Identification and characterization of starch and inulin modifying network of Aspergillus niger by functional genomics
Yuan, X.L.
Citation
Yuan, X. L. (2008, January 23). Identification and characterization of starch and inulin modifying network of Aspergillus niger by functional genomics.
Institute of Biology Leiden (IBL), Group of Molecular Microbiology, Faculty of Science, Leiden University. Retrieved from
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Chapter 8
A novel screening method for isolation of mutants involved in inulin signalling in Aspergillus niger
XiaoLianYuan,MarkArentshorst,CeesA.M.J.J.vandenHondel,ArthurF.J.Ram
Abstract
Among the inulinolytic genes present in A.niger, we identified the inuE gene as the most
strongly induced gene in the presence of the pathway inducing poly and disaccharides
inulin and sucrose, respectively. Using the inuE promoter region, we present a positive
screeningmethodfortheisolationofmutantsthatdonotrespondtothepresenceofinulin
orsucrose.ReporterstrainswereconstructedinwhichtheinuEpromoterwasfusedtothe
racAG12VmutantgeneortotheeGFPreporter.ExpressionoftheracAG12Vmutantgene
from the inuE promoter resulted in reduced growth and reduced conidiation under inuE
inducing conditions. Expression from the inuE promoter was also visible using the
PinuEeGFP reporter and resulted in strong GFP expression under inducing conditions.
The growth phenotype of the PinuEracAG12V strain allows a genetic screen to identify
mutants defective in the activation of inulin signalling pathway. The rationale of this
screeningisthatamutationinagenewhichisinvolvedinaninulinsignallingpathway,e.g.
theinuRtranscriptionfactormutant,abolishestheinductionoftheinuEgene,resultingin
normalgrowthandnormalconidiationofthemutantstraininthepresenceoftheinducer.
Thefunctionalitiesofthetworeporterswereexaminedseparatelyandcanbecombinedinto
asinglereporterstraininthefuturetoallowtheselectionfortransactingmutationsinthe
inulinsignallingpathway.Whereasinthewildtypestrain,inductionofthereportersleads
toimpairedgrowthandconidiation(PinuEracAG12V)orGFPexpression(PinuEeGFP)on
inducingcarbonsources,bothreporterswerenotinducedintheinuRbackground.These
resultsindicatethatthetworeportersystemsarefunctioningwellandcanbecombinedto
identifygenesinvolvedinaninulinsignallingpathway.
Introduction
The filamentous fungus Aspergillus niger is well known for secreting a large spectrum of
enzymesinvolvedinthedegradationofplantpolysaccharidessuchasxylan,pectin,starch,
and inulin (De Vries et al., 2003; Yuan et al., 2006; MartensUzunova et al., 2006). The
expression of these enzymes is controlled by Zn(II)2Cys6 type transcription factors. For
example,XlnRandAmyRactivatethetranscriptionofgenesinvolvedinxylanandstarch
degradationrespectively(vanPeij1998;Petersenetal.,1999andGomietal.,2000).Anewly
isolated InuR transcription factor controls the expression of inulinolytic genes in A. niger
(Yuanetal.,2008).
Activation of Zn(II)2Cys6 transcription factors regulating genes encoding sugar
polymer degrading enzymes is triggered by pathway inducing molecules. For example
isomaltosetriggersAmyRmediatedamylaseinductioninAspergillusspecies(Tsukagoshiet
al., 2001; Kato et al., 2002). Galactose triggers activation of Gal4 mediated transcription of
GalpathwaygenesinSaccharomycescerevisiae(Siletal.,1999).Themechanismofactionof
theinducingmoleculesisthoughttobeviainducingconformationalchangesinthetarget
transcriptionfactors,whichleadstoachangeintheiractivityorinterandintramolecular
interactions (Sharrocks, 2000). Obviously, activation of transcription factors is a complex
process.Itrequiresproteinstosensetheavailabilityofthenutrients,proteinstogeneratean
inducing molecule, proteins to transport the inducer and to promote the assembly of
higherorderactivatorcomplexes.TheGal4systemissofarthebestunderstoodregulatory
mechanismrelatedtosugarmetabolism.InS.cerevisiae,theinteractionsandassociationsof
three proteins, Gal4, Gal80 and Gal3, control the regulation of Gal pathway genes. The
capacityofGal4toactivatetranscriptionisinhibitedbyassociationoftheGal80proteinto
theactivationdomainofGal4intheabsenceofgalactose.Inthepresenceofgalactose,Gal3
associates with Gal80, relieving Gal80 inhibition of Gal4, thereby switching on the Gal4
activationsystem(Pilaurietal.,2005;Diepetal.,2006).
