

a

considerable

number

are

related

to

transcription

translation

and

amino

acid

metabolism

The

increase

in

expression

of

those

genes

might

indicate

an

overall

increase

of

metabolic

activity

on

sucrose

compared

to

xylose

and

a

subsequent

higher

expression

of

the

protein

transcription/translation

machinery

The

genome

wide

analysis

indicated

that

three

extracellular

inulinolytic

genes

and

four

putative

sugar

transporter

genes

were

strongly

induced

on

sucrose

in

an

InuR

dependant

way

To

identify

possible

elements

in

the

promoter

regions

of

these

genes

that

could

mediate

InuR

binding

an

in

silico

analysis

of

upstream

promoter

sequences

of

these

seven

genes

was

performed

As

shown

in

Fig.

optimal

alignment

of

the

promoter

sequences

revealed

a

consensus

sequence

-LRB-

-RRB-

which

is

present

at

least

once

in

the

promoter

region

of

all

genes

At

least

one

of

the

triplets

consistently

is

preceded

by

a

T

resulting

in

a

conserved

TCGG

motif

in

every

proposed

element

Within

the

second

triplet

the

conservation

of

the

GG

is

consistent

but

conservation

of

the

C

is

not

This

putative

binding

site

of

the

InuR

protein

-LRB-

-RRB-

is

identical

to

the

binding

site

of

the

AmyR

transcription

factor

which

binds

and

induces

the

expression

of

amylolytic

genes

-LRB-

Petersen

et

al.

-RRB-

To

examine

whether

AmyR

is

also

involved

in

the

regulation

of

inulinolytic

genes

the

ΔamyR

mutant

was

constructed

and

analysed

for

growth

on

various

carbon

sources

As

shown

in

Fig.

deletion

of

the

amyR

transcription

factor

did

not

result

in

a

growth

defect

on

inulin

sucrose

or

other

carbon

sources

The

ΔamyR

strain

did

show

a

strong

growth

reduction

on

maltose

-LRB-

data

not

shown

-RRB-

To

examine

a

possible

overlapping

role

of

the

InuR

and

AmyR

transcription

factors

for

inulin

and/or

sucrose

utilization

the

ΔamyR/ΔinuR

double

mutant

was

constructed

Growth

analysis

of

the

double

mutant

on

inulin

showed

a

minor

further

reduction

in

growth

and

sporulation

on

inulin

and

sucrose

-LRB-

Fig.

-RRB-

indicating

AmyR

and

InuR

have

independent

functions

Fig.

silico

prediction

of

putative

InuR

binding

sites

in

the

promoter

of

sucrose

induced

genes

Conserved

nucleotides

are

coloured

in

gray

Numbers

beside

the

sequences

indicate

the

position

relative

to

the

ATG

of

the

downstream

located

gene

Discussion

We

previously

showed

that

the

genes

encoding

extracellular

inulinolytic

enzymes

in

A.

niger

are

co-regulated

at

the

transcription

level

and

identified

sucrose

but

not

fructose

or

glucose

as

an

efficient

inducer

of

inulinolytic

genes

It

was

proposed

that

there

might

be

a

common

transcription

factor

to

activate

the

expression

of

inulinolytic

genes

in

response

to

the

presence

of

inducing

carbon

sources

-LRB-

Yuan

et

al.

-RRB-

similar

to

that

which

has

been

found

in

other

carbohydrate

degrading

enzymes

networks

such

as

the

amylolytic

genes

-LRB-

AmyR

-RRB-

or

the

xylanolytic

genes

-LRB-

XlnR

-RRB-

In

this

study

we

reported

the

successful

identification

of

an

inulinolytic

transcriptional

activator

InuR

Previous

studies

to

isolate

the

AmyR

and

XlnR

transcription

factors

were

based

on

genetic

screens

to

identify

mutants

with

impaired

expression

of

amylolytic

and

xylanolytic

enzymes

-LRB-

Petersen

et

al.

Gomi

et

al.

van

Peij

et

al.

-RRB-

Here

we

used

a

different

approach

which

made

use

of

the

full

genome

sequence

of

A.

niger

The

approach

is

based

on

the

observation

that

in

some

cases

e.g.

in

the

case

of

the

AmyR

transcription

factor

the

target

genes

of

the

transcription

factor

are

adjacent

to

the

transcription

factor

itself

In

the

case

of

the

inulinolytic

genes

three

possible

inulinolytic

gene

clusters

were

identified

each

of

which

contains

a

gene

encoding

a

putative

transcription

factor

-LRB-

Fig.

-RRB-

Disruption

mutants

were

successfully

obtained

for

two

of

the

three

transcription

factors

and

subsequent

phenotypic

analysis

indicated

that

only

InuR

-LRB-

-RRB-

was

involved

in

the

inulin

utilization

pathway

The

ΔinuR

mutant

showed

severe

reduced

growth

on

agar

plates

and

in

submerged

cultures

containing

sucrose

or

inulin

as

sole

carbon

source

Furthermore

we

showed

that

the

induction

of

the

inulinolytic

genes

by

inulin

and

sucrose

is

dependant

on

the

presence

of

InuR

In

addition

to

the

transcription

factor

itself

the

inulinolytic

gene

cluster

identified

in

A.

niger

consists

of

a

sugar

transporter

and

sucB

which

encodes

an

intracellular

invertase

with

transfructosylating

activity

-LRB-

Goosen

et

al.

