EUKARYOTIC CELL, Aug. 2003, p. 708–717 Vol. 2, No. 4
1535-9778/03/$08.00⫹0 DOI: 10.1128/EC.2.4.708–717.2003
Copy igh © 2003, Ame ican Socie y o Mic obiology. All Righ s Rese ed.
Glucose Up ake in T ichode ma ha zianum: Role o g 1
Jesu´s Delgado-Ja ana,† Miguel A
´ngel Mo eno-Ma eos, and Tahía Bení ez*
Depa amen o de Gene´ ica, Facul ad de Biología, 41012 Se ille, Spain
Recei ed 27 Janua y 2003/Accep ed 9 May 2003
Using a di e en ial display echnique, he gene g 1, which codes o a high-a ini y glucose anspo e , has
been cloned om he mycopa asi e ungus T ichode ma ha zianum CECT 2413. The deduced p o ein sequence
o he g 1 gene shows he 12 ansmemb ane domains ypical o suga anspo e s, oge he wi h ce ain
esidues in ol ed in glucose up ake, such as a conse ed a ginine be ween domains IV and V and an a oma ic
esidue (Phe) in he sequence o domain X. The g 1 gene is ansc ip ionally egula ed, being ep essed a high
le els o glucose. When ca bon sou ces o he han glucose a e u ilized, g 1 ep ession is pa ially alle ia ed.
Full de ep ession o g 1 is ob ained when he ungus is g own in he p esence o low ca bon sou ce concen-
a ions. This egula ion pa e n co ela es wi h he ole o his gene in glucose up ake du ing ca bon
s a a ion. Gene exp ession is also con olled by pH, so ha he g 1 gene is ep essed a pH 6 bu no a pH
3, a ac which ep esen s a no el aspec o he in luence o pH on he gene exp ession o anspo e s. pH also
a ec s glucose anspo , since a s ongly acidic pH p o okes a 40% dec ease in glucose anspo eloci y.
Biochemical cha ac e iza ion o he anspo shows a e y low K
m
alue o glucose (12 ␮M). A ans o man
s ain ha o e exp esses he g 1 gene shows a h ee old inc ease in glucose bu no galac ose o xylose up ake,
a inding which con i ms he ole o he g 1 gene in glucose anspo . The cloning o he i s ilamen ous
ascomyce e glucose anspo e is he i s s ep in elucida ing he mechanisms o glucose up ake and ca bon
ep ession in ae obic ungi.
Filamen ous ungi a e ubiqui ous o ganisms able o ob ain
ene gy om e y di e en subs a es. Consequen ly, some o
hem a e impo an pa hogens o o he ungi, insec s, plan s,
and animals, and some a e qui e ele an in bio echnology.
The wide use o s ains o he genus T ichode ma is based on
hei abili y o deg ade plan polyme s, such as cellulose (20),
and o an agonize o he ungi (18). Fo his eason, s ains o
T ichode ma ha zianum ha e been commonly used as agen s
o he biocon ol o plan pa hogenic ungi. Se e al mecha-
nisms ha e been conside ed o be key ac o s in an agonis ic
in e ac ions: lysis o hos cell walls, an ibiosis, compe i ion o
nu ien s, induced esis ance in plan s, and inac i a ion o hos
enzymes (15). Bo h he lysis o ungal cell walls and he e i-
cien use o a ailable nu ien s a e based on he abili y o
T ichode ma s ains o ob ain ATP om he me abolism o
di e en suga s, such as hose ob ained om polyme s wide-
sp ead in ege al and ungal sou ces: cellulose, hemicellulose,
xylan, mannan, a abinan, and chi in, among o he s.
The majo i y o he enzymes in ol ed in he deg ada ion o
hese polyme s a e ep essed by glucose (2, 20, 24). The mech-
anism(s) by which glucose ep ession is igge ed emains un-
disco e ed, despi e he la ge amoun o in o ma ion ob ained
om Saccha omyces ce e isiae (13). Unlike ha o he unicel-
lula ascomyce e S.ce e isiae, he me abolism o mycelial ungi
is p e e en ially espi a o y in he p esence o oxygen. S.ce -
e isiae and o he e men a i e yeas s usually g ow on glucose-
en iched subs a es, such as g apes. Fe men a i e me abolism
co ela es pe ec ly wi h he a ailabili y o high glucose con-
cen a ions. Mos ilamen ous ungi, howe e , ha e e ol ed a
p e e ence o espi a o y me abolism and a low- a e bu high-
ATP-yield p ocess (35). The impac o his di e ence on gen-
e al ca bon ep ession mechanisms is no ye known due o he
sca ci y o da a a ailable on glucose me abolism in easily man-
ageable o ganisms wi h espi a o y me abolism. Taking ad an-
age o exp essed sequence ag analysis and mic oa ays,
Chambe go e al. (5) ha e epo ed gene al changes in he
egula ion o c i ical genes ha con ol he me abolic lux o
e men a i e o espi a o y beha io in T ichode ma eesei.
Howe e , whe he i is glucose anspo in o he cell o suga
phospho yla ion ha p o okes ca bon ep ession (13) is s ill a
ques ion ha emains o be answe ed, and he ole o he
glucose anspo sys em ha p o ides enough glucose o sup-
po g ow h when low- a e espi a o y me abolism is u ilized
also emains o be disco e ed.
Suga anspo in S.ce e isiae has been ex ensi ely s udied
(4, 22, 28, 32). The hexose anspo e s belong o a anspo e
supe amily e med he majo acili a o s supe amily (28).
Se en een genes ha e been cloned and cha ac e ized as being
in ol ed in glucose and uc ose anspo . The ac i i ies o all
o hese anspo e s gene a e wo glucose up ake sys ems: a
cons i u i e low-a ini y sys em (K
m
, 15 o 20 mM) and a glu-
cose- ep essed high-a ini y sys em (K
m
, 1 o 2 mM) (22, 32). A
simple si ua ion is obse ed in Kluy e omyces lac is, which has
only one low-a ini y (K
m
, 20 o 50 mM) inducible glucose
anspo e (56) and only one high-a ini y (K
m
, 1 mM) cons i-
u i ely exp essed glucose anspo e (3).