IntheinulindegradationpathwayinA.niger,sucrosewasidentifiedasapathway
inducingmolecule,probablyviatheactivationoftheInuRtranscriptionfactor(Yuanetal.,
2006;2008).Inthisstudywesetupascreeningmethodtoidentifygenesencodingproteins
that are involved in the inulin signalling pathway. The screening was based on our
observation that exoinulinase gene inuE is pathway specifically regulated: induced on
inulin,sucroseandrepressedonglucose,notexpressedonfructoseandxylose(Yuanetal.,
2006). The racAG12V is a mutant of a small GTPase involved in actin polarization. Its
expressionleadstocessationofcellgrowth(ArentshorstandRam,unpublishedresults).A
second reporter, eGFP, was also constructed to examine the activity of theinuE promoter
microscopically. We reasoned that mutants defective in the induction of the inuE gene
would grow relative normally in the presence of the inducer, while the wildtype strain
expressing the racAG12V mutant protein from the inuE promoter would have a strong
growthphenotype.
ThescreeningmethodwasevaluatedusingtheinuRdisruptionstrain.Theresults
demonstratethatthisscreeningmethodworksandcanbesuccessfulfortheidentificationof
genes involved in inulin signalling. Such a screening method might also be useful for the
identification of regulators from which the transcription factor has not been successful so
far.
Materials and methods
Strains,cultureconditionsandtransformations
A.niger strainN402, a cspA1derivative of the wildtype strain A.niger van Tieghem (CBS
120.49,ATCC 9029) (Bos etal., 1988) and strain AB4.1, a pyrG negative derivative of N402
(van Hartingsveldt et al., 1987) were used in this study. A.niger strainswere grown in an
Aspergillus minimal medium (MM) (Bennett and Lasure, 1991), or an Aspergillus complete
medium (CM) consisting of a MM medium with the addition of 0.5% (w/v) yeast extract
and0.1%(w/v)casaminoacids.Thegrowthmediumwassupplementedwith10mMuridine
(Serva) when required. For the submerged culture, A. niger strains were grown in MM
supplemented with 1% (w/v) carbon source and 0.1% (w/v) casamino acids. The
conidiospores were inoculated at a concentration of 2x106 spores per ml. Glucose, sucrose
and raffinose (BDH chemicals), xylose, fructose and maltose (SigmaAldrich) and inulin
(Sensus Frutafit, Cosun) were used as carbon sources. Conidiospores were obtained by
harvesting spores from CM plates after 46 days of growth at 30 °C, using a 0.9% NaCl
solution. Transformation of A.niger AB4.1was as described by Punt and van den Hondel
(1992) using lysing enzymes (L1412, Sigma) for protoplast formation. The bacterial strain
used for transformation and amplification of recombinant DNA was Escherichia coli XL1
Blue(Stratagene,LaJolla,CA).TransformationofXL1Bluewasperformedaccordingtothe
heatshockprotocolasdescribedbyInoueetal.,(1990).
Microarrayexperimentsanddataanalysis
The microarray experiments and data analysis were performed as described in Chapter 7
(Yuanetal.,2008).
Molecularbiologicaltechniques
Ligation of DNA fragments was performed with the Rapid DNA ligation kit (Roche).
Plasmid DNA was isolated from the E. coli transformants using Plasmid miniprep or
midiprepKit(Qiagen).Restrictionenzymedigestion(Invitrogen)wasperformedaccording
tothesupplier’smanual.FungalchromosomalDNAwasisolatedasdescribedbyKolaret
al.(1988).ForSouthernanalysis,10PlofgenomicDNAwasdigestedwith20Uofrestriction
enzymes at 37 °C for 3h, respectively. [-32P]dCTP labelled probes were synthesized using the Rediprime II DNA labelling System (Amersham Pharmacia Biotech, UK) according to the instructions of the manufacturer.
Constructionofreporterplasmidsandstrains
The recombinant plasmid PinuEracAG12V was constructed by a three way ligation. The
first fragment containing the promoter of inuE was amplified by PCR using primers
PinuEP1f (ataagaatgcggccgcttcccaccacacggacagt with the NotI site underlined) and
PinuEP2r (catgccatggctaatgagccctttgaga with the NcoI site underlined). The resulting PCR
fragment was cloned into a NotINcoI opened pUC21 vector and verified by sequence
analysis. A second fragment containing racAG12VTtrpC was isolated as an NcoIXbaI
fragment from plasmid pPglaAracAG12VTtrpC (Arentshorst and Ram, unpublished
vector).ThethirdfragmentcontainingthevectorbackbonewasisolatedasNotIXbaIfrom
pAN521 NotI (Punt, unpublished vector). The three fragments were ligated together
resulting in plasmid pPinuEracAG12VTtrpC. The resulting vector wasthen opened with
XbaItointroduceapyrG*gene(Goukaetal.,1995),isolatedasa3.8kbXbaIfragmentfrom
plasmid ppyrG*XbaI (Arentshorst and Ram, unpublishedresults)to give a finalconstruct
ofpPinuEracAG12VpyrG*(namedpMA61.1).