-RRB-

To

examine

whether

the

position

and

clustering

of

the

InuR

orthologs

was

conserved

among

aspergilli

we

compared

the

genes

adjacent

to

the

InuR

homologous

transcription

factors

in

A.

nidulans

A.

oryzae

and

A.

fumigatus

The

clustering

of

the

three

genes

inuR

-LRB-

-RRB-

-LRB-

putative

sugar

transporter

-RRB-

and

sucB

-LRB-

-RRB-

in

A.

niger

was

conserved

in

A.

nidulans

-LRB-

e-value

e-value

and

e-value

-RRB-

and

A.

oryzae

-LRB-

e-value

e-value

and

e-value

respectively

-RRB-

In

A.

fumigatus

the

InuR

orthologue

-LRB-

e-value

-RRB-

is

located

next

to

the

SucB

orthologue

-LRB-

e-value

-RRB-

but

no

homolog

to

the

putative

sugar

transporter

-LRB-

-RRB-

was

present

in

the

gene

cluster

in

A.

fumigatus

The

initial

gene

model

of

the

inuR

gene

in

the

A.

niger

annotation

was

lacking

the

ZnII

-LRB-

-RRB-

binuclear

cluster

domain

Repositioning

the

intron/exon

positions

in

N-terminal

region

resulted

in

the

prediction

of

a

amino

acid

long

protein

containing

a

complete

Zn

-LRB-

-RRB-

binuclear

cluster

domain

BlastP

searches

with

the

A.

niger

InuR

protein

revealed

orthologous

genes

in

the

three

Aspergillus

species

examined

-LRB-

A.

nidulans

A.

oryzae

and

A.

fumigatus

-RRB-

The

closest

homologs

in

A.

oryzae

and

A.

nidulans

did

not

contain

a

Zn

-LRB-

II

-RRB-

domain

and

the

A.

fumigatus

homolog

only

a

partial

Zn

-LRB-

II

-RRB-

cluster

The

gene

models

of

the

other

Aspergilli

were

manually

inspected

and

by

improving

the

gene

model

a

Zn

-LRB-

II

-RRB-

domain

was

found

to

be

present

in

the

proteins

Interestingly

it

turned

out

that

the

gene

model

of

A.

niger

was

completely

conserved

in

the

other

Aspergili

Thus

the

gene

model

of

the

A.

nigerinuR

gene

could

be

used

as

a

template

for

the

prediction

of

the

inuR

gene

models

in

the

other

Aspergilli

Both

the

numbers

of

introns

as

well

as

the

intron/exon

positions

were

conserved

among

the

four

Aspergilli

This

result

implies

that

gene

model

prediction

in

Aspergilli

can

be

improved

by

comparison

of

the

gene

models

of

orthologous

genes

The

inuR

gene

is

situated

in

the

genome

next

to

a

putative

sugar

transporter

gene

-LRB-

-RRB-

and

sucB

We

recently

showed

that

the

sucB

gene

encodes

an

intracellular

invertase

with

transglycosylating

activity

-LRB-

Goosen

et

al.

-RRB-

As

depicted

in

Fig.

the

putative

sugar

transporter

-LRB-

-RRB-

in

the

cluster

shares

the

same

promoter

region

with

the

sucB

gene

The

Northern

analysis

and

microarray

analysis

showed

that

the

two

genes

are

not

coregulated

indicating

that

the

common

promoter

region

does

not

act

bidirectionally

as

has

been

observed

e.g.

in

the

case

of

the

niiA

and

niaD

genes

-LRB-

Punt

et

al.

-RRB-

Expression

of

the

gene

was

highly

induced

by

sucrose

or

inulin

and

the

induction

was

dependent

on

InuR

-LRB-

Fig.

-RRB-

while

the

sucB

gene

was

lowly

expressed

on

various

carbon

sources

and

hardly

induced

on

sucrose

and

inulin

The

function

of

remains

to

be

elucidated

but

it

is

tempting

to

suggest

that

it

involved

in

the

transport

of

sucrose

or

its

hydrolysis

products

fructose

or

glucose

The

function

of

SucB

in

relation

to

inulin

or

sucrose

metabolism

is

also

currently

unknown

We

showed

that

deletion

of

the

sucB

did

not

result

in

a

phenotype

related

to

growth

on

inulin

or

sucrose

-LRB-

Goosen

et

al.

-RRB-

The

constitutive

low

level

of

expression

of

the

sucB

gene

might

suggest

that

SucB

acts

as

a

scouting

enzyme

to

sense

the

presence

of

a

useful

carbon

source

by

generating

an

inducer

molecule

which

can

further

activate

transcription

of

additional

inulinolytic

genes

A

possible

explanation

for

the

lack

of

phenotype

of

the

sucB

deletion

mutant

is

the

presence

of

an

addition

intracellular

invertase

encoded

by

the

sucC

gene

Both

the

enzymatic

activity

of

the

SucC

protein

as

well

as

the

effect

of

deletion

of

sucC

and

simultaneously

deletion

of

sucB

and

sucC

will

be

the

subject

of

future

research

The

genome

wide

transcription

analysis

using

Affymetrix

microarrays

identified

a

limited

number

of

genes

that

were

strongly

induced

by

sucrose

in

an

InuR

dependent

way

These

genes

encode

the

extracellular

inulinolytic

enzymes

and

four

genes

encoding

putative

transporters

The

specificity

of

the

sugar

transporters

should

be

determined

biochemically

as

has

been

done

previously

for

A.

niger

sugar

transporters

-LRB-

vanKuyk

et

al.