Fo Neu ospo a c assa, wo glucose up ake sys ems ha e
been desc ibed: a cons i u i e low-a ini y sys em (K
m
, 8 mM)
and an inducible high-a ini y sys em (K
m
,10␮M) (44–47).
F uc ose and galac ose anspo sys ems ha e also been iden-
i ied (36, 37). The di e ences among he alues o e men-
* Co esponding au ho . Mailing add ess: Depa amen o de Ge-
ne´ ica, Facul ad de Biología, A . Reina Me cedes 6, 41012 Se ille,
Spain. Phone: (34)-95-4557109. Fax: (34)-95-4557104. E-mail: ahia@
us.es.
† P esen add ess: Ins i u o de Biología Vege al y Fo osín esis, Con-
sejo Supe io de In es igaciones Cien í icas, Uni e sidad de Se illa,
41092 Se ille, Spain.
708
on July 13, 2017 by USE/BCTA.GEN UNIVERSITARIAh p://ec.asm.o g/Downloaded om
a i e yeas s and ungi wi h espi a o y me abolism may e lec
he di e en a ailabili ies o glucose in he en i onmen s o a
e y di e se g oup o ascomyce es. DNA sequences om a
pu a i e anspo e in ilamen ous ascomyce es ha e no been
epo ed, bu a pu a i e glucose senso gene, co3, has been
cloned (27). Only wo genes encoding glucose anspo e s in
ilamen ous ungi ha e been cloned and cha ac e ized o da e;
bo h a e om basidiomyce e species: hx 1 om U omyces abae
(53) and AmMs 1 om Amani a musca ia (30).
In his wo k, we epo he isola ion o a gene, g 1, which
codes o a glucose anspo e in T.ha zianum CECT 2413.
When o e exp essed, g 1 p o okes a h ee old enhancemen
o glucose anspo eloci y, a ac which makes g 1 he i s
glucose anspo e iden i ied in an ascomyce e- ela ed spe-
cies. We show ha he g 1 gene is ep essed in he p esence o
high glucose concen a ions and s ongly induced in he p es-
ence o ca bon s a a ion. We discuss a possible ole o g 1
du ing glucose assimila ion in mycopa asi ism.
MATERIALS AND METHODS
S ains, media, and g ow h condi ions. T.ha zianum CECT 2413 was ob ained
om he Coleccio´n Espan˜ola de Cul i os Tipo (Bu jasso , Valencia, Spain). T.
ha zianum was main ained on po a o dex ose aga (2% [w / ol] comme cial
mashed po a oes [dehyd a ed po a o lakes], 2% [w / ol] dex ose, 2% [w / ol]
aga ). Fo liquid cul u es, mycelia we e g own in 250-ml lasks con aining 100 ml
o minimal sal medium (MM) (34) supplemen ed wi h a ious ca bon sou ces
and 0.5% (w / ol) ammonium sul a e as a ni ogen sou ce and incuba ed a 22°C
on a o a y shake (200 pm). When needed, media we e bu e ed wi h ei he 0.2
M sodium ci a e (pH 3) o 0.2 M 2-(N-mo pholino)e hanesul onic acid–KOH
(pH 6). All media used o ans o ma ion we e as desc ibed p e iously (34).
Mycelia o bo h No he n expe imen s and he di e en ial display echnique
we e ob ained in wo s eps as ollows. MM wi h 2% glucose was inocula ed wi h
a spo e suspension ( inal concen a ion o 10
6
spo es pe ml) and incuba ed o
36 h as desc ibed abo e. Mycelia we e collec ed, washed ex ensi ely wi h 2%
MgCl
2
and dis illed wa e , and used o einocula e medium con aining he
ca bon sou ce desc ibed o each expe imen . A e 8ho incuba ion, mycelia
we e collec ed and ozen a ⫺80°C un il used o RNA ex ac ion.
Cloning o he g 1 gene, di e en ial display echnique, and cDNA isola ion.
The di e en ial display echnique was pe o med as ollows. Mycelia we e p e-
cul u ed, collec ed, and washed as desc ibed abo e and used o einocula e lasks
con aining MM wi h 2 o 0.2% glucose and bu e ed a ei he pH 3 o pH 6. The
ou esul an cul u es we e incuba ed o 8 h. One hund ed mic og ams o RNA
om each o he ou cul u es was isola ed and ea ed wi h 10 U o RNase- ee
DNase I. Finally, he RNA was ea ed wi h phenol, p ecipi a ed, and s o ed a
1␮g/␮la ⫺80°C.
Th ee di e en e e se ansc ip ion eac ions we e pe o med, each wi h a
di e en oligo(dT) (AAGCT
11
M, 2 ␮M, whe e M is G, A, o C; p o ided by
GenHun e , Nash ille, Tenn.). Two hund ed nanog ams o each RNA p epa a-
ion and 20 ␮M deoxynucleoside iphospha es we e also used in a 20-␮l eac ion
wi h Moloney mu ine leukemia i us Supe sc ip II (Gibco BRL, Paisley, Uni ed
Kingdom) in acco dance wi h he manu ac u e ’s ins uc ions. Two mic oli e s o
each eac ion mix u e (12 di e en eac ion mix u es) was ampli ied by PCR
ca ied ou wi h 0.2 ␮M concen a ions o bo h he a bi a y p ime s (H-AP
p ime s, se 10; GenHun e ) and he oligo(dT) p ime s (GenHun e ), 2 ␮M
deoxynucleoside iphospha es, and 0.2 ␮lo [␣-
33
P]dATP. Fo y cycles (94°C o
30 s, 40°C o 2 min, and 72°C o 30 s) we e comple ed by using an Ampli-Taq
polyme ase PCR ki (Roche Molecula Sys ems, B anchbu g, N.J.). This p o o-
col yielded 96 di e en eac ions, since eigh di e en a bi a y p ime s we e
used wi h he 12 di e en eac ion mix u es.