ToconstructplasmidPinuEeGFPTtrpC,thefirstfragmentcontainingeGFPTtrpC
andthepUC18backbonesequencewasisolatedasanNcoINotIfragmentfromPgpdAH2B
GFPTtrpC(ArentshorstandRam,unpublishedvector)toremovePgpdAH2B.Thisopened
vector was then ligated with a NotINcoI cut PinuE fragment to obtain the plasmid
PinuEeGFPTtrpC. Finally a 3.8 kb XbaI fragment containing the pyrG* gene was inserted
into XbaI opened plasmid PinuEGFPTtrpC and resulted in the final plasmid PinuEGFP
TtrpCpyrG*(namedpMA62.1).
The obtained final plasmids pPinuEracAG12VpyrG* and pPinuEGFPTtrpC
pyrG* were independently transformed into an A. niger pyrG strain AB4.1 (van
Hartingsveldt et al., 1987) or an pyrG derivative strain of the inuR strain (Yuan et al.,
2008).Uridineprototrophictransformantswereselectedbyincubatingprotoplastsonagar
platescontainingMMwithouturidine.TransformantswerescreenedbyPCRusingprimers
PinuEP1fandSagDP4(cgccggggaaagcgttgg)forracAG12VreporterstrainsorpinuEp1fand
GFPP5 (ggatgttgccgtcctcctt) for GFP reporter strains. Only transformants harboring a
targeted plasmid should result in the amplification of a 2.1 or 1.7 kb PCR fragment for
pinuEracAG12V and pinuEeGFP, respectively. PCR positive transformants were verified
bySouthernblotanalysisasdescribedbySambrooketal.(1989).
PhenotypeanalysisofA.nigerreporterstrains
For the growth assays on plates, equal amounts of freshconidiospores were incubatedon
agarplatescontainingMMsupplementedwith1%(w/v)variouscarbonsourcesandgrown
at30°Cfor3days.Forcoverslipassays,1x104freshconidiosporeswereinoculatedontwo
glasscoverslipsin5cmPetridishes.EachPetridishcontained8mlofMMsupplemented
with 1% (w/v) carbon source and 0.1% (w/v) casamino acids and incubated at 37°C for 6
hours and then switched to 25°C for 16 hours before microscope observation. Light and
GFPimagesofthethinmyceliafragmentsattachedtothecoverslipswereanalyzedbyDIC
orfluorescentmicroscopyusingaZeissAxioplan2Opticalmicroscopeatafixedexposure
timesandstandardfiltersettings.
Results and discussion
Expressionanalysisofinulinolyticgenesusingmicroarrays
A. niger contains five inulinolytic genes including inuE (exoinulinase), inuA (endo
inulinase), sucA (invertase), sucB (intracellular invertase, Goosen et al., 2007) and sucC
(putative intracellular invertase) in A. niger genome (Yuan et al., 2006). In this study the
expressionoftheseinulinolyticgeneswasstudiedinrelationtovariouscarbonsourcesand
theInuRtranscriptionalactivator(Yuanetal.,2008)usingAffymetrixmicroarrays.
A. niger strains were pregrown on MM containing 2% xylose for 18 h and the
mycelia were transferred to fresh MM containing 1% various carbon sources. After 2 h or
8h,myceliawereharvestedandthecorrespondingRNAsampleswereisolatedandusedin
microarray experiments tomonitor the transcriptionlevelsofinulinolytic genes.Based on
ourpreviousNorthernanalysis(Yuanetal.,2006),theexpressionofinulinolyticgeneson
sucrose was highly induced 2 h after the transfer, and the induction was dramatically
reduced after 8 hours. Therefore, for the sucrose culture, RNA from the 2 h growth time
points after the transfer was used for microarray analysis. The results are summarized in
Fig. 1. After the transfer to xylose or maltose, the expression of inulinolytic genes were
showntobeeitherverylow(inuE,sucB)ornotdetectable(inuA,sucA,sucC),similarasin
theexpressionlevelsinthexylosepreculture.Incontrast,thetransfertosucroseresultedin
astronginducedexpressionfortheinuE,sucAandinuAgenes.Afterthetransfertoinulin,
inuE was modestly expressed at an early stage (2 h) and displayed strong induction
(63fold)atlaterstage(8h).TheexpressionofsucAwasonlymodestlyinducedoninulinat
laterstage(8h)(Fig.1).TheexpressionofinuAwasnotinduced2hoursafterthetransferto
inulin,andonlyslightlyhigherafter8hours.Theseresultssupportpreviousdatathatthe
induction of the inulinolytic genes on sucrose was very rapid and more rapid than the
inductiononinulin(Yuanetal.,2006).Asexpectedtheexpressionoftheinulinolyticgenes
islowonmaltoseandxylose.Asreportedearlier(Yuanetal.,2006),theexpressionofsucB
and sucC is low under the various carbon sources and not induced in the presence of
sucroseorinulin.Thesedataalsoindicatethat,amongthefiveinulinolyticgenes,inuEwas
themosthighlyexpressedandmoststronglyinduciblegeneinresponsetothepresenceof
bothsucroseandinulin.