-RRB-

since

phylogenetic

comparison

to

suggest

possible

substrates

is

not

reliable

-LRB-

Patricia

vanKuyk

personal

communication

-RRB-

The

limited

number

of

genes

that

are

induced

by

sucrose

in

an

InuR

dependant

way

suggests

a

relatively

simple

regulatory

mechanism

regarding

sucrose

catabolism

in

A.

niger

Unpublished

additional

microarray

data

showed

that

the

same

three

extracellular

enzymes

were

induced

on

inulin

in

an

inuR

dependant

way

The

expression

of

the

putative

sugar

transporters

on

inulin

differed

from

the

pattern

observed

on

sucrose

Two

of

the

four

transporters

-LRB-

and

-RRB-

were

not

induced

on

inulin

after

and

h

of

growth

on

inulin

The

transporter

encoded

by

the

gene

was

four

to

fivefold

induced

on

inulin

compared

to

xylose

after

h

of

the

transfer

Expression

of

this

sugar

transporter

is

also

high

on

xylose

-LRB-

Table

-RRB-

and

maltose

Yuan

et

al.

unpublished

results

-RRB-

indicating

that

the

expression

of

this

sugar

transporter

is

not

very

tightly

regulated

and

suggest

a

broad

specificity

of

the

sugar

transporter

The

fourth

transporter

-LRB-

-RRB-

was

more

highly

expressed

on

inulin

h

after

the

transfer

than

on

maltose

and

xylose

-LRB-

four

to

fivefold

induction

-RRB-

and

the

induction

was

InuR

dependant

The

difference

between

the

observed

expression

patterns

of

the

extracellular

enzymes

and

the

putative

sugar

transporters

suggest

that

under

the

different

growth

conditions

the

fungus

expresses

the

same

set

of

extracellular

enzymes

but

that

the

fungus

fine-tunes

the

expression

of

the

sugar

transporters

in

order

to

allow

efficient

uptake

Such

a

fine-tuning

mechanism

could

explain

why

such

a

large

number

of

sugar

transporters

is

present

in

the

genome

of

A.

niger

as

proteins

have

been

identified

that

belong

to

the

Major

Facilititator

Superfamily

of

transporters

-LRB-

Pel

et

al.

-RRB-

Such

a

mechanism

allows

the

fungus

to

express

the

sugar

transporters

with

the

optimal

properties

in

relation

to

substrate

diversity

substrate

concentration

environmental

pH

and

other

environmental

factors

and

nutrient

conditions

The

InuR

transcription

factor

is

a

member

of

the

Zn

-LRB-

II

-RRB-

DNA

transcription

factor

family

which

is

a

fungal

specific

type

of

transcription

factor

In

Aspergilli

this

family

of

transcription

factors

has

expanded

significantly

when

genome

data

is

compared

to

that

of

the

yeast

S.

cerevisiae

or

Neuropora

crassa

In

S.

cerevisiae

and

N.

crassa

and

genes

respectively

-LRB-

Akache

et

al.

Borkovich

et

al.

-RRB-

have

been

identified

that

contain

the

Zn

-LRB-

II

-RRB-

DNA

binding

domain

the

genomes

of

Aspergilli

contain

-LRB-

A.

nidulans

-RRB-

-LRB-

A.

fumigatus

-RRB-

or

-LRB-

A.

niger

-RRB-

Zn

-LRB-

II

-RRB-

DNA

transcription

factors

The

actual

number

of

proteins

containing

a

Zn

-LRB-

II

-RRB-

DNA

binding

domain

might

still

increase

in

Aspergilli

and

other

fungi

We

showed

that

the

predicted

gene

model

for

the

inuR

gene

in

several

Aspergilli

was

incorrect

resulting

in

a

protein

lacking

the

Zn

-LRB-

II

-RRB-

DNA

binding

domain

Closer

examination

of

the

A.

nidulans

and

A.

niger

genome

annotations

revealed

that

the

difficulty

of

the

automatic

annotation

in

predicting

the

correct

gene

model

for

this

class

of

transcription

factors

is

not

limited

to

inuR

gene

but

is

a

more

general

problem

Manual

improvement

of

the

gene

models

identified

Zn

-LRB-

II

-RRB-

DNA

binding

domain

containing

proteins

in

A.

nidulans

and

Zn

-LRB-

II

-RRB-

DNA

binding

domain

containing

proteins

in

A.

