The expression of selected non-ribosomal peptide synthetases in Aspergillus fumigatus is controlled by the availability of free iron

The exp ession o selec ed non- ibosomal pep ide syn he ases in Aspe gillus umiga us is con olled by he a ailabili y o ee i on Ka h in Reibe a , Eme P. Ree es a , Clai e M. Ne ille a , Robe Winkle a,b , Pe e Gebha d a,b , Ke in Ka anagh a , Sean Doyle a,* a Na ional Ins i u e o Cellula Bio echnology, Depa men o Biology, Na ional Uni e si y o I eland, Maynoo h, Co. Kilda e, I eland b Hans-Knoell-Ins i u e o Na u al P oduc Resea ch, Depa men o Bios uc u e Chemis y, Jena, Beu enbe gs . 11, D-07745, Ge many Recei ed 7 Ap il 2005; ecei ed in e ised o m 12 May 2005; accep ed 16 May 2005 Fi s published online 31 May 2005 Edi ed by G.M. Gadd Abs ac Th ee non- ibosomal pep ide syn he ase genes, e med sidD,sidC and sidE, ha e been iden ified in Aspe gillus umiga us. Gene exp ession analysis by RT-PCR confi ms ha exp ession o bo h sidD and Cwas educed by up o 90% unde i on- eple e condi- ions indica i e o a likely ole in side opho e biosyn hesis. SidE exp ession was less sensi i e o i on le els. In addi ion, wo p o eins pu ified om mycelia g own unde i on-limi ing condi ions co esponded o SidD (200 kDa) and SidC (496 kDa) as de e mined by MALDI ToF pep ide mass finge p in ing and MALDI LIFT-ToF/ToF. Side opho e syn he ases a e unique in bac e ia and ungi and ep esen an a ac i e a ge o an imic obial chemo he apy. 2005 Fede a ion o Eu opean Mic obiological Socie ies. Published by Else ie B.V. All igh s ese ed. Keywo ds: NRPS; I on; Side opho e; MALDI ToF; Seconda y me aboli es; P o eomics; 40-PPTase 1. In oduc ion The ungus Aspe gillus umiga us is ecei ing inc eas- ing a en ion as a significan cause o mo ali y in immu- nocomp omised indi iduals such as bone ma ow and s em cell ansplan pa ien s [1]. The nea -comple ion o he A. umiga us genome sequencing effo and esul- an da a [2] has g ea ly acili a ed he sea ch o genes which may be in ol ed in media ing o ganismal pa ho- genici y and a numbe o epo s ha e shown ha ei he specific gene dele ion o silencing may down egula e he i ulence o A. umiga us [3–5]. Hissen e al. [6] ha e con incingly a gued ha side opho es p oduced by he ungus a e in ol ed in i on acquisi ion om ans e in and ha , a leas in i o, hey may be esponsible o o ganism g ow h and su i al in human se um. Mos membe s o he amily Ascomyco a (e.g., A. umiga us) p oduce hyd oxama e- ype side opho es. Hyd oxama es can be sub-di ided in o ou g oups: ho- do o ulic acid, usa inines, cop ogens and e ich omes. A. umiga us p oduces e ich ome class side opho es (e.g., e ic ocin), which a e cyclic pep ides [7]. In addi- ion, a numbe o side opho es (p ima ily iace yl usa - inine C and e ic ocin) we e pu ified om A. umiga us cul u e medium a e 8 h o g ow h in medium con ain- ing human se um [6]. In Aspe gillus nidulans, side opho e biosyn hesis commences wi h he N 5 -hyd oxyla ion o o ni hine, ol- lowed by ansacyla ion and subsequen co alen assem- bly o he modified o ni hine esidues wi h o wi hou u he amino acids (e.g., se ine, alanine and glycine), 0378-1097/$22.00 2005 Fede a ion o Eu opean Mic obiological Socie ies. Published by Else ie B.V. All igh s ese ed. doi:10.1016/j. emsle.2005.05.028 * Co esponding au ho . Tel.: +353 1 7083858; ax: +353 1 7083845 E-mail add ess: [email p o ec ed] (S. Doyle). www. ems-mic obiology.o g FEMS Mic obiology Le e s 248 (2005) 83–91 ARTICLE IN PRESS ca alyzed by non- ibosomal pep ide syn he ases (NRPS) [8,9]. NRPS a e mul i unc ional enzymes, which ope a e ia a hio empla e mechanism [10] and consis o semi- au onomous uni s o modules (Fig. 1), co-linea ly a - anged o he s uc u e o he pep ide p oduc . A ypical module minimally consis s o an adenyla ion domain (A-domain), esponsible o he ac i a ion o i s cogna e subs a e amino acid as an amino acyl adenyla e, a downs eam loca ed pep idyl ca ie p o ein (PCP o P) o hiola ion domain and an ups eam posi ioned condensa ion domain (C-domain) [11]. Side opho es a e p edominan ly eleased by cycliza ion o he pep ide p oduc , o en ca alyzed by cyclo ime izing TE- domains (e.g., en e obac in, ye siniabac in) o a ian s o he condensa ion domains, e med as cycliza ion do- mains [12,13]. Fu he mo e, he i e a i e ( epe i i e) use o he enzyme empla e, as well as a non-linea s uc u e (e.g., –P–C–P–C–), seems o be cha ac e is ic o side o- pho e syn he ases. SidC, an NRP syn he ase in ol ed in e ic ocin biosyn hesis in A. nidulans, encodes a 525.5 kDa p o ein comp ising h ee adenyla ion, fi e condensa ion and fi e hiola ion domains [9,14]. In spi e o only h ee comple e modules, he en i e hexapep ides o he cyclic hyd oxama e- ype side opho es, such as iace yl usa inine C and e ic ocin a e o med by e- pea ed use o modules. Fu he mo e, sidA encodes an L-o ni hine-N 5 -monooxygenase which ca alyzes he fi s s ep in he side opho e biosyn hesis pa hway in A. nidu- lans [9]. Dele ion o he sidA gene comple ely inhibi ed side opho e biosyn hesis in A. nidulans and esul ed in se e ely diminished ungal iabili y. The sidA o holog in A. umiga us has been ecen ly epo ed and dele ion mu an s show a dis inc educ ion in i ulence [3].Us i- lago maydis, an in ec ious agen o maize, sca enges i on using ex acellula hyd oxama e side opho es, whe e side opho e p oduc ion is ini ia ed by he sid1 gene. Howe e , analysis o side opho e mu an s in U. maydis sugges s ha he side opho e biosyn he ic pa hway is no in ol ed in he in ec ion o maize [15]. Mo e e- cen ly, Yuan e al. [16] ha e iden ified an NRP syn he- ase gene (sid2) in ol ed in e ich ome biosyn hesis in U. maydis. Exp ession o sid2 was up egula ed (·2.5) in low-i on compa ed o high-i on media. We ha e de ec ed a numbe o pu a i e NRPS open eading ames in he genome o A. umiga us and e- cen ly demons a ed in i o NRPS ( e med Pes1/SidB) ac i a ion by a unc ional 40-phosphopan e heinyl ans- e ase [17]. E idence is now p esen ed ha a leas h ee dis inc NRPS genes a e suscep ible o egula ion o exp ession by he le el o ee i on p esen in he cul u e medium. 2. Ma e ials and me hods 2.1. Mic oo ganisms and cul u e media Aspe gillus umiga us ATCC 26933 (ob ained om he Ame ican Type Cul u e Collec ion, Manasas, VA, USA) was used in his s udy and main ained as spo e sus- pensions con aining 50% ( / ) glyce ol a 80 C. The ungus was g own in a mine al sal medium (pH 6.8) composed o 25 g/l glucose, 3.5 g/l (NH 4 ) 2 SO 4 , 2.0 g/l KH 2 PO 4 , 0.5 g/l MgSO 4 and 8 mg/l ZnSO 4 and supple- men ed wi h diffe en concen a ions o Fe(III)Cl 3 , as e- qui ed. A. umiga us cul u es we e se up in 500 ml shaking flasks a 230 pm, p e iously ea ed o ensu e ha all aces o i on we e emo ed om he glasswa e [6]. Medium (250 ml) was inocula ed wi h A. umiga us conidia a a final concen a ion o 10 7 pe ml and flasks we e incuba ed a 37 C and 230 pm. 2.2. Bioin o ma ic