Fig.1.TranscriptionalexpressionofA.nigerinulinolyticgenes.Accessionnumbersofthegenenames
are given in Table 1. Strains and time points after transfer from the preculture are indicated below,
inulinolyticgenesareindicatedontheright.ThevaluesandPresent/Absentcallsfromtheexpression
dataareprovidedasSuppl.Table1.
AsimilartransferexperimentwasalsoperformedfortheinuRstrain.AsshowninFig.1,
theinductionofinulinolyticgenesonsucrose(forinuE,sucA,inuAat2haftertransfer)and
inulin (for inuE and sucA at 8 h after transfer) was abolished in the inuR mutant and
confirms our previous Northern observations that the induced expression of inulinolytic
genesrequiresInuR(Yuanetal.,2008).
Setupofageneticscreentoidentifyinulinsignallingmutants
To screen for mutants affected in the activation of the InuRmediated induction of
inulinolyticgenes,areportersystembasedoninuEpromoterwasdesigned.TheinuEgene
was selected because it displayed the strongest induction in expression on inulin and
sucrose(Fig.1).Asareporterforthegeneticscreen,theinuEpromoterwasclonedintoin
front of the A.niger racAG12V gene. racAG12V is a dominant active mutant form of the
racA gene. racA encodes a small GTPase involved in actin organization and the induced
expressionofracAG12Vresultsinthelossofhighlypolarizedtipgrowthandsubsequent
swellingofthehyphaltip(ArentshorstandRam,unpublisheddata).
Therefore,thereporterstraincontainingthePinuEracAG12Vfusionconstructisexpected
tocauserestrictedgrowthoninducingcarbonsourcesforinuEexpressionsuchassucrose
and inulin, but to display normal growth on noninducing or repressing carbon sources.
Suchascreenallowsapositiveselectiontoisolatemutantsthatdisplaynormalgrowthon
inducingcarbonsources,indicatinginthismutanttranscriptionactivationoftheinuEgene
isimpaired.Totestthescreeningmethod,wealsotransformedthereporterconstructina
inuRstrain.Here,thehypothesisisthatintheinuRbackground,inuEisnotinducedand
therefore this strain is expected to grow normally in the presence of inducing carbon
sources. A second reporter gene was constructed in which the inuE promoter was cloned
upstream to eGFP. In this study, the usefulness of this reporter was evaluated and
transformedseparatelyfromthePinuEracAG12Vconstructtoboththewildtypeandthe
inuR.Infuturework,astrainshouldbeconstructedcontainingbothreporterstrainswhich
allowthescreeningfortransactingmutants,insteadofcisactingmutants.Inthefollowing
section,theprocedureoftheconstructionofthestrainsisdescribedindetail.
ConstructionofA.nigerreporterstrains
To construct the racAG12V reporter strain the PinuEracAG12V plasmid was made as
described in Materials and methods (Fig. 2A). Briefly, the 1.0 kb promoter of inuE was
clonedupstreamoftheracAG12VgeneanddownstreamoftheA.nidulanstrpCgeneasa
terminatorsequence.ThepyrG*wasintroducedasaselectionmarker,allowingthereporter
plasmid to be inserted at the pyrG locus (Gouka et al., 1995). Since xylose acts as a non
inducingcarbonsourceforinuEexpression(Yuanetal.,2006),primarytransformantswere
grown on xylose and screened by PCR for the presence of racAG12V (see Materials and
methods).TwoputativereporterstrainsMA61.24andMA61.25werefurtherexaminedby
Southern blot analysis to verify the proper insertion of the construct. Using an XhoI
fragmentcontaininganA.nigerpyrGgeneasprobe,BamHIdigestedgenomicDNAshowed
three fragments, 2.3, 2.7 and 9.7 kb in the reporter strain MA61.24 and two fragments 2.1
and 2.3 kb in WT strain N402 as anticipated. Strain M61.25 displayed extra fragments 2.1
and 7.6 kb instead of 9.7 kb, suggesting the pyrG* point mutation on the BamHI site was
repaired during transformation (Fig. 2C). Strain MA61.24 was used for further
characterization.