niger

-LRB-

Ram

and

Punt

unpublished

data

-RRB-

Thus

it

is

likely

that

other

fungal

genomes

contain

more

as

yet

unidentified

Zn

-LRB-

II

-RRB-

DNA

binding

domain

containing

proteins

which

could

be

revealed

with

improved

gene

models

or

manual

annotation

Transcription

factors

containing

the

Zn

-LRB-

II

-RRB-

DNA

binding

domain

usually

bind

the

promoter

region

of

their

target

genes

via

a

conserved

DNA

binding

site

Often

but

not

always

the

binding

site

consists

of

conserved

terminal

trinucleotides

usually

in

symmetrical

configuration

-LRB-

direct

repeat

everted

repeat

or

an

inverted

repeat

-RRB-

and

spaced

by

an

internal

variable

sequence

of

a

defined

length

Examples

are

the

and

transcription

factors

that

bind

to

and

UaY

that

bind

to

and

that

binds

to

-LRB-

Todd

and

Andrianopoulos

-RRB-

The

binding

sites

for

the

AmyR

transcription

factor

have

been

studied

in

detail

Several

promoter

regions

of

amylolytic

genes

contain

a

-LRB-

A/C

-RRB-

GG

site

in

their

promoter

region

to

which

AmyR

can

bind

and

activate

transcription

-LRB-

Tani

et

al.

Ito

et

al.

-RRB-

Comparison

of

the

upstream

promoters

of

the

strongly

InuR

dependent

sucrose

induced

genes

revealed

that

a

common

consensus

sequence

-LRB-

-RRB-

is

present

in

the

promoters

of

all

genes

Strikingly

in

every

putative

element

at

least

one

repeat

consists

of

a

TCGG

sequence

-LRB-

Fig.

-RRB-

This

observation

might

be

explained

by

a

speculative

but

testable

model

of

InuR

binding

and

activation

We

propose

that

InuR

will

bind

as

a

dimer

to

the

motif

and

that

the

first

InuR

molecule

will

bind

with

high

affinity

to

the

TCGG

DNA

sequence

This

binding

may

recruit

a

second

InuR

molecule

to

the

site

that

will

bind

to

the

second

motif

possibly

with

a

weaker

affinity

for

the

DNA

because

of

the

sequence

variation

Such

a

model

for

cooperative

binding

of

a

dimeric

transcription

factor

has

been

experimentally

substantiated

for

the

AmyR

transcription

factor

-LRB-

Ito

et

al.

-RRB-

The

proposed

InuR

binding

site

-LRB-

-RRB-

might

be

conserved

among

filamentous

fungi

as

the

shared

promoter

of

the

inuC

and

inuD

genes

in

Penicillium

sp.

strain

contains

a

similar

site

-LRB-

Moriyama

et

al.

-RRB-

The

proposed

InuR

binding

site

is

very

similar

to

the

AmyR

binding

site

which

is

present

in

the

promoter

of

many

amylolytic

genes

in

A.

niger

-LRB-

Yuan

et

al.

unpublished

results

-RRB-

and

raises

immediately

the

question

of

how

the

specificity

of

the

InuR

and

AmyR

transcription

factors

is

accomplished

Comparison

of

the

Zn

-LRB-

II

-RRB-

domain

of

several

Zn

-LRB-

II

-RRB-

transcription

factors

revealed

that

the

Zn

-LRB-

II

-RRB-

domain

of

InuR

and

AmyR

is

not

more

related

to

each

other

than

they

are

related

to

unrelated

Zn

-LRB-

II

-RRB-

transcription

factors

-LRB-

XlnR

and

FacB

-RRB-

Interestingly

the

MHD

of

InuR

and

AmyR

showed

a

high

degree

of

identity

and

suggest

that

the

MHD

domain

may

assist

in

binding

to

the

DNA

target

sequence

After

establishing

that

the

conserved

motif

in

the

promoter

of

inulinolytic

genes

is

involved

in

the

binding

and

activation

of

expression

of

the

target

genes

the

AmyR

and

the

InuR

transcription

factors

are

interesting

subjects

for

further

research

aimed

to

understand

how

Zn

-LRB-

II

-RRB-

transcription

factors

recognize

their

target

genes

and

what

determines

the

specificity

of

the

binding

Analysis

of

the

ΔamyRΔinuR

double

mutant

indicated

that

both

transcription

factors

have

independent

functions

The

slightly

decreased

growth

and

sporulation

of

the

ΔamyRΔinuR

double

mutant

on

sucrose

compared

to

growth

of

the

ΔinuR

mutant

on

sucrose

might

be

explained

by

the

activity

of

α-glucosidases

that

are

under

control

of

the

AmyR

transcription

factor

It

is

well

established

that

sucrose

can

be

hydrolyzed

either

by

β-fructosidase

that

splits

sucrose

by

attacking

the

terminal

non-reducing

α1

β2

linked

fructose

moiety

but

also

by

an

α-glucosidase

catalyzing

the

hydrolysis

of

sucrose

at

the

terminal

non-reducing

α1

linked

glucose

residue

-LRB-

Geber

et

al.

Kelly

and

Kwon-Chung

Alberto

et

al.