Fou mic oli e s o each eac ion mix u e was sepa a ed by 6% polyac ylamide
gel elec opho esis in T is-bo a e-EDTA bu e . Elec opho esis was pe o med
wi h Genomyx LR equipmen (Beckman Coul e Inc., Fulle on, Cali .) in ac-
co dance wi h he manu ac u e ’s ins uc ions. The d ied gel was exposed o
Kodak Biomax MR ilm (Ame sham Biosciences, Ba celona, Spain). Gel bands
showing di e en ial exp ession we e excised, ehyd a ed, eampli ied wi h he
co esponding pai o p ime s, and cloned by using pGEM-T-easy (P omega,
Madison, Wis.). Bands we e sequenced comme cially. One o hem, which
ma ched yeas anspo e s, was used o sc een a ␭-ZAP-II lib a y (S a agene,
La Jolla, Cali .) cons uc ed wi h mRNA om a cul u e con aining ungal cell
walls as he sole ca bon sou ce (B. Sua´ ez, unpublished da a) in acco dance wi h
he manu ac u e ’s ins uc ions. The comple e cDNA was isola ed, sequenced,
and named g 1.
Sequence analysis. P o ein sequences we e aligned by using he CLUSTAL W
algo i hm (17). Alignmen was ea ed as desc ibed p e iously (39). B ie ly,
sequences wi hou homology o any o he o he aligned sequences we e dele ed
om he alignmen , and a single base was le o cause a gap. All gaps exceeding
a single amino acid we e conside ed missing in o ma ion and we e eplaced by
ques ion ma ks. This edi ed alignmen was analyzed wi h he PHYLIP package,
e sion 3.5.c (h p://e olu ion.gene ics.washing on.edu/phylip). To ob ain s a is-
ical suppo o he b anches, 100 da a se s we e c ea ed by using SEQBOOT.
Dis ance ma ices we e calcula ed wi h he PROTDIST p og am, b anch leng hs
we e e alua ed by he neighbo -joining me hod (42), and he ee consensus
sequence was gene a ed wi h CONSENSE. To ob ain un oo ed pa simony anal-
ysis, he PROTPARS p og am was used (jumble op ion wi h n⫽25), and he
ee consensus sequence also was gene a ed wi h CONSENSE. The GTR1 se-
quence om humans was used as an ou g oup.
DNA p ocedu es and Sou he n analysis. S anda d molecula echniques we e
pe o med h oughou hese s udies (43). Genomic DNA isola ion and analysis
we e ca ied ou as desc ibed p e iously (8, 23). Sou he n blo analysis o
genomic DNA was pe o med wi h he comple e cDNA o g 1 as a p obe, and
a high-s ingency hyb idiza ion solu ion (50% o mamide) and a washing solu-
ion we e used as desc ibed p e iously (43).
RNA ex ac ion and No he n analysis. Mycelia we e lysed by using a Mini-
Bead Bea e (Biospec P oduc s, Ba les ille, Okla.) wi h 2.3-mm-diame e s eel
beads. RNA was isola ed by he acid phenol ex ac ion p ocedu e (6). Ten
mic og ams o o al RNA om each sample was sepa a ed on 1.2% aga ose–
o maldehyde gels, blo ed on o nylon memb anes, and hyb idized (43). Blo s
we e p obed wi h he comple e g 1 cDNA. P obes we e labeled wi h
[␣-
32
P]dCTP by using an oligolabeling ki (Ame sham Pha macia Bio ech). The
loading con ol o No he n blo s was checked by using adish 18S RNA as a
p obe.
T ans o ma ion p ocedu e. P o oplas p epa a ion and ans o ma ion we e
ca ied ou as desc ibed by Pen ila¨e al. (34). T.ha zianum was co ans o med
wi h plasmids pLMRS3::g 1 and p3SR2 (34); he la e ca ies he A.nidulans
amdS gene as a selec ion ma ke , allowing g ow h in media wi h ace amide as he
sole ni ogen sou ce. Plasmid pLMRS3::g 1 was cons uc ed by using g 1 cDNA
(1.9 kb) o he pki (py u a e kinase) p omo e and o he cbh2 (cellobiohyd olase
II) e mina o , bo h om T. eesei (26). The cDNA was ob ained wi h p ime s
G 1U (5⬘-ATGGTCAAGGTCCTCTAGACAAAGCATCAA-3⬘) and G 1L
(5⬘-AGCCTACCGCCATGCATAAGATTATCATCG-3⬘), which in oduced
XbaI and NsiI es ic ion enzymes si es. PCR was ca ied ou o 35 cycles o 30 s
a 94°C,30sa 55°C, and 90 s a 72°C wi h Expand high- ideli y polyme ase
(Roche Molecula Sys ems). The DNA was ea ed wi h XbaI and NsiI, and he
agmen was cloned in o plasmid pLMRS3 (26) ha had been ea ed wi h he
same enzymes. Co ans o ma ion was conduc ed wi h a 1:10 (amdS::g 1) plasmid
a io. Colonies ob ained a e ans o ma ion and selec ion we e allowed o g ow
on po a o dex ose aga in o de o p oduce spo es, which we e collec ed and
sp ead on o selec i e medium pla es. This p ocess was epea ed wice. T ans o -
man s we e hen checked o mi o ic s abili y, and hose ha showed 100% o
spo es g owing on selec i e medium we e chosen o u he s udy.
[
14
C]glucose up ake s udies. T.ha zianum was used o inocula e MM wi h 2%
glyce ol as a ca bon sou ce a a inal concen a ion o 10
7
conidia pe ml.
Cul u es we e incuba ed o 19 h a 22°C. Spo es we e swollen, bu less han 1%
showed a ge m ube p o usion. A his poin , cells we e collec ed by cen i u-
ga ion a 4°C and washed ou imes wi h MM wi hou a ca bon sou ce. Finally,
cells we e wo old concen a ed in MM. When needed, MM was bu e ed wi h
0.1 M phospha e bu e a pH 3, 4, 5, o 6. The assay was pe o med wi h 250-␮l
aliquo s. Equal olumes o 2 mM, 1 mM, 200 ␮M, 100 ␮M, 20 ␮M, 10 ␮M, and
2␮M glucose solu ions (each con aining 2 ␮M[
14
C]glucose [31 Ci/mmol]) we e
added o p ewa med cells, and anspo eac ions we e ca ied ou o 5, 30, 60,
and90sa 30°C. Reac ions we e s opped by he addi ion o 1.5 ml o ice-cold 200
mM non adioac i e glucose. Cells we e il e ed h ough HAWP 02500 ni ocel-
lulose il e s (Millipo e, Bed o d, Mass.) and washed wice wi h he same glucose
solu ion. Radioac i i y was measu ed by using a Wallac 1409 scin illa ion
coun e . Cell iabili y was ob ained by pla ing se ial dilu ions o 250-␮l aliquo s
on po a o dex ose aga plus 0.1% ( ol/ ol) T i on X-100. Colonies we e coun ed
a e 3 days o incuba ion. T anspo expe imen s we e pe o med a leas h ee
imes in duplica e, wi h simila esul s. The double- ecip ocal plo was plo ed
wi h da a ela i e o wild- ype minimal alues in o de o minimize a iabili y
among expe imen s.