PinuE-racA-G12V transformants A.niger pyrG mutant
9.7 kb B
2.3 kb 2.1 kb
B B B
PinuE-racA-G12V plasmid
pyrG-
R R
B 2.3 kb B 2.7 kb B
5181
1
PInuX-RacA-G12V-pyrGster 9749bp XbaI
XhoI
XbaI
5181 pMA61
PinuE
TtrpC NotI
XhoI BamHI
R R
racA-G12V
pyrG*
NcoI
- 2.1 kb - 2.3 kb - 2.7 kb - 7.6 kb MA61.24
MA61.25 N402
M - 9.7 kb
XbaI
PinuE-GFP transformants A.niger pyrG mutant PinuE-GFP plasmid NotI
pMA62 9504 bp
PinuE eGFP
TtrpC
pyrG*
XbaI
XhoI XhoI
BamHI
R R
NcoI
2.1 kb 2.3 kb
B B B
pyrG-
4.7 kb 6.9 kb 2.3 kb
B B B B
R R
- 2.3 kb - 4.7 kb - 6.9 kb
- 2.1 kb MA62.1
MA62.2 N402
M
A B
C D
PinuE-racA-G12V transformants A.niger pyrG mutant
9.7 kb B
2.3 kb 2.1 kb
B B B
PinuE-racA-G12V plasmid
pyrG-
R R
B 2.3 kb B 2.7 kb B
5181
1
PInuX-RacA-G12V-pyrGster 9749bp XbaI
XhoI
XbaI
5181 pMA61
PinuE
TtrpC NotI
XhoI BamHI
R R
racA-G12V
pyrG*
NcoI
- 2.1 kb - 2.3 kb - 2.7 kb - 7.6 kb MA61.24
MA61.25 N402
M - 9.7 kb
XbaI
PinuE-GFP transformants A.niger pyrG mutant PinuE-GFP plasmid NotI
pMA62 9504 bp
PinuE eGFP
TtrpC
pyrG*
XbaI
XhoI XhoI
BamHI
R R
NcoI
2.1 kb 2.3 kb
B B B
pyrG-
4.7 kb 6.9 kb 2.3 kb
B B B B
R R
- 2.3 kb - 4.7 kb - 6.9 kb
- 2.1 kb MA62.1
MA62.2 N402
M
A B
C D
Fig. 2. Schematic representation of the construction of the reporter strains containing construct
PinuEracAG12V(A)orPinuEeGFP(B).SouthernanalysisofthereporterstrainsindicatedinAandB
areshowninCandD,respectively.
The second reporter construct (pPinuEeGFP) was made as describedinthe materials and
method section. Transformants with a targeted integration of the PinuEeGFP construct at
the pyrG locus were identified by PCR analysis. PCR positive transformants MA61.1 and
MA61.2 were verified by Southern analysis as shown in Fig. 2B. Using an XhoI digested
A.niger pyrG gene fragment as a probe, BamHI digested genomic DNA showed three
fragments 2.3, 4.7 and 6.9 kb in the reporter strain containing PinuEeGFP and two
fragments2.1and2.3kbinWT,asanticipated(Fig.2D).StrainMA62.1wasusedforfurther
characterization.
PhenotypecharacterizationofreporterstraincontainingpinuEracAG12V
Colonymorphologyassayinagarplates
ToexaminethegrowtheffectofthePinuEracAG12Vreporterstrain(MA61.24)ondifferent
carbon sources, this strain was grown on MM agar plates containing 1% different carbon
sources.AsshowninFig.3A,bothgrowthandconidiationofthereporterstrainMA61.24
were affected on inulin, sucrose, raffinose, and maltose indicating that racAG12V was
overexpressed by the activated inuE promoter at these carbon sources. The effect was
anticipatedforinulinandsucrose.TheexpressionoftheinuEgenehasnotpreviouslybeen
examinedonraffinose,buttheresultsfromthereporterstrainindicatethattheinuEgeneis
also expressed on raffinose. This is not complete surprising as the action of alpha
galactosidasessecretedbyA.nigermightresultintheformationofsucrosewhichcanactas
an inducer for the inulinolytic enzymes (Yuan et al., 2006). Interestingly, on the maltose
plate, the growth of MA61.24 was also affected, suggesting that growth on maltose on
plates induces inuE expression. Northern and array analysis (Fig. 1) showed a minor
expressionoftheinuEonmaltoseundersubmergedconditionandpossiblytheexpression
oftheinuEgeneonmaltoseishigheronplate.OnxyloseandglucoseMA61.24growthand
conidiation is similar as in the wild type strain (N402), indicating xylose and glucose are
repressingcarbonsourcesforinuE,whichisconsistentwithprevioustranscriptionanalysis
(Yuanetal.,2006;2008).FructoseseemstomodestlyinduceinuEexpressionasthegrowth
of reporter strain was slightly affected when compared to N402. On sorbitol and glycerol,
which were considered as noninducing carbon sources, MA61.24 also shows a growth
phenotype (reduced conidiation), indicating that these two carbon sources are inducing
inuEexpressionaswell.Takentogether,thesedatasuggestthatbesidestheknowninducers
(inulin, sucrose) raffinose, maltose, fructose, sorbitol and glycerol also lead to inuE
expression on solid agar plates. To demonstrate that this reporter system can be used for
identifyingpathwaysignallinggenes,thereporterconstructwasalsotransformedintothe
inuR strain.A inuR strain containing the PinuEracAG12V construct(strain XY7.1)was
isolatedbyPCRandconfirmedbySouthernanalysis(datanotshown).AsshowninFig.3B,
on all tested conditions, including inducing, noninducing and repressing carbon sources,
no differences of either growth or conidiation was observed between strain XY7.1
containinganinuEracAG12VconstructandtheinuRstrain.Thisindicatesthatdeletingof
inuRabolishedtheexpressionofinuE,therebyresultinginnoexpressionofracAG12V.