-RRB-

A.

niger

contains

several

α-glucosidase

encoding

gene

of

which

two

of

them

are

under

control

of

the

AmyR

transcription

factor

Disruption

of

the

amyR

transcription

factor

further

decreases

the

level

of

agdA

and

agdB

transcripts

-LRB-

Yuan

et

al.

unpublished

results

-RRB-

which

might

result

in

lower

α-glucosidase

activity

and

a

subsequent

decrease

in

the

efficiency

by

which

sucrose

can

be

hydrolyzed

In

conclusion

we

have

identified

InuR

as

the

transcriptional

activator

required

for

utilization

of

inulin

and

sucrose

The

results

in

this

paper

suggest

that

A.

niger

metabolizes

inulin

and

sucrose

by

inducing

the

expression

of

a

limited

number

of

extracellular

enzymes

and

sugar

transporters

that

are

under

transcriptional

control

of

a

single

transcription

factor

InuR

The

mechanism

by

which

the

InuR

transcription

factor

is

activated

in

response

to

the

presence

of

inulin

and

sucrose

and

the

mechanism

that

determines

the

specificity

of

the

binding

to

the

InuR

target

genes

are

the

subjects

of

future

research

Electronic

supplementary

material

Below

is

the

link

to

the

electronic

supplementary

material

Supplementary

Tables

-LRB-

DOC

kb

-RRB-

sucrose
affymetrix genechip arrays
transcription factor
invertase


Microtitre plate-based antibacterial assay incorporating resazurin as an indicator of cell growth , and its application in the in vitro antibacterial screening of phytochemicals The resazurin assay utilising microtitre-plate , described by Drummond and Waigh in 2000 , has been modified to achieve more accuracy in the determination of the minimum inhibitory concentration -LRB- MIC -RRB- values of natural products , including crude extracts , chromatographic fractions or purified compounds against various bacterial strains . This modified resazurin method is simple , sensitive , rapid , robust and reliable , and could be used successfully to assess antibacterial properties of natural products . 1 Introduction Antibiotics have revolutionised mankind 's health status , allowing treatment of life threatening infections . However with the increasing occurrence of bacterial resistance against available antibiotics , it has now become essential to look for newer antibiotics . Most of the antibiotics available today come from natural origin , especially from various microbial or marine sources . Plants also produce compounds to protect themselves from microbial attacks . For screening natural products , e.g. crude extracts , chromatographic fractions or purified compounds for antibacterial activities , it is essential to employ an in vitro antibacterial assay that is simple , rapid , efficient , reliable , sensitive , safe and cost-effective . Moreover , most often the small quantities of natural products , especially purified compounds , that are available for antibacterial screening , can be a limiting factor in any viable screening programme . The conventional methods , e.g. disc diffusion method , may be time consuming and require significant quantities of the test materials , and there are also a few other problems associated with this method as discussed by Drummond and Waigh -LSB- 3 -RSB- . At the beginning of our research project , aimed at screening Scottish plants for antibacterial activities -LSB- 1,2 -RSB- , the resazurin assay utilising a microtitre-plate as described by Drummond and Waigh in 2000 -LSB- 3 -RSB- seemed useful . This was because this method uses an indicator , resazurin , which allows the detection of microbial growth in extremely small volumes of solution in microtitre plates without the use of a spectrophotometer . However , it was soon realised that this published method incorporated changes in the concentration of both the test material as well as the bacterial suspension . Thus , although there is a serial dilution of the test materials , this method results in a decrease in the bacterial concentration serially , and as a consequence can not be a ` true ' indicator of the minimum inhibitory concentration -LRB- MIC -RRB- which is supposed to be assessed in this assay . Moreover , in a number of other antibacterial assays , the bacterial concentration is only approximate as they are compared to the Macfarland standard . This approach is subjective and results are often not reproducible in two different laboratories . To overcome these drawbacks , we have modified the resazurin assay , especially the dilution protocols , and utilised a standard concentration of bacterial suspension so that a ` true ' MIC value can be obtained . By challenging the test materials with a standardised inoculum , reproducible and meaningful results can be obtained . We now present a detailed description of the modified resazurin method , which is simple , sensitive , rapid , robust and reliable , and could be used successfully to assess antibacterial properties of natural products . 2 Method 2.1 General Incubator at 35 and 37 °C ; pipettes of various sizes -LRB- Gilson -RRB- ; sterile tips , 100 , 200 , 500 , and 1000 μL ; 5 mL multichannel pipette -LRB- Fischer Supplies -RRB- ; centrifuge tubes -LRB- Fischer Supplies -RRB- ; vortex mixer -LRB- Fischer Supplies -RRB- ; centrifuge -LRB- Fisons -RRB- ; petridishes -LRB- Fischer Supplies -RRB- ; sterile universal bottles -LRB- Fischer Supplies -RRB- ; UV spectrophotometer -LRB- Shimadzu -RRB- ; sterile resazurin tablets -LRB- BDH Laboratory Supplies -RRB- ; sterile normal saline ; sterile isosensitest broth -LRB- Oxoid -RRB- ; sterile isosensitest agar -LRB- Southern Group Laboratory , SGL -RRB- ; antibiotic solutions -LRB- Sigma -- Aldrich -RRB- ; sterile solution of 10 % -LRB- v/v -RRB- DMSO in water -LRB- Sigma -- Aldrich -RRB- . 2.2 Medium Isosensitest medium was used throughout this assay , as it is pH buffered . Although NCCLS recommends the use of Mueller Hinton medium for susceptibility testing -LSB- 4 -RSB- , the isosensitest medium had comparable results for most of the tested bacterial strains -LSB- 5 -RSB- . 