analyzes P elimina y A. umiga us sequence da a we e ob- ained om The Ins i u e o Genomic Resea ch websi e a h p://www. ig .o g. The unanno a ed A. umiga us genome was in e oga ed, ia a BLAST p og am, o non- ibosomal pep ide syn he ase encoding open ead- ing ames (ORFs) using he Ac emonium ch ysogenum Fig. 1. Modula o ganiza ion o h ee non- ibosomal pep ide syn he- ase genes om Aspe gillus umiga us:sidD,Cand E, and ele an o hologs (i.e., sid2 o A. o yzae and sidC o A. nidulans). Whe e a ailable, he deg ee o iden i y and simila i y is indica ed. Single ca aly ic uni s o NRPS a e indica ed as ollows: A, adenyla ion; P, hiola ion and C, condensa ion domain. The posi ion o PCR amplified and sequenced egions is ma ked by a ows and has been deposi ed in GenBank. P elimina y da a we e ob ained om h p:// www. ig .o g. 84 K. Reibe e al. / FEMS Mic obiology Le e s 248 (2005) 83–91 ARTICLE IN PRESS alpha-aminoadipyl-cys einyl-D- aline-syn he ase (ACVS) encoding gene (GenBank Accession No. E05192) and Aspe gillus nidulans pep ide syn he ase (sidC) gene (Gen- Bank Accession No. AY223812). Pu a i e NRPS-encod- ing ORFs, om he A. umiga us genome, we e aligned o o he pep ide syn he ases using Clus al W (h p:// www.ebi.ac.uk/clus alw/). Boo s ap neighbo joining phylogene ic ees, using he esul ing alignmen , we e d awn using he ee finde p og am (h p:// axon- omy.zoology.gla.ac.uk/ od/ ee iew.h ml). 2.3. De ec ion and quan ifica ion o side opho es Side opho e p oduc ion in A. umiga us was in es i- ga ed using he Ch ome Azu ol S (CAS) assay [18,19]. 2.4. DNA/RNA isola ion and RT-PCR Fil e ed and washed A. umiga us hyphae, collec ed a app op ia e cul u e ime-poin s, we e c ushed using a mo a and pes le unde liquid ni ogen. All DNA and RNA manipula ions we e ca ied ou as ecen ly desc ibed [20]. To acili a e equal amoun s o RNA o cDNA syn hesis and RT-PCR, densiome ic mea- su emen s o RNA on RNA aga ose gels we e pe - o med. PCR o genomic o cDNA was pe o med using AccuTaq polyme ase (Sigma–Ald ich), 1–10 ng genomic DNA and 0.5 lM each o o wa d and e- e se p ime in a o al olume o 20 ll. PCR condi- ions we e as ollows: 96 C dena u a ion o 3 min; (93 C dena u a ion o 30 s, 65 C annealing o 90 s, 68 C ex ension o 90 s) ·45 cycles; 68 C ex en- sion o 7 min. PCR was pe o med using p ime s sidD- o wa d (50-ACGCAACCGACTGGTTGTT-30), sidD- e e se (50-ATTCGTGCGAGACTCGGAT-30); sidC- o wa d (50-CCGATTATGCACATCCTCTTCC- 30), sidC- e e se (50-CCAGAATCTTCGGGTCTC-30), sidE- o wa d (50-GAGCCAGCTGACGAGATTGAT- 30)sidE- e e se (50-GGAGCGCTTGTTAAACAAC- CT-30). The gene encoding calmodulin, which is cons i- u i ely exp essed in A. umiga us, se ed as a con ol in RT-PCR expe imen s and was amplified ac oss an in on–exon bounda y [21]. Op imal cDNA amplifica- ion was ound o equi e 45 cycles o PCR. PCR- amplified DNA was elec opho esed, isualized and subjec ed o semi-quan i a i e exp ession analysis as ecen ly desc ibed [20]. 