Hyphalmorphologyinsubmergedculture
We also examined the hyphal morphology of the PinuEracAG12V reporter strain during
sporegermination.A.nigerstrainsMA61.24(expressingPinuEracAG12V)andN402(wild
typestrain)weregrownoncoverslipsinasubmergedculture(seeMaterialsandmethods)
containing different carbon sources, followed by microscope observation. As shown in
Fig.3C,underinducingconditions,suchasinulin,sucroseandraffinose,thereporterstrain
MA61.24 containing PinuEracAG12V showed extreme swelling and short hyphae. This
indicates that expression from the inuE promoter of racAG12V in the submerged culture
occursalreadyduringsporegerminationandcausesdefectivedevelopmentofhyphae.This
is remarkable different from expression of the racAG12V protein from the glucoamylase
promoterwhichonlygivesaneffectonthemorphologyofthegermlingsafter16hoursof
germination(ArentshorstandRam,unpublishedresults).Themorphologyofthehyphaeof
theracAG12Vreporterstrainontheothercarbonsourceswasnormal(Fig.3C),indicating
thatracAG12Visnotexpressed.Thisresultisdifferentfromthepreviousagarplateassay,
in which growth on maltose, fructose, sorbitol and glycerol induced inuE expression.
Obviously there are other factors involved in the physiological growth conditions
(e.g.carbon source concentration, pH, age of the mycelium) that affect the inulinolytic
system. The results indicate that the expression from the PinuE is tightly regulated in
responsetocarbonsourcesinasubmergedcultureduringgerminationandthatracAG12V
isagoodandsensitiveindicatortodetectexpression.AsshowninFig.3D,reporterstrain
XY7.1 (inuR strain containing inuEracAG12V) displays normal hyphae similar as N402
and the inuRmutant, suggesting no expression of racAG12V from the inuEpromoter in
theinuRmutant(Fig.3D).
A B C D
MA61.24 N402 XY7.1 inuR MA61.24 N402 XY7.1 inuR
inulin
sucrose
raffinose
maltose
xylose
glucose
fructose
sorbitol
glycerol
Fig.3.EffectsofoverexpressionofracAG12VfromtheinuEpromoteronA.nigergrowthmorphology
duringconidiation(AandB)andduringgermination(CandD).MA61.24isaN402(pyrG)containing
aPinuEracAG12Vconstruct;XY7.1isainuR(pyrG)straincontainingPinuEracAG12Vconstruct.
From both the agar plate assay for colony morphology and cover slip assay for hyphal
morphology, we conclude that inulin, sucrose and raffinose are strong inducers for inuE
expressionandleadtostrongphenotypesfromthereporterracAG12V.Complicatingfactor
for a mutant screen is our observation that the inuRstrain can not grow or only shows
limited growth on inulin and sucrose plates (Fig. 3B). Interestingly, we found that in the
inuR mutant harboring the racAG12V reporter, the colony size and the degree of
conidiation on raffinose were much larger than those on inulin and sucrose. Although
growthofN402onraffinoseinducestheexpressionofinuE,growthoftheinuRmutanton
raffinose still occurs, indicating that A. niger has an InuR independent mechanism to
metabolize raffinose. As mentioned above, raffinose can be metabolized by alpha
galactosidase to galactose and sucrose. The galactose will assist growth of the potential
mutantreporterstrainswhilesucroseinducestheInuRmediatedexpressionofinuE.From
this result, we conclude that raffinose would be a suitable carbon source for mutant
screening. Bygrowing the potential mutant on raffinose,we expect that the mutant strain
containsthe reporter construct to grow normally, whereas the wild typestrain containing
inuEracAG12VwillnotgrowduetothehighexpressionofracAG12V.
fructose inulin
sucrose
xylose
MA62.1 N402 XY8.1 inuR
glucose
sorbitol
glycerol raffinose
maltose
A B
fructose inulin
sucrose
xylose
MA62.1 N402 XY8.1 inuR
glucose
sorbitol
glycerol raffinose raffinose
maltose maltose
A B
Fig.4.EffectsofoverexpressionofeGFPfromtheinuEpromoteronfluorescenceofA.nigergrownin
submerged culture. MA62.1 is a N402 (pyrG) containing a PinuEeGFP construct; XY8.1 is a inuR
(pyrG)straincontainingPinuEeGFPconstruct.