2.3 Use of standardised bacterial colony numbers The method wherein turbidity is compared to Macfarland standards usually 0.5 is not able to give a standardised number of CFU for all strains , because this is operator driven and is thus subjective . It also makes it difficult to compare different bacterial species as they have differing optical densities . Thus to ensure that a uniform number of bacteria were always used , a set of graphs of killing/viability curves for each strain of bacterial species was prepared . A final concentration of 5 × 105 cfu/mL was adopted for this assay . Thus different strains and different bacterial species could be compared . 2.4 Preparation of bacterial culture Using aseptic techniques a single colony was transferred into a 100 mL bottle of isosensitest broth , capped and placed in incubator overnight at 35 °C . After 12 -- 18 h of incubation , using aseptic preparation and the aid of a centrifuge , a clean sample of bacteria was prepared . The broth was spun down using a centrifuge set at 4000 rpm for 5 min with appropriate aseptic precautions . The supernatant was discarded into an appropriately labelled contaminated waste beaker . The pellet was resuspended using 20 mL of sterile normal saline and centrifuged again at 4000 rpm for 5 min . This step was repeated until the supernatant was clear . The pellet was then suspended in 20 mL of sterile normal saline , and was labelled as Bs . The optical density of the Bs was recorded at 500 nm , and serial dilutions were carried out with appropriate aseptic techniques until the optical density was in the range of 0.5 -- 1.0 . The actual number of colony-forming units was calculated from the viability graph . The dilution factor needed was calculated and the dilution was carried out to obtain a concentration of 5 × 106 cfu/mL . 2.5 Preparation of resazurin solution The resazurin solution was prepared by dissolving a 270 mg tablet in 40 mL of sterile distilled water . A vortex mixer was used to ensure that it was a well-dissolved and homogenous solution . 2.6 Preparation of the plates Plates were prepared under aseptic conditions . A sterile 96 well plate was labelled -LRB- Fig. 1 -RRB- . A volume of 100 μL of test material in 10 % -LRB- v/v -RRB- DMSO or sterile water -LRB- usually a stock concentration of 1 mg/mL for purified compounds , and 10 mg/mL for crude extracts -RRB- was pipetted into the first row of the plate . To all other wells 50 μL of nutrient broth or normal saline was added . Serial dilutions were performed using a multichannel pipette . Tips were discarded after use such that each well had 50 μL of the test material in serially descending concentrations . To each well 10 μL of resazurin indicator solution was added . Using a pipette 30 μL of 3.3 × strength isosensitised broth was added to each well to ensure that the final volume was single strength of the nutrient broth . Finally , 10 μL of bacterial suspension -LRB- 5 × 106 cfu/mL -RRB- was added to each well to achieve a concentration of 5 × 105 cfu/mL . Each plate was wrapped loosely with cling film to ensure that bacteria did not become dehydrated . Each plate had a set of controls : a column with a broad-spectrum antibiotic as positive control -LRB- usually ciprofloxacin in serial dilution -RRB- , a column with all solutions with the exception of the test compound , and a column with all solutions with the exception of the bacterial solution adding 10 μL of nutrient broth instead -LRB- see Fig. 2 -RRB- . The plates were prepared in triplicate , and placed in an incubator set at 37 °C for 18 -- 24 h . The colour change was then assessed visually . Any colour changes from purple to pink or colourless were recorded as positive -LRB- Fig. 1 -RRB- . The lowest concentration at which colour change occurred was taken as the MIC value . The average of three values was calculated and that was the MIC for the test material and bacterial strain . 3 Results Resazurin is an oxidation -- reduction indicator used for the evaluation of cell growth , particularly in various cytotoxicity assays -LSB- 6 -RSB- . It is a blue non-fluorescent and non-toxic dye that becomes pink and fluorescent when reduced to resorufin by oxidoreductases within viable cells . Resorufin is further reduced to hydroresorufin -LRB- uncoloured and nonfluorescent -RRB- . A resazurin reduction test has also been used for decades to demonstrate bacterial and yeast contamination of milk -LSB- 6,7 -RSB- . The effectiveness of this modified resazurin assay has been demonstrated with methanol extracts of selected Scottish plants , purified compounds -LRB- Fig. 1 -RRB- , and the positive control ciprofloxacin -LRB- Table 1 -RRB- , and a direct comparison of the MIC determination of the antibiotics using the old and modified resazurin methods -LRB- Table 2 -RRB- using the antibiotics norfloxacin , cefotaxime , and amoxicillin . 4 Concluding remarks Choosing the correct assay to assess the antimicrobial potential of extracts and compounds is important for generating high-quality data with the greatest accuracy , speed and efficiency , enabling the addition of potential new antimicrobial compounds and extracts to our armamentarium . This modified resazurin assay corrected the dilution inaccuracies , especially in relation to the MIC determinations , as described by Drummond and Waigh -LSB- 3 -RSB- , and enabled results to be comparable for the test material for different bacterial strains . The method described here is easy to follow and accurate . The generation of an accurate MIC value , which can be compared to existing antibiotics , empowers the scientist with the knowledge to decide whether the extracts and compounds are worth pursuing further in terms of their antimicrobial potential .

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

antibacterial assay
resazurin
serial dilution


Strategies

to

Reduce

Alcohol-Exposed

Pregnancies

Introduction

Prenatal

alcohol

exposure

remains

a

leading

preventable

cause

of

birth

defects

and

developmental

disabilities

in

the

United

States

with

the

prevalence

of

alcohol

consumption

by

women

in

childbearing

age

remaining

high

and

unchanged

over

time

-LRB-

Fig.