2.5. P o ein pu ifica ion All p o ein pu ifica ion s eps we e pe o med a 4 C. Mycelia we e ha es ed by fil a ion and washed wice in phospha e-buffe ed saline (PBS, pH 7.4). Washed myce- lia we e esuspended in Buffe A (100 mM T is, 50 mM NaCl, 20 mM EDTA, 30 mM DTT, 44.71 mg/l PMSF, 2 mg/l DNaseI, 300 mg/l lysozyme, 21 mg/l leupep in, 10 mg/l TLCK, 10 mg/l peps a in A and 10% ( / ) glyc- e ol, pH 7.3 [22]) and passed h ough a F ench p ess (700–1000 ba ). Cell deb is was emo ed by cen i uga ion a 40,000g o 30 min. Supe na an was concen a ed by Q-Sepha oseeanion-exchange ch o- ma og aphy (load/wash: Buffe A wi hou NaCl; elu- ion: Buffe A + 1 M NaCl; bed olume: 20 ml; p o ein load: 80 mg). Selec ed ac ions, iden ified by SDS– PAGE (da a no shown), con aining high molecula mass p o eins we e u he pu ified by gel pe mea ion ch oma og aphy o e a Supe ose 6 esin (10 ·300 mm; flow a e 1.0 ml/min; 100 mM T is, 50 mM NaCl, 20 mM EDTA, 30 mM DTT and 10% ( / ) glyce ol, pH 7.3.) using an A ¨KTA pu ifie 100 sys em (Ame - sham Bioscience, Sweden). P o ein concen a ions we e de e mined using he B ad o d assay [23] wi h BSA as s anda d. SDS–PAGE was pe o med acco ding o he me hod o Laemmli [24] using high molecula mass p o ein calib a ion (Sigma–Ald ich). 2.6. Two-dimensional elec opho esis A e gel pe mea ion ch oma og aphy, selec ed p o- ein-con aining ac ions we e also subjec o sepa a ion by 2D-PAGE. P o ein (200 g) was p ecipi a ed wi h 5 olumes o ice-cold 100% ace one and ai -d ied. Pelle s we e esuspended in 250 ll IEF-buffe (10 mM T is, 8 M u ea, 2 M hiou ea, 4% (w/ ) CHAPS, 1% ( / ) T i- on X-100, 65 mM DTT and 0.8% (w/ ) ampholy e), loaded on o linea immobilized pH g adien (IPG) s ips (13 cm, pH ange: 3–10) and isoelec ic ocussing (IEF) was pe o med a 16 C using an E aneIPGpho IIe ins umen (Ame sham Bioscience). A e a condi ion- ing s ep a 50 V o 10 h, 250 V was held o e 15 min, ollowed by ol age amping o 8000 V in 5 h, which was held o u he 8 h. IPG s ips we e hen incuba ed wice in 15 ml equilib a ion buffe (50 mM T is, 30% ( / ) glyce ol, 2% (w/ ) SDS and 6 M u ea, pH 6.8), sup- plemen ed wi h 2% (w/ ) DTT and subsequen ly wi h 2.5% (w/ ) iodoace amide o 20 min. The s ips we e hen placed on a 7.5–10% (w/w) SDS–PAGE gel and elec opho esed o e nigh a 80 V a 4 C. 2.7. Mass spec ome y P o ein samples o pep ide mass de e mina ion we e sepa a ed by SDS–PAGE o 2D-PAGE, excised om he gels and diges ed wi h ypsin (P omega sequencing g ade; 5–20 lg, o e nigh ). The esul an pep ide mix- u es we e ex ac ed om gel pieces, mixed wi h sa u- a ed a-cyano-4-hyd oxycinnaminic acid as p e iously desc ibed [17] and mix u es (0.5 ll) applied o MTP 384 g ound s eel mass spec ome y a ge s (B uke ) and allowed o d y. Ma ix Assis ed Lase Deso p ion/ Ioniza ion-Time o Fligh Mass Spec ome y (MALDI ToF MS) was pe o med using a B uke ul aflex K. Reibe e al. / FEMS Mic obiology Le e s 248 (2005) 83–91 85 ARTICLE IN PRESS LIFT-ToF/ToF (UK). De e mina ion o yp ic finge - p in s was ca ied ou on diffe en spo s o one sample, and de ice pa ame e s, especially lase powe , adjus ed un il an op imal signal/noise a io was ound. Mass spec a wi h high signal in ensi y we e subjec ed o LIFT-ToF/ToF analysis [25]. Spec a we e p ocessed using FlexAnalysis so wa e (B uke ), using yp ic ag- men s o ypsin o in e nal calib a ion o spec a. Da abase sea ches and sequence compa isons we e ca - ied ou ia Masco in-house se e (Ma ixScience) and Bio ools (B uke ), espec i ely. 3. Resul s and discussion 3.1. Molecula cha ac e iza ion and phylogene ic analysis o h ee NRPS-encoding genes sidD,Cand E A e in e oga ion o he A. umiga us genome by BLAST analysis, using he ACVS- and A. nidulans pep- ide syn he ase (sidC) encoding genes, se e al ORFs, encoding NRPS we e iden ified. A usidD and C(Gen- Bank Accession Nos. DQ013888 and DQ011870) clus e ed wi h known and p oposed side opho e syn he- ases upon phylogene ic analysis (da a no