VOL. 2, 2003 GLUCOSE TRANSPORTER IN T.HARZIANUM 709
on July 13, 2017 by USE/BCTA.GEN UNIVERSITARIAh p://ec.asm.o g/Downloaded om
Nucleo ide sequence accession numbe . The g 1 nucleo ide sequence was
assigned GenBank accession numbe AJ269534.
RESULTS
The sequence o he g 1 gene, isola ed by di e en ial dis-
play, shows ea u es o suga anspo e genes. An agonis ic
in e ac ions be ween T ichode ma and ungal hos s ake place
in poo en i onmen s wi h a low a ailabili y o nu ien s,
mainly ca bon sou ces. The e o e, we we e in e es ed in iso-
la ing genes which we e di e en ially exp essed in cul u es
g own in he p esence o high o low glucose concen a ions.
We used a di e en ial display echnique wi h RNA isola ed
om mycelia ob ained by cul i a ing T.ha zianum CECT 2413
in media con aining ei he 2 o 0.2% glucose and bu e ed a
ei he pH 3 o pH 6 as desc ibed in Ma e ials and Me hods.
Media we e bu e ed because a numbe o genes in ol ed in
he an agonism o T.ha zianum, such as genes o p o eases
(9) and glucanases and chi inases (M. A. Mo eno-Ma eos and
T. Bení ez, unpublished da a), a e pH con olled. Gene ex-
p ession con olled by pH has also been desc ibed o ungal
pa hogens o insec s and mammals, such as Me a hizium
anisopliae and Candida albicans (7, 48). One o he clones
exp essed only in medium con aining a low glucose concen a-
ion was chosen o u he cha ac e iza ion in his s udy. The
DNA agmen ob ained was used as a p obe o sc een a
␭⫺ZAP-II cDNA lib a y.
The 1,972-bp cDNA isola ed was named g 1 and had a
deduced p oduc o 561 amino acids wi h a calcula ed molec-
ula mass o 62.1 kDa. The g 1 gene is p esen in he genome
o T.ha zianum as a single copy (Fig. 1), and no o he hyb id-
iza ion signals we e de ec ed unde low-s ingency condi ions
(da a no shown). A high deg ee o simila i y o ungal glucose
anspo e s was obse ed by means o he BLASTX algo i hm
(1). Some o hese p o eins we e aligned by using he
CLUSTAL W algo i hm (17), and he co esponding dis ance
and pa simony phylogene ic ees we e ob ained. Only he
dis ance phylogene ic ee is displayed (Fig. 2), since pa si-
mony me hods ga e e y simila esul s. The G 1 p o ein
g ouped wi h Hg 1 om K.lac is (3) and Hg 1 om C.albicans
(52); hese esul s seem o indica e ha hese p o eins cons i-
u e a di e en sub amily o anspo e s (4). The es o he
p o eins g ouped wi h yeas senso s (Rg , Sn 3, and Rag4;
g oup A) o yeas anspo e s (g oup B), whe eas he ilamen-
ous ungal p o eins ep esen ed sepa a e b anches wi hou
s a is ical suppo o being g ouped oge he . Rco3, a pu a i e
senso p o ein om N.c assa (27), did no g oup wi h he
o he senso s. The G 1 p o ein showed 53 and 54% iden i ies,
espec i ely, wi h high-a ini y glucose anspo e s, such as
Hg 1 om K.lac is (3) and Hg 1 om C.albicans (52). The
G 1 p o ein deduced sequence was also e y simila (44%) o
ha o suga anspo e Ms A om he basidiomyce e A.
musca ia (30) and Hx 1 om U. abae (53), glucose anspo -
e s om Schizosaccha omyces pombe (ca. 44%) (16), and he
pu a i e glucose senso Rco3 om N.c assa (42.5%) (27). The
G 1 p o ein lacks he long C- e minal ail ha unc ions as a
signal ansduce in he yeas p o eins and ha is also p esen
in Rco3 (31) (Fig. 3). The amino- and ca boxyl- e minal e-
gions p edic ed o be on he cy osolic side did no show a high
deg ee o homology in he sequences analyzed (Fig. 3).
Twel e pu a i e ansmemb ane domains we e de ec ed by
using he TopP ed algo i hm (54) and he hyd opa hy p o ile
algo i hm o Ky e and Dooli e (21) (da a no shown). These
domains a e dis ibu ed wi h a long ex acellula loop p esen
be ween domains I and II, an in acellula loop loca ed be-
ween segmen s VI and VII, and ano he in acellula loop
loca ed be ween domains IX and X (Fig. 3). This model has
also been desc ibed o mammalian glucose anspo e GHT1
(14, 29). Some speci ic amino acids, such as se e al leucines
and isoleucines be ween domains I and II, which a e p obably
in ol ed in he oligome iza ion o anspo e p o eins, a e
well conse ed (32). The ime ic domain GRR is p esen in
he loops in he cy oplasm ha connec domains II and III and
domains VIII and IX, al hough he second one is dis up ed in
he G 1 p o ein by he dime FT. A g151 is conse ed in all o
he glucose anspo e s s udied (Fig. 3). The sequence o G 1
also shows a Phe412 esidue well conse ed in o he glucose
anspo e s, such as Hx 2 om S.ce e isiae, whe e i s ele-
ance in suga speci ici y has been p o ed (19). The same ole
has been a ibu ed o Ty 440 in he Hx 2 sequence. The se-
quence o Gal2, a yeas galac ose anspo e , shows subs i u-
ions o hese Phe and Ty esidues by Ty 446 and T p455,
espec i ely. The G 1 sequence also shows a yp ophan es-
idue (T p425) ins ead o he y osine esidue o Hx 2 (Fig. 3).