-RRB-

This

paper

provides

a

summary

of

current

knowledge

and

information

on

recognition

and

prevention

of

an

alcohol-exposed

pregnancy

including

overall

alcohol

exposure

burden

among

women

of

child-bearing

age

diagnostic

criteria

for

fetal

alcohol

syndrome

-LRB-

FAS

-RRB-

and

recommendations

for

screening

assessment

and

interventions

to

reduce

alcohol

exposed

pregnancies

Burden

of

alcohol

exposure

in

pregnancy

Most

women

reduce

their

alcohol

use

substantially

when

they

realize

they

are

pregnant

but

significant

numbers

continue

to

drink

at

levels

that

can

be

hazardous

to

the

fetus

-LSB-

-RSB-

Of

particular

concern

are

the

group

of

sexually

active

women

who

are

not

planning

to

become

pregnant

-LRB-

about

half

of

all

pregnancies

in

the

US

-RRB-

but

do

so

and

continue

alcohol

use

during

the

early

stages

of

embryonic

and

fetal

development

-LSB-

-RSB-

For

many

women

pregnancy

recognition

does

not

occur

until

the

week

of

gestation

-LSB-

-RSB-

According

to

national

survey

data

in

about

of

women

aged

to

years

were

sexually

active

fertile

not

using

any

form

of

birth

control

and

at

risk

of

becoming

pregnant

-LSB-

-RSB-

This

group

of

women

also

report

high

rates

of

binge

drinking

with

in

in

the

age

category

of

to

years

reporting

multiple

episodes

of

binge

drinking

and

are

therefore

at

risk

for

an

alcohol-exposed

pregnancy

Fig.

percentage

of

alcohol

consumption

for

non-pregnant

women

aged

to

years

during

the

previous

days

BRFSS

United

States

Data

were

not

collected

in

and

Frequent

refers

to

drinks/week

or

binge

Binge

drinking

refers

to

drinks

on

one

occasion

Adverse

fetal

outcomes

Prenatal

alcohol

exposure

can

result

in

a

wide

range

of

adverse

outcomes

including

fetal

alcohol

syndrome

-LRB-

FAS

-RRB-

although

not

all

infants

exposed

to

alcohol

in

utero

develop

the

same

severity

of

effects

as

do

others

The

term

fetal

alcohol

spectrum

disorder

-LRB-

FASD

-RRB-

has

been

introduced

to

describe

the

range

of

physical

mental

and

behavioral

effects

that

can

occur

in

an

individual

exposed

to

alcohol

in

utero

Because

diagnostic

criteria

are

not

available

for

all

conditions

along

the

spectrum

there

are

no

prevalence

rates

available

for

the

full

spectrum

Prevalence

rates

of

FAS

range

from

to

cases

per

live

births

-LSB-

-RSB-

depending

on

the

methodology

used

and

the

sub-populations

assessed

Reported

rates

are

higher

among

infants

born

to

women

who

are

American

Indian/Alaska

Native

or

African

American

unmarried

smokers

have

low

incomes

and

have

a

history

of

previous

drug

use

or

mental

health

conditions

-LSB-

-RSB-

In

an

effort

to

promote

the

more

complete

recognition

of

FAS

guidelines

for

identifying

and

referring

persons

with

FAS

have

been

collaboratively

developed

and

disseminated

recently

-LSB-

-RSB-

-LRB-

-RRB-

Approaches

to

reducing

alcohol-exposed

pregnancies

A

report

from

the

Institute

of

Medicine

addressing

prevention

of

FAS

recommended

implementation

of

preventative

actions

at

multiple

levels

including

individual

group

and

universal

levels

-LSB-

-RSB-

To

date

most

of

the

effective

prevention

strategies

identified

have

focused

on

the

individual

level

In

the

U.S.

Preventive

Services

Task

Force

-LRB-

USPSTF

-RRB-

released

a

report

recommending

screening

and

behavioral

interventions

to

reduce

alcohol

misuse

in

adults

in

primary

care

settings

-LSB-

-RSB-

The

report

concluded

that

effective

interventions

include

brief

counseling

comprised

of

feedback

regarding

screening

and

assessment

information

advice

goal-settings

and

follow-up

assistance

The

report

further

cited

complementary

practices

of

motivational

interviewing

assessing

readiness

to

change

and

use

of

the

A

's

behavioral

counseling

framework

of

assess

advice

agree

assist

and

arrange

Validated

screening

instruments

are

available

for

screening

pregnant

and

non-pregnant

women

of

reproductive

age

including

the

T-ACE

TWEAK

and

AUDIT

-LSB-

-RSB-

More

information

on

these

instruments

is

available

at

the

following

website

Primary

healthcare

providers

can

play

a

pivotal

role

in

identifying

women

of

reproductive

age

-LRB-

during

pregnancy

and

before

pregnancy

occurs

-RRB-

who

are

at

high

risk

for

an

alcohol-exposed

pregnancy

and

providing

them

with

advice

counseling

and

referral

as

appropriate

This

strategy

has

widespread

support

among

professional

organizations

including

the

American

College

of

Obstetricians

and

Gynecologist

-LRB-

ACOG

-RRB-

and

the

American

Academy

of

Pediatrics

-LRB-

AAP

-RRB-

as

well

the

U.S.