MicroscopicanalysisofreporterstrainscontainingPinuEeGFP
ToexaminethereporterstraincontainingthePinuEeGFPconstruct,thestrainMA62.1was
grown on cover slips in a submerged culture and observed by fluorescent microscopy.
Similar as observed for the PinuEracAG12V reporter, inulin, sucrose and raffinose
triggeredexpressionfromtheinuEpromoterresultinginastronggreenfluorescenceinthe
hyphae of GFP reporter strain MA62.1 (Fig. 4A). Due to several layers of hyphae in one
image,thefluorescenceshownherewasonlyhighlightedatonelayerwithacertainfocus.
The observed fluorescence was specific for the PinuEGFP construct as very low
backgroundfluorescencewasobservedintheN402strain.Thefluorescenceofthereporter
strain in the other carbon sources was comparable to the fluorescence in the N402 strain,
indicating that these carbon sources do not induce inuE under these conditions. These
results for the PinuEeGFP are consistent with the results for the expression of thePinuE
racAG12V in germination morphology assay. This result further proves that PinuE is
tightly regulated and that the PinuEeGFP construct is also a good reporter to detect
expression from the inuE promoter. The PinuEeGFP reporter construct was also
transformedintoainuRstrainandconfirmedbySouthernanalysis(datanotshown).As
expected only basal background fluorescence was observed in both the reporter strain
XY8.1andthecontrolstraininuR(Fig.4B).
Takentogetherourresultsindicatethatboththereporterconstructs,PinuEracAG12Vand
PinuEeGFP,aresuitableforidentifyingmutantsaffectedininuEexpression.Bycombining
these two reporter constructs in a single strain, we can easily discriminate between cis
actingmutantsandtransactingmutantsthatareaffectedintheinuEexpression.Fromthe
cisacting mutants, we might be able to identify InuR binding sites. Transacting mutants
probably represent mutants in which are disturbed in the activation of the InuR
transcription factor or the InuR transcription factor itself. Such a genetic screen and the
identificationofthemutantswouldallowtheidentificationofimportantproteinsinvolved
in activation of the inulinolytic system in A.niger. These proteins might include proteins
that generate inducing molecules, transport or sense the inducing molecules and proteins
requiredfortheactivationoftheInuRtranscriptionfactor.
WorkingmodeloftheinulinsignallingpathwayinA.niger
The recent identification of inulinolytic genes in the A. niger genome (Yuan et al., 2006,
Chapter5)andstudiesontheirtranscriptionalcontrol(Yuanetal.,2006and2008,Chapter5
and 7) as well as the identification of putative sugar transporter encoding genes that are
inducedinsucrosemetabolism(Yuanetal.,2008,Chapter5),allowedustobuildaworking
modeloftheinulinsignallingpathwaywhichispresentedinFig.5.
Fig. 5. A working model of the inulin signalling pathway. Solid line indicates inulin degradation
pathway, broken line indicates raffinose pathway. Abbreviations stand for: STP, sucrose transporter;
FTP,fructosetransporter;GTP,glucosetransporter;GTPa,galactosetransporter;I,inducer.
Wepresumethatinulinisbrokendowntooligofructoses,sucrose,glucoseandfructoseby
the combined action of InuA (endoinulinase), InuE (exoinulinase) and SucA (invertase).
These enzymes are likely to be produced in the presence of inulin as the genes encoding
these proteins are highly expressed on inulin (Fig. 1 and Yuan et al., 2006). Similarly,
raffinosewhichhasbeenshowntoinduceinuE(thischapter)canbehydrolyzedtogenerate
galactose and sucrose by the action of alphagalactosidase (GalA). The sucrose can be
furtherhydrolyzedtoglucoseandfructosebyInuEandSucA.Inchapter5itisshownthat
sucrose act as an inducer for the inulinolytic genes and we therefore propose that the
sucrose which is formed from inulin or raffinose induces expression of the inulinolytic
genes.Duringthegrowthonsucrose,thesucrosecandirectlyactasaninducer.Although
we can not exclude the possibility that extracellular sucrose can activate the InuR
transcriptionfactor,wecurrentlyfavortheideathatthesucroseistransportedintothecell
byaproposedsucrosetransporter(STP).Candidatesforsuchasucrosetransporter,butalso
forotherpredictedtransporters(FructoseTransporter(FTP),GlucoseTransporters(GTP)),
have been identified in the genome wide array analysis but future functional analysis
should be performed to show the specificity of the different transporters. The function of
sucBandsucC,bothencodingintracellularinvertasesisnotyetclear.InChapter6weshow
that sucB encodes an invertase with transglycosylation activity, and is able to form
1kestose. Disruption of the sucB gene did not affect growth of the disruption strain on
inulin or sucrose, indicating that SucB itself is not required to activate the inulinolytic
system (Chapter 6). Possibly, SucC and SucB have redundant functions and a definitive
conclusion on SucB/SucC functioning awaits the disruption of both genes simultaneously.