The p esence o yp ophan is also obse ed in Hg 1 om K.
lac is and Hx 1 om U. abae, as well as o he glucose ans-
po e s.
No he n blo analysis o g 1.No he n blo expe imen s
we e ca ied ou in o de o in es iga e he ela ionship be-
ween he a ailabili y o a ca bon sou ce and he le el o
exp ession o g 1. Fi s , di e en hexoses, such as glucose,
uc ose, and galac ose, we e used a high (2%) and low (0.2%)
concen a ions (Fig. 4A). When high suga concen a ions
we e used, weak signals we e de ec ed wi h uc ose and ga-
lac ose bu no glucose. g 1 mRNA accumula ion was s ong in
he p esence o all o he suga s a low concen a ions. Thus,
g 1 ansc ip ion was ully de ep essed when suga s we e
p esen a low concen a ions. Speci ically, he lack o glucose,
e en when ano he ca bon sou ce was p esen a a high con-
cen a ion, p o oked a sligh de ep ession.
FIG. 1. Sou he n blo analysis. Genomic DNA was diges ed wi h
he ollowing es ic ion enzymes: SalI (lane 1), XhoI (lane 2), XbaI
(lane 3), ClaI (lane 4), and BamHI (lane 5). Comple e cDNA o g 1
was used as a p obe. Single bands we e de ec ed, unless enzymes which
cu he g 1 sequence we e used, such as SalI and XhoI.
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A second se o expe imen s was designed o de e mine he
ole o o he ca bon sou ces, such as glyce ol, on g 1 mRNA
accumula ion (Fig. 4B). The use o glyce ol as he sole ca bon
sou ce ga e ise o a basal le el o g 1 mRNA compa ed o
wha was seen unde de ep ession condi ions (0.2% glucose).
The addi ion o small amoun s o suga (s) o glyce ol-con ain-
ing media did no induce o ep ess he exp ession o g 1,
excep o pen oses. When 0.2% xylose o a abinose was used
in combina ion wi h glyce ol as ca bon sou ces, a small in-
c ease ( h ee old wi h xylose) in he le el o exp ession o g 1
was obse ed. Howe e , his inc ease was almos negligible
compa ed o he exp ession obse ed in cul u es wi h glucose
a a low concen a ion. The use o xylose a a high concen a-
ion in combina ion wi h glyce ol did no inc ease he exp es-
sion o g 1 compa ed o ha seen in glyce ol-con aining media
(Fig. 4C). The e o e, g 1 was sligh ly de ep essed when glyc-
e ol was used as a sole ca bon sou ce, and his basal le el was
inc eased when pen oses, such as xylose o ( o a lesse ex en )
a abinose, bu no hexoses we e p esen a low concen a ions.
The de ep ession o g 1 was maximal when glucose (o o he
suga s) was used a a e y low concen a ion as he sole ca bon
sou ce, ha is, unde ca bon s a a ion condi ions.
Ano he se o No he n blo expe imen s was ca ied ou in
o de o in es iga e he hie a chy be ween pH and ca bon
sou ce egula ion o g 1 exp ession (Fig. 4D). Cul u es con-
aining glucose o galac ose a high (2%) and low (0.2%)
concen a ions and a pH 6 o 3 we e used. As expec ed, g 1
was ep essed a high glucose le els and induced a low le els.
Howe e , he g 1 mRNA le el was lowe when he pH o he
low-glucose cul u e was nea neu ali y (pH 6) han in he
p esence o an acidic pH (pH 3). This pH e ec on gene
exp ession was mo e no iceable when galac ose was used as he
ca bon sou ce, since exp ession was obse ed a pH 3 e en
wi h a high galac ose concen a ion. Bu e ing cul u es a pH 6
seems o ep ess he exp ession o g 1, e en wi h a low galac-
ose concen a ion. In conclusion, acidic pH condi ions p o-
oked g 1 mRNA accumula ion wi h high galac ose bu no
high glucose concen a ions.
The G 1 p o ein is a glucose anspo e . Due o simila i ies
be ween g 1 and o he sequences ound in sequence da abases
and o i s ansc ip ion pa e n, a po en ial ole in he anspo
o hexoses could be assigned o he G 1 p o ein. To examine
his hypo hesis, we i s ied o complemen yeas mu an s,
such as s ains RE700 and EBY.W4000, which a e unable o
FIG. 2. Homology o he G 1 p o ein o o he ungal anspo e s. A dis ance phylogene ic ee was ob ained as desc ibed in Ma e ials and
Me hods (pa simony me hods ga e e y simila esul s). Sequences we e aligned by using he CLUSTAL W algo i hm (17), and a dis ance ee
o 100 boo s apped da a se s was gene a ed by using he PROTDIST p og am and he neighbo -joining me hod (42). Each numbe on he ee
ep esen s he numbe o imes he g oup loca ed o he igh occu ed in 100 di e en ees (12). B anches no suppo ed (⬍90) we e collapsed.
Designa ions in pa en heses a e GenBank accession numbe s. Pe cen ages in pa en heses indica e he pe cen simila i y o he G 1 p o ein. A and
B indica e g oups.
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g ow in he p esence o glucose due o he lack o glucose o
hexose anspo e s (38, 57). Vec o pAJ401, which con ains
he yeas pgk p omo e , was used o g 1 exp ession in yeas s.
The ans o man s ob ained we e es ed o hei abili y o
g ow in he p esence o glucose o uc ose. Plasmid
pAJ401::g 1 did no appea o complemen he hexose up ake
de ec s in ei he o he wo s ains, in spi e o he ac ha g 1
mRNA was being p ope ly de ec ed (da a no shown). Fo his
eason, we decided ha he unc ion o G 1 should a he be
es ed wi h ilamen ous ungi. We ha e ied ex ensi e gene
dis up ion when analyzing he g 1 gene and o he genes (T.
Bení ez, pe sonal communica ion). Howe e , we ha e ob-
ained no null mu an s o T.ha zianum CECT 2413 o da e.