Office

of

the

Surgeon

General

and

the

U.S.

Department

of

Health

and

Human

Services

A

useful

example

of

an

intervention

targeting

groups

of

women

using

many

of

the

components

described

in

the

recommendation

of

the

USPSTF

is

found

in

a

CDC

sponsored

study

Project

CHOICES

a

feasibility

pilot

study

that

targeted

non-pregnant

women

at

high

risk

for

an

alcohol-exposed

pregnancy

-LSB-

-RSB-

Project

CHOICES

was

conducted

in

diverse

sub-populations

of

women

determined

to

have

higher

proportions

of

individuals

at

risk

for

an

alcohol-exposed

pregnancy

Compared

to

the

overall

estimated

of

childbearing

women

at

risk

for

an

alcohol-exposed

pregnancy

the

Project

CHOICES

intervention

settings

-LRB-

e.g.

a

jail

alcohol

and

drug

treatment

facilities

and

primary

care

clinics

-RRB-

had

an

overall

rate

of

of

women

at

risk

for

an

alcohol-exposed

pregnancy

A

unique

aspect

of

the

intervention

is

that

it

focused

not

only

on

reducing

risk

drinking

but

also

addressed

pregnancy

postponement

as

a

route

for

avoiding

an

alcohol-exposed

pregnancy

The

intervention

consisted

of

brief

motivational

interventions

sessions

and

consultation

visit

to

a

family

planning

provider

All

participants

were

at

risk

for

an

alcohol-exposed

pregnancy

in

that

they

were

sexually

active

fertile

risky

drinkers

and

not

taking

effective

measures

to

avoid

pregnancy

At

the

month

follow-up

assessment

were

at

reduced

risk

because

they

had

either

changed

their

risk

drinking

instituted

effective

contraception

or

both

Subsequently

the

Project

CHOICES

Research

Group

completed

a

randomized

controlled

trail

to

test

the

efficacy

of

the

intervention

with

a

report

of

the

study

findings

currently

underway

Universal

level

interventions

have

not

received

recent

attention

with

the

exceptions

of

the

release

of

U.S.

Surgeon

General

's

Advisory

on

Alcohol

Use

and

Pregnancy

This

advisory

drew

attention

to

the

continuing

problem

of

FASD

and

the

continuing

high

rates

of

alcohol

use

including

binge

drinking

among

childbearing

aged

women

in

the

U.

S

The

impact

of

the

advisory

has

provided

support

for

those

seeking

to

inform

and

educate

the

public

healthcare

system

overall

to

this

important

public

health

concern

Summary

In

addition

to

the

adverse

effects

of

alcohol

on

the

fetus

alcohol

use

can

leads

to

other

adverse

effects

on

the

health

of

women

including

reproductive

health

conditions

that

are

not

addressed

in

this

brief

Though

the

approaches

mentioned

here

are

primarily

aimed

at

reduction

of

prenatal

alcohol-related

pregnancies

any

reduction

in

hazardous

alcohol

consumption

among

women

will

add

to

improvements

in

the

general

health

of

women

Multiple

federal

and

non-fedral

health

agencies

and

organizations

recommend

that

pregnant

women

and

those

planning

a

pregnancy

should

abstain

from

alcohol

use

Although

assessment

and

interventions

are

valuable

tools

to

recognize

and

address

alcohol

use

and

secondary

pregnancy

outcomes

they

are

underutilized

in

primary

care

settings

To

reduce

the

burden

of

alcohol

exposed

pregnancies

and

alcohol

's

impact

on

the

health

of

families

it

is

critical

for

physicians

and

health

care

providers

to

consistently

screen

childbearing-aged

women

for

alcohol

use

with

validated

screening

tools

which

can

be

embedded

in

the

patient

screening

protocol

Tools

are

now

available

for

pediatricians

and

child

health

care

providers

to

enhance

early

recognition

of

FASD

and

reduce

secondary

conditions

that

often

accompany

physical

maturation

-LSB-

-RSB-

Brief

clinician-delivered

behavioral

interventions

to

women

and

their

partners

counseling

regarding

effective

contraceptive

options

when

not

planning

a

pregnancy

and

improving

access

to

such

services

for

those

who

are

unable

reduce

their

alcohol

intake

can

help

more

women

reduce

their

risk

for

an

alcohol

exposed

pregnancy

-LSB-

-RSB-

Referral

to

needed

social

services

should

complement

health

services

in

order

to

achieve

maximum

benefits

of

primary

care-based

attempts

to

reduce

alcohol-exposed

pregnancies

Given

the

levels

of

alcohol

use

among

childbearing

aged

women

primary

care-based

individual

level

assessment

and

intervention

continues

to

remain

a

critical

prevention

strategy

for

reducing

alcohol-exposed

pregnancies

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 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 B O O O B 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 B O O O O O B O O B 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 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 B 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 B O O O O O O O O O O O O B 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 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 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 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 B O O B 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 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 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 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 B 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 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 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 B O O B 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 B 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 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 B O O O B O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O B 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 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 B 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 B 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 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