shown). Acco ding o he modula o ganiza ion (Fig. 1) and s ong esemblance, sidD (GenBank Accession No. DQ011871) is likely o be an o holog o sid2, a pu a i e side opho e syn he ase encoding gene o Aspe gillus o y- zae (GenBank Accession No. AB087617). Bo h se- quences showed 75% iden i y (85% simila i y). Like sid2,sidD encodes a bi-modula NRPS consis ing o wo adenyla ion, wo hiola ion and one condensa ion domain. SidD comp ises 1915 amino acids co espond- ing o an es ima ed molecula mass o 210 kDa. The eigh -amino acid mo i wi hin adenyla ion domains, which has a p edominan influence on he o ma ion o he subs a e-specific pocke [26,27], was de e mined o module 1 o he pu a i e SidD and Sid2 (Table 1). Bo h mo i s a e iden ical (DVDGGGGI), he eby p e- dic ing ha 5-hyd oxy-o ni hine is ecognized and ac i- a ed by he fi s adenyla ion domain ([28];h p:// aynam.chm.jhu.edu/~n ps/ins uc ions.h ml). 5-Hyd o- xy-o ni hine is he main cons i uen o hyd oxama e sid- e opho es. The A-domain in module 2 o sidD and sid2 (Table 1) seemed o be degene a e and only 51 amino acids (ins ead o a ound 450) show sequence simila i y o o he adenyla ion domains. Hence, no p edic ion wi h espec o amino acid mo i was possible and i is p oposed ha only he fi s module is capable o amino acid inco po a ion. The p esence o a hiola ion domain on he las posi ion o a NRPS is unusual. I may se e as a ‘‘linke ’’ domain o acili a e epe i i e use o SidD- domains o in e ac ion wi h ano he NRPS, o which he pa ially o med pep ide is ans e ed o u he modifica ion [29]. A simila wo king p inciple wi h wo in e ac ing NRP syn he ases was sugges ed o pep aibol o ma ion in Sepedonium ampullospo um [30]. In addi- ion, a mul i-subuni NRP syn he ase complex is p esen in C. pu pu ea, which di ec s e go pep ide o ma ion and was he fi s iden ified ungal NRP syn hesis sys em con aining diffe en subuni s [31]. The ORF encoding sidC in A. umiga us appea ed o be an o holog o sidC, which encodes an NRPS in- ol ed in hyd oxama e side opho e ( e ic ocin) biosyn- hesis in A. nidulans [8]. Bo h desc ibe a simila modula s uc u e (Fig. 1) wi h sequence iden i y o 55% (simila - i y: 71%). The 13,551 bp ORF o A usidC p edic ed a p o ein o 4517 amino acids (molecula mass: 496 kDa). A uSidC clus e s oge he wi h bo h AniSidC and Sid2, a e ich ome side opho e pep ide syn he ase gene by U. maydis (GenBank Accession No. U62738), indica ing a unc ional ela ionship. Simila amino acid codes we e ound o he co esponding adenyla ion modules (Table 1) in AniSidC and A uSidC. Thus, almos he same amino acids we e p edic ed o be Table 1 P edic ion o he heo e ical subs a e amino acids ac i a ed by he single adenyla ion domains o SidD, C and E om A. umiga us P edic ed amino acid inco po a ion Module (m) Signa u e mo i 12345678 5(OH)O n A uSidD-m1 DVDGGGGI 5(OH)O n Ao Sid2-m1 DVDGGGGI – A uSidD-m2 No p edic able Ao Sid2-m2 No p edic able Ala A uSidC-m1 DPMMWMAI Ala AniSidC-m1 DPMMWMAI – A uSidC-m2 DVQ H T I T I – AniSidC-m2 DVQ H T I T V Gly/Se A uSidC-m3 DVL S S G AI Ty /T p/Gly AniSidC-m3 DPL S T G AI – A uSidE-m1 No p edic able Val A uSidE-m2 DVY F T G GV Compa ison wi h pu a i e o holog sequences Sid2 (A. o yzae) and SidC (A. nidulans) acco ding o he NRPS p edic ion blas se e ([28];h p:// aynam.chm.jhu.edu/~n ps/ins uc ions.h ml). 