These esul s led us o design a s a egy o he han dele ion o
de e mine which suga (s) was anspo ed by G 1. A cons uc
in which he g 1 gene was placed unde he con ol o he
cons i u i e py u a e kinase gene p omo e om T. eesei was
made. This p omo e allows a high le el o exp ession when
glyce ol is used as he sole ca bon sou ce (8). T.ha zianum
CECT 2413 was ans o med, and a s ain, designa ed T1, ha
ca ied one ex a copy o he g 1 gene (Fig. 5A) was isola ed
and cha ac e ized. S ain T1 showed a highe g 1 exp ession
le el han he wild- ype s ain when ei he 2% glucose (Fig.
5B) o 2% glyce ol (da a no shown) was used. Hence, he use
o glyce ol as a sole ca bon sou ce allowed us o analyze wo
isogenic s ains, one o which showed enhanced exp ession o
g 1. S ain T1 did no show ei he g ow h o an agonis ic
de ec s compa ed o he wild- ype s ain (da a no shown).
To measu e anspo capaci y, spo es o bo h s ains we e
ge mina ed in MM con aining 2% glyce ol as a ca bon sou ce
o 19 h. Glyce ol was used ins ead o glucose because o he
possibili y ha pos ansc ip ional mechanisms could a ec he
s abili y and/o ac i i y o he p o ein. Since he g 1 gene was
exp essed when glyce ol was used, i was assumed ha hose
mechanisms would be ac i e unde hese condi ions. Low glu-
cose concen a ions we e no used because he high g 1
mRNA exp ession le els in he wild- ype s ain we e e y sim-
ila o hose in s ain T1 unde s ong de ep ession condi ions
(da a no shown). Almos none o he spo es showed p o u-
sion o he ge m ube, bu all we e swollen. These cells we e
used o measu e he anspo o glucose, xylose, and galac ose.
Xylose was chosen because o he possibili y ha enhanced
exp ession in media con aining glyce ol plus xylose could e-
lec a ole in pen ose anspo . We also used galac ose be-
cause G 1 sha es a conse ed amino acid (T p425) in ol ed in
anspo speci ici y wi h Gal2, and we ied o assess whe he
G 1 plays a ole in he up ake o galac ose.
S ain T1 was able o anspo glucose wo o h ee imes
as e han he wild- ype s ain when low concen a ions o
glucose we e used in measu emen s (1 ␮M o 1 mM) (Fig. 6A).
When he da a we e plo ed as double- ecip ocal plo s (Lin-
ewea e -Bu k ans o ma ion), a K
m
o abou 12 ␮M could be
calcula ed o he high-a ini y glucose anspo componen o
T.ha zianum (Fig. 6). The calcula ed V
max
(ob ained wi h he
double- ecip ocal plo ) o s ain T1 was abou 1.9 ⫻10
⫺3
nmol/10
6
cel/s, whe eas he alue o he wild- ype s ain was
abou wo o h ee old lowe : 0.9 ⫻10
⫺4
nmol/10
6
cel/s. No
enhancemen o anspo capabili y o ei he xylose o galac-
ose was obse ed o s ain T1 unde he condi ions es ed
(Fig. 6A). These esul s led us o conclude ha g 1 codes o
a high-a ini y glucose anspo e .
Glucose anspo is dis u bed a an acidic pH. One o he
mos in e es ing aspec s o g 1 was he pH con ol o gene
exp ession (Fig. 4C). Acidic media, wi h a high a ailabili y o
p o ons, could gi e ise o he induc ion (o de ep ession) o
g 1 ( h ough PacC o a simila ansc ip ion ac o ) (11) in
o de o u ilize his g adien o p o ons o anspo glucose.
Al e na i ely, G 1 ac i i y migh be dis u bed a a s ongly
acidic pH. Then, he lack o glucose up ake migh igge he
de ep ession o induc ion o he g 1 gene by a C eA-like
mechanism (10, 41). To in es iga e hese hypo heses, we mea-
su ed he anspo eloci y o s ain T1 g own o 19 h in
glyce ol-con aining medium bu e ed a di e en pHs (pH 3, 4,
5, o 6) by using a anspo assay (Fig. 7). The up ake o
glucose was diminished abou 20% a pH 4 wi h espec o pH
5 and abou 40% a pH 3. Since cells we e g own unde he
same condi ions, his e ec can be explained only by a slowing
down o he glucose anspo e unc ion a an acidic pH.
DISCUSSION
We epo he isola ion and cha ac e iza ion o he g 1 gene,
he i s glucose anspo e gene desc ibed o an ascomyce e-
ela ed ilamen ous ungus. Al hough we ha e highly de ailed
in o ma ion abou he anspo o hexoses in yeas s such as S.
ce e isiae and K.lac is, only wo anspo e p o eins ha e been
desc ibed o ilamen ous ungi, bo h in basidiomyce es:
AmMs A om A.musca ia (30) and Hx 1 om U. abae (53).
Twen y HXT genes in ol ed in hexose anspo ha e been
isola ed om S.ce e isiae, all o hem wi h signi ican simila -
i ies in hei sequences. Howe e , g 1 seems o be p esen in a
single copy in he T.ha zianum genome. S.ce e isiae has o
ca y ou e men a i e me abolism when glucose is a ailable a
a high concen a ion (g ape mus ). Fe men a ion is a p ocess
ha yields low amoun s o ene gy (2 mol o ATP/mol o glu-
cose), so a high lux o glucose, anspo ed by a numbe o
pe meases, is necessa y o ensu e he gene a ion o enough
ene gy du ing p ocesses such as enological e men a ion, when
suga a ailabili y changes cons an ly (25). En i onmen s in-
habi ed by T ichode ma s ains a e nu ien poo , and hei
esou ces a e exploi ed by hei ex acellula hyd olases.
T ichode ma has espi a o y me abolism (5), which p oduces
highe ATP yields (36 mol o ATP/mol o glucose). Fo hese
easons, wo anspo e s wi h di e en K
m
alues may be su -
FIG. 3. Alignmen o a ious ungal anspo e s and anspo e -like p o eins. (A) Alignmen o deduced p o ein sequences, om op o
bo om, o G 1 om T.ha zianum, Hg 1 om K.lac is, HXT1 om U. abae, Sn 3, Gal2, and Hx 2 om S.ce e isiae, and Rco3 om N.c assa.