AnattractivepossibilityisthatSucBand/orSucCactsontheintracellularsucrosepossibly
astransglycosylasesformingtransglycosylationproducts(e.g.1kestose)whichmightactas
an inducer molecules. Alternatively, SucB/SucC might act as invertases to hydrolyze
intracellular sucrose, without having a function in the formation of inducer. Sucrose, or a
derivativeofsucrose,isthoughttoactivatetheInuRtranscriptionfactorandthisactivation
might require addition proteins (InuX1InuX2) which are currently unknown. The genetic
screendescribedinthischapterallowstheidentificationofsuchproteinsaswellasproteins
that are involved in the transport or formation of the inducer. An important limitation of
such a genetic screen is that redundant functions, e.g. the presence of multiple sucrose
transporters, or multiple proteins that can form the inducer make the identification of
mutantsverydifficult,ifnotimpossible.Thepresenceofinulin,sucroseorraffinoseinthe
mediumandthesubsequentinductionoftheinulinolyticgenesareprobablyaccomplished
by the constitutive expression of genes encoding proteins that generate the inducer. The
methods used in this study to determine expression levels (Northern blot and microarray
analysis) are not the preferred methods to quantify expression levels of lowly expressed
genes.QuantitativeRTPCRexperimentsorenzymeactivityassaysshouldbeperformedto
determinethegenes/enzymesthatareconstitutivelyexpressed.
Finally,theracAG12Vreporterbasedscreeningmethodcanbegenerallyusedfor
the identification of transcription factor mutants. Prerequisite for such a screen is the
identification of a strongly induced gene, with a relative low basal expression level. Since
microarray data with A.niger will become more and more available, the identification of
such genes is rather straightforward and the strategy discussed in this paper can be an
interesting followup experiment after performing genome wide array analysis to identify
regulatorymutants.
Supplementary Data
Suppl.Table1:ExpressionprofilesofinulinolyticgenesofA.niger.
gene N402 (xyl 18h) N402 (xyl 2h) N402 (mal 2h) N402 (inu 2h) N402 (suc 2h) N402 (xyl 8h) N402 (mal 8h) N402 (inu 8h) inuR (inu 8h) inuR (suc 2h) inuE 1.82 P 1.81 ± 0.28 a P,M 3.93 ± 2.10 P 7.96 ± 2.56 P 145.30 ± 27.90 P 1.89 ± 0.01 P,M 3.88 ± 0.22 P 114.60 ± 2.40 P 1.59 ± 0.36 P 1.97 ± 0.21 P,M inuA 0.58 A 0.63 ± 0.23 A 0.26 ± 0.07 A 0.77 ± 0.11 A 29.27 ± 0.80 P 0.89 ± 0.07 A 0.99 ± 0.29 A 2.14 ± 0.18 P 0.50 ± 0.16 A 0.74 ± 0.06 A sucA 0.14 A 0.11 ± 0.01 A 0.07 ± 0.04 A 0.95 ± 0.65 A 103.40 ± 21.54 P 0.11 ± 0.08 A 0.29 ± 0.60 A 6.07 ± 0.51 P 0.17 ± 0.16 A 0.26 ± 0.45 A sucB 2.05 P 1.94 ± 0.21 P 2.01 ± 0.17 P 2.23 ± 0.16 P 3.66 ± 0.49 P 1.89 ± 0.06 P 2.06 ± 0.08 P,M 2.25 ± 0.43 P 2.55 ± 0.11 P 2.08 ± 0.53 P sucC 0.45 A 0.34 ± 0.09 A 0.31 ± 0.19 A 0.33 ± 0.16 A 0.34 ± 0.30 A 0.47 ± 0.16 A 0.26 ± 0.02 A 0.66 ± 0.02 A 0.25 ± 0.06 A 0.26 ± 0.31 A
aTheexpressionlevelwasbasedonthegeometricmeanvalueoftheduplicatesamplesandthedeviationvaluesbetweentheduplicate
samplesareindicated.P,MorA,representingdetectioncallsforpresent,marginalorabsentrespectively.