86 K. Reibe e al. / FEMS Mic obiology Le e s 248 (2005) 83–91 ARTICLE IN PRESS ecognized and ac i a ed by he adenyla ion domains o module one (alanine) and h ee (glycine o se ine) o bo h genes acco ding o NRPS adenyla ion domain p e- dic ion [28]. Alanine, glycine and se ine migh be cha ged by he domains o A uSidC, which a e he eby cons i uen s o he hyd oxama e side opho e e ic ocin, p oduced by A. umiga us [32]. SidE comp ises a 6330 bp ORF and encoded a heo- e ical bi-modula NRPS-p o ein o 2109 amino acids. Like A uSidD, his hypo he ical p o ein (SidE) ap- pea ed o con ain wo adenyla ion and hiola ion domains and one condensa ion domain (Fig. 1). SidE sha ed 24% amino acid iden i y (42% simila i y) wi h A uSidC and bo h p o eins may be de i ed om a com- mon ances o . In addi ion, SidE clus e ed wi h A. nidu- lans SidC and Sid2 o U. maydis ollowing phylogene ic analysis whe eby 25% iden i y/42% simila i y and 23% iden i y/40% simila i y, espec i ely, is e iden . While no p edic ion was possible o he subs a e amino acid encoded by module 1 o SidE, module 2 con ains he eigh -amino acid non-linea mo i , DVYFTGGV, and p edic ed inco po a ion o aline (Table 1)[28]. 3.2. Side opho e p oduc ion Fig. 2 shows ha op imal side opho e p oduc ion oc- cu ed when A. umiga us was g own in he absence o i on. Fu he mo e, significan ly highe side opho e amoun s we e appa en a he 72 h ime-poin ollowing cul u e unde i on- ee condi ions compa ed o ea lie ime-poin s (p< 0.05). Significan diminu ion o side o- pho e le els was appa en (p< 0.05) unde 20 lM i on condi ions (up o 2- old), al hough o al le els p esen a 72 h ime-poin we e app oxima ely 50% o hose e i- den unde i on- ee condi ions. Low side opho e p o- duc ion was e iden , and minimal diffe ences in side opho e le els we e de ec able, a any ime-poin , unde high i on (300 lM) cul u e condi ions. Mo eo e , maximum side opho e p oduc ions unde high i on con- di ions we e be ween 2- and 15- old less han hose obse ed unde i on ee o low i on cul u e condi ions. I has ecen ly been demons a ed ha sidA exp es- sion in A. umiga us is up egula ed unde condi ions o i on s a a ion (12–24 h cul u e ime) [3]. Mo eo e , d ama ic inc eases in he le els o ex acellula TAFC and in acellula des e i e ic ocin we e concomi an ly obse ed [3]. Hissen e al. [6] ha e demons a ed ha side opho e p oduc ion by A. umiga us is significan ly g ea e in he p esence o holo- ans e in, as opposed o se um, in minimal essen ial medium (MEM) o e a 19-h incuba ion ime and ha Fe(III) (20 lM) al eady causes i on limi a ion which esul s in induc ion o en- hanced side opho e o ma ion and exc e ion. In he p esen s udy, whe e minimal media con aining glucose and se e al sal s we e supplemen ed wi h diffe en con- cen a ions o Fe(III), his finding was confi med. These da a clea ly demons a e ha side opho e p oduc ion is enhanced in he absence o ee i on. 3.3. Exp ession analysis o sidD,Cand E Exp ession o sidD,Cand Ewas assessed by semi- quan i a i e RT-PCR analysis. RNA loading was used as a con ol o ensu e equal amoun s o RNA used o cDNA syn hesis (Fig. 3A). The p esence o genomic DNA was excluded by bo h DNAse ea men o iso- la ed RNA p io o RT-PCR and analysis o he size di - e ence be ween he genomic and cDNA amplicon o calm om which in ons ha e been excised (Fig. 3B). I can be seen ha sidD and Cexp ession was e iden a all ime poin s du ing A. umiga us g ow h om T= 24–72 h in he absence o ee i on and ha diminu- ion o exp ession occu s bo h unde low (20 lM) and high (300 lM) i on condi ions (Fig. 3C and D). Exp