Sequences we e aligned by using he CLUSTAL W algo i hm (17). Ho izon al black ba s indica e he ansmemb ane domains o G 1 ob ained
by using he TopP ed algo i hm (54). Black shading indica es highly conse ed esidues, and g ey shading indica es lesse deg ees o simila i y. The
black a ow indica es A g151, he s iped a ow indica es Phe412, and he g ey a ow indica es T p425 (posi ions ela i e o hose o he G 1
sequence).
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icien o sus ain he espi a o y me abolism and g ow h o T.
ha zianum unde ield condi ions, wi hou la ge oscilla ions in
suga concen a ions. The second anspo e p obably is no
ela ed o G 1, since low-s ingency hyb idiza ion did no
show addi ional signals. This si ua ion esembles ha in K.
lac is, in which only wo anspo e s ha e been iden i ied: one
wi h a high a ini y and one wi h a low a ini y.
Dis ance phylogene ic ee analysis, which places he G 1
p o ein nea o he high-a ini y hexose anspo e s om asco-
myce es, such as K.lac is and C.albicans, and sequence ea-
u es indica ed a ole o G 1 in glucose anspo in T.ha -
zianum. Mo eo e , he pa e n o g 1 gene ansc ip ion is e y
simila o ha desc ibed o high-a ini y hexose anspo e
genes. A high exp ession le el is obse ed only when glucose is
p esen a a e y low concen a ion (0.2%). The same esul
has been obse ed wi h hx 2 om S.ce e isiae (55). The g 1
gene is also ep essed when T.ha zianum is cul i a ed in media
wi h 2% glucose, a ea u e ypical o he genes o anspo e s
in ol ed in he high-a ini y up ake o hexoses, such as HXT2,
HXT4,HXT6, and HXT7 (32). These wo cha ac e is ics p o-
ide a use ul mechanism o ensu ing ha a gene will be an-
sc ibed when needed (4). In K.lac is,hg 1 gene egula ion is
di e en : his high-a ini y anspo e gene is ansc ibed con-
s i u i ely, whe eas a low-a ini y anspo e gene is induced by
la ge amoun s o glucose. The same pa e n has been desc ibed
o Aspe gillus nige (51). These s a egies ha e a common
elemen : one o he genes is cons i u i ely ansc ibed, whe eas
he o he one is egula ed. These indings allow us o sugges
ha , on he basis o he model o K.lac is,T.ha zianum e y
likely has ano he gene coding o a hexose anspo e ; his
gene will be a low-a ini y anspo e gene and p obably will be
cons i u i ely exp essed.
g 1 is ully ep essed only when high le els o glucose a e
p esen . Pa ial de ep ession is obse ed when he suga used
is o he han glucose (glyce ol, uc ose, galac ose, xylose, and
a abinose). De ep ession is ully ob ained when suga s a e
a ailable only a low le els (0.2%). Tha is, g 1 seems o be
only ep essible and de ep essible bu no inducible, since
small amoun s o glucose combined wi h glyce ol did no aise
g 1 mRNA le els. Glyce ol in T.ha zianum is p obably me-
abolized o py u a e ia glycolysis o o glucose ia glucone-
ogenesis (13). T.ha zianum equi es abundan suga phos-
pha es and depends on gluconeogenesis o p o iding
ca bohyd a es mos ly o cell wall biosyn hesis and hyphal
g ow h (51). I glyce ol is apidly me abolized, hen ca abolic
ep ession could be exe ed by glucose syn hesized om glyc-
e ol h ough gluconeogenesis. The a e o g ow h o T.ha zia-
num in glyce ol is simila o ha in glucose (da a no shown),
so ha he e iciency o glyce ol ca abolism migh in luence
FIG. 4. No he n blo analysis. RNA was ex ac ed om mycelia
g own unde he condi ions indica ed in Ma e ials and Me hods. Va -
ious ca bon sou ces we e used a 2 o 0.2% (w / ol): glucose (Gluc),
uc ose (F c), galac ose (Gal), a abinose (A a), xylose (Xyl), and
glyce ol (Gly). In panels B and C, he p esence and absence o 2%
glyce ol a e indica ed by plus and minus signs, espec i ely. In panel D,
media we e bu e ed a he indica ed pHs. Radish 18S RNA was used
as a loading con ol.
FIG. 5. Cha ac e iza ion o s ain T1. (A) Sou he n blo analysis.
Genomic DNA was diges ed wi h he XbaI enzyme. w , wild- ype
s ain. (B) No he n blo analysis. RNA was ex ac ed om mycelia
g own wi h 2% glucose as a ca bon sou ce as desc ibed in Ma e ials
and Me hods. Fo bo h panels A and B, he comple e g 1 gene was
used as a p obe, and adish 18S RNA was used as a loading con ol.
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FIG. 6. Kine ics o up ake o D-glucose. (A) Up ake o D-glucose, D-xylose, and D-galac ose (nanomoles o glucose anspo ed pe million li ing
cells) o T ichode ma s ains CECT 2413 (wild- ype s ain) and T1 (s ain ha o e exp esses he g 1 gene). The amoun o suga used in each
anspo assay is indica ed abo e each g aph. (B) Double- ecip ocal plo s used o ob ain he K
m
and V
max
alues o high-a ini y D-glucose
anspo . The inse is an ampli ica ion o he 1/S alues om 0 o 0.02 ␮M. V, eloci y. E o ba s indica e s anda d de ia ions.
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bo h he g ow h a e and he glucose concen a ion inside he
cell; ca bon ca aboli e egula ion migh be media ed by he
ep esso C e1p in a manne simila o ha o Mig1p (10, 13,
40, 41). Al e na i ely, he esul s migh indica e a mechanism
o ca bon ep ession di e en om ha in yeas s and ilamen-
ous ungi, which is ela ed o he pa icipa ion o he ep esso
p o eins Mig1 and C eA, as has been sugges ed by o he au-
ho s (11). g 1 egula ion di e s subs an ially om ha shown
o he mos simila anspo e genes. While g 1 exp ession
seems o be ep essible, HGT1 om K.lac is (3) and AmMs A
om A.musca ia (30) we e highly exp essed in media con ain-
ing high glucose concen a ions. The exp ession o he HXT1
gene om U. abae was es ic ed o haus o ia (53), al hough
s udies wi h shake lask cul u es o analyze ca bon egula ion
o gene exp ession ha e no been done o da e, as ha e been
done o he HGT1 gene om C.albicans (52).