es- sion o sidD diminished by be ween 25% and 60% unde low i on condi ions and by up o 90% unde high i on condi ions (24 and 48 h), upon compa ison o sidD exp ession in he absence o ee i on. Al hough sidD exhibi s 75% sequence iden i y wi h sid2 o A. o yzae, no in o ma ion is a ailable on he exp ession pa e n o sid2 and so a compa a i e analysis was no possible. No I on Low I on High I on 0 50 100 150 200 250 300 350 400 450 24 h 48h 72h 24h 48h 72h 24h 48h 72h Side opho e/P o ein Ra io (mg side opho e/mg mycelial p o ein) Fig. 2. Quan i a ion o side opho e p oduc ion by A. umiga us unde diffe en ial i on concen a ions (0, 20 and 300 lM). Maximum side opho e p oduc ion was induced when A. umiga us was g own in he absence o i on. Diminu ion o side opho e p oduc ion is appa en unde low i on condi ions (20 lM). Low side opho e le els we e e iden when he ungus was g own in he p esence o 300 lM Fe(III). K. Reibe e al. / FEMS Mic obiology Le e s 248 (2005) 83–91 87 ARTICLE IN PRESS Exp ession o sidC appea ed o be main ained unde low i on condi ions compa ed o ha o sidD, bu was educed by app oxima ely 65–80% unde high i on con- di ions (24 and 48 h) ela i e o exp ession in i on- ee cul u e condi ions. Yuan e al. [16] ha e obse ed a sim- ila phenomenon when U. maydis was cul u ed in low i on medium whe eby a 2.5- old inc ease in sid2 exp es- sion was de e mined by No he n hyb idiza ion. In e - es ingly, exp ession o bo h sidD and Cappea ed o be up egula ed unde high i on condi ions a he 72 h ime-poin , possibly due o deple ion o i on in he med- ium as ungal g ow h p oceeds. Cons i u i e and con- s an exp ession o sidE was obse ed unde all expe imen al condi ions, excep du ing he ea ly g ow h phase unde high i on condi ions (24 h) whe e a 65% de- c ease in exp ession is e iden ela i e o cul u e in he absence o i on a 24 h. Al hough p e ious wo k has also shown ha he exp ession o A. nidulans sidC is nega i ely egula ed by he p esence o i on (10 lM) [33], he esul s p esen ed he e a e he fi s demons a- ion ha he exp ession o up o h ee NRPS-encoding genes in A. umiga us is diffe en ially egula ed by i on a ailabili y. Fig. 3. RT-PCR analysis o he egula i e esponse o non- ibosomal pep ide syn he ase genes in A. umiga us (sidD,Cand E) in he p esence and absence o ee i on. Lanes 1–9 con ain RNA o cDNA amplicons ob ained a e 24, 48 and 72 h o cul i a ion in no i on supplemen ed WB-medium (1–3), low i on supplemen ed WB-medium (20 lM, 4–6) and high i on concen a ions in WB-medium (300 lM, 7– 9). PCR on genomic DNA (gDNA) se ed as a con ol in RT-PCR expe imen s. (A) Equi alen amoun s o RNA we e used o all cDNA syn hesis and amplica ion eac ions as judged by RNA. (B) RT-PCR analysis o he house-keeping gene (calmodulin) confi med he absence o DNA (in on excision) and u he confi med mRNA/cDNA in eg i y. (C) The pu a i e side opho e syn he ase encoding gene, sidD, demons a es a diminu ion o exp ession wi h inc easing i on a ailabili y, as judged by RT-PCR analysis. (D) SidC shows a weake , bu simila , egula ion esponse. (E) Exp ession o sidE appea s o be unaffec ed by a ailable Fe(III), unlike ha o sidD o sidC, unde all in es iga ed cul u e condi ions excep a he 24 h ime-poin unde high i on condi ions. Fig. 4. (A) SDS–PAGE analysis o high molecula mass (HMM) p o eins ex ac ed om A. umiga us cul u es g own unde s ong i on limi a ion (no addi ional i on supply) and sufficien (300 lM) i on supply. Equi