The use o galac ose allowed us o iden i y a no el aspec o
anspo e egula ion in T.ha zianum: he ole o pH. g 1
seems o be ully ep essed in he p esence o 2% glucose a
neu al o acidic pH, al hough he de ep ession obse ed a
low glucose le els was highe a acidic pH han a neu al pH.
This e ec was obse ed in a mo e ex eme manne when
galac ose was used, since g 1 exp ession was ob ained e en
when galac ose was used a high concen a ions a acidic pH
bu no a neu al pH. T anspo expe imen s showed a de-
c ease in glucose anspo a acidic pH, as has been desc ibed
o A.nige (51). G 1 ac i i y seems o be dis u bed a a
s ongly acidic pH compa ed o he anspo ob ained a pH 5.
This dec ease p o okes a slow educ ion in in acellula glu-
cose le els and may be he eason why acidic pH ep esses g 1
exp ession in cul u es wi h low glucose concen a ions. The
dec ease in glucose up ake obse ed a pH 6 could be due o
he educ ion in g 1 exp ession (Fig. 4D).
In he p esen s udy, he deduced K
m
o high-a ini y ans-
po in T.ha zianum g own in glyce ol was abou 12 ␮M. g 1
is p esen in a single copy, and he e a e no closely ela ed
genes in he genome o T.ha zianum CECT 2413. These ac s,
oge he wi h he enhancemen o glucose up ake in s ain
T1—which ca ies one addi ional copy—indica e a signi ican
ole o G 1 in he anspo o glucose when T.ha zianum is
g own in he p esence o low le els o suga s. Un o una ely,
he low K
m
alue ob ained p e en ed us om measu ing pos-
sible di e ences in g ow h a es be ween s ain T1 and he
wild- ype s ain a mic omola concen a ions o glucose. No
di e ences in g ow h a es be ween s ain T1 and he wild- ype
s ain we e de ec ed a 0.5, 5, and 25 mM glucose (da a no
shown). Howe e , a hese concen a ions, a second, low-a in-
i y glucose anspo e migh be ac i e. The K
m
alue is lowe
han hose measu ed o S.ce e isiae (32) and K.lac is (3), bo h
ca. 1 o 2 mM, and S.pombe (16), A.musca ia (30), and U.
abae (53) (all ca. 0.4 mM). Howe e , mos o hese da a we e
ob ained wi h he e ologous sys ems, by aking ad an age o
s ain RE700 o S.ce e isiae (38, 57) and/o g owing mic oo -
ganisms a high glucose concen a ions, which may in ol e he
ansc ip ional induc ion o only low-a ini y anspo e s. A
e y low K
m
(10 ␮M) has been es ima ed o he anspo o
glucose in N.c assa g own in media wi h low concen a ions o
his suga (44, 45). Simila kine ic da a (Km,15␮M) ha e also
been ob ained o Candida u ilis g own in he p esence o
glyce ol (33). Bo h eac ions a e media ed by ac i e anspo ,
p obably also he case o T.ha zianum.U. abae—a bio ophic
basidiomyce e—possesses an H
⫹
ATPase and a p o on-cou-
pled glucose anspo sys em loca ed a he haus o ial in e -
ace du ing he p ocess o in ec ion o Vicia aba (49, 50, 53,
58). This in o ma ion sugges s a e y a ac i e idea: an ago-
nism combined wi h a e y-low-K
m
anspo e could allow T.
ha zianum no only o ob ain ene gy om hyd olyzed polyme s
bu also o apidly ake suga molecules in o he cells, p o-
cesses which could ep esen e y use ul mechanisms o com-
pe ing o nu ien s du ing mycopa asi ic in e ac ions. We
ha e de e mined ha g 1 mRNA le els a e inc eased when T.
ha zianum is con on ed wi h he ungus Rhizoc onia solani
(da a no shown). Howe e , i is di icul o de e mine speci ic
exp ession due o he s ong signal obse ed du ing he ca bon
s a a ion condi ions needed o p o oke an an agonis ic in e -
ac ion.
The isola ion o g 1, he i s high-a ini y glucose ans-
po e gene isola ed om an ascomyce e- ela ed ilamen ous
ungus, p o ides a new ool o help lea n whe he mechanisms
o glucose ep ession simila o hose desc ibed o e men a-
i e yeas s a e p esen in ae obic ungi and whe he hese
ep esen key me abolic di e ences be ween e men a i e
yeas s and ungi wi h espi a o y me abolism.
ACKNOWLEDGMENTS
We a e g a e ul o Daniel Ramo´n and And ew MacCabe (Ins i u o
de Ag oquímica y Tecnología de Alimen os, Valencia, Spain) o help
wi h ini ial suga up ake measu emen s; o Ra ael C. Jime´nez Dome-
nech o help wi h sequence analysis; o Bele´n Sua´ ez o p o iding us
wi h he ␭-ZAP-II lib a y; and o Ana M. Rinco´n, Hamza El-Do y,
and Rosa io Lagunas (Ins i u o de In es igaciones Biome´dicas, Ma-
d id, Spain) o c i ical eading o he manusc ip .
M.A.M.-M. is he ecipien o a g an om he Minis e io de Ciencia
y Tecnología. This wo k was suppo ed by g an s PAI CVI-107 and
CICYT IFD97-0668, IFD97-0820, and AGL2000-0524.
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FIG. 7. Dec eased up ake o D-glucose a acidic pH. The up ake o
D-glucose was measu ed by using 10 ␮MD-glucose and di e en phos-
pha e bu e s a pH 3, 4, 5, o 6. T anspo eloci y was ob ained by
using slopes o he linea eg ession o D-glucose up ake a 0, 5, 30, 60,
and 90 s. E o ba s indica e s anda d de ia ions.
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