alen p o ein amoun s (376 lg) we e loaded o examine diffe en ial p o ein exp ession o e a ime cou se o 3 days (24, 48 and 72 h). Two high molecula mass p o eins, e med HMMP1 and 2, we e up egula ed unde condi ions o i on limi a ion, especially a he 48 h ime-poin and we e subsequen ly analyzed by MALDI-ToF mass spec ome y. (B) 2D-PAGE u he esol ed a numbe o p o eins (a leas h ee) which appea a a molecula size o app ox. 200 kDa. The spo (ci cled) was analyzed by MALDI-ToF mass spec ome y. (C) The MALDI LIFT-ToF/ToF mass spec um o a pep ide o 1498.744 Da, co esponding o a SidD agmen . The amino acid (aa) sequence, analyzed by PSD (pos -sou ce decay) agmen a ion, confi ms N- (2 aa) and C- e minal (4 aa) sequence: C- e minal (y- agmen s): RARL; N- e minal (b- agmen s): VE, confi ming a pep ide sequence: VEXXXXXXXLRAR. In e nal agmen s co e he emainde o he pep ides: LG (y11b4) and GEIESQLRA (y10b12). Thus, he ollowing amino acid sequence VELGEIESQL- RAR o HMMP1 ma ches 100% o he heo e ical one o SidD and confi ms HMMP1 as he sidD-encoded NRPS. Inse : pep ide mass finge p in o ypsin-diges ed SidD. (D) Pep ide mass finge p in o ypsin-diges ed HMMP2. 88 K. Reibe e al. / FEMS Mic obiology Le e s 248 (2005) 83–91 ARTICLE IN PRESS 3.4. P o ein exp ession analysis P o ein ex ac s o A. umiga us cul u es g own wi h and wi hou ee i on o e a ime cou se o 3 days we e p epa ed and subjec ed o SDS–PAGE analysis. Two p o eins, one o app oxima e molecula mass 200 kDa, e med high molecula mass p o ein (HMMP)1, and an- o he o 300–400 kDa, e med HMMP2, we e p edomi- nan ly exp essed a 48 h, in he absence o i on (Fig. 4A). Exp ession o HMMP1 was s ill e iden a 72 h, mo eo e , his p o ein also seems o appea a a la e ime-poin (72 h) in he high-i on con aining medium. The p o ein exp ession p ofile o HMMP1 indica ed esemblance o he ansc ip cou se o sidD, which en- codes a 210 kDa NRP syn he ase (Fig. 3). HMMP1 (app ox. 200 kDa) was pa ially pu ified by means o Fig. 4. (con inued) K. Reibe e al. / FEMS Mic obiology Le e s 248 (2005) 83–91 89 ARTICLE IN PRESS ion-exchange and gel pe mea ion ch oma og aphy (Fig. 4B). Pooled gel pe mea ion column ac ions, con aining HMMP1, we e u he sepa a ed by 2D-PAGE. As can be seen in Fig. 4B, up o h ee p o eins o equi alen molecula mass (app ox. 200 kDa) we e p esen . Pep ide mass finge p in ing o he mos abundan p o ein (indi- ca ed by a ci cle) confi med ha o 126 de ec ed pep- ides, up o 40 ma ched wi h he heo e ical yp ic pep ides o he sidD encoded NRP syn he ase, p o iding sequence co e age o HMMP1/SidD up o 22.8% (m/z- ole ance: 1.5 Da) (Fig. 4C). Se e al pep ides, which co esponded o heo e ical yp ic agmen s o he SidD, we e sequenced by MALDI LIFT-ToF/ToF mass spec ome y [25] (Fig. 4C; Table 2). Pos -sou ce decay (PSD) agmen a ion o he pep ide wi h a mono-iso o- pic mass o 1498.744 Da e ealed he amino acid se- quence VELGEIESQLRAR and ma ched 100% wi h he heo e ical sequence o a SidD pep ide o 1498.810 Da (Fig. 4C). All esul an amino acid se- quences co ela ed wi h he co esponding heo e ical pep ides o SidD, he eby confi ming HMMP1 as he sidD-encoded NRP syn he ase, which is e y likely o be in ol ed in side opho e biosyn hesis in A. umiga us. A 48 h and absence o i on, ano he HMMP is appa en a a molecula mass o 300–400 kDa (HMMP2). Due o i s size and appa en i on esponsi e- ness, HMMP2 was p oposed o co ela e o ano he NRPS, which may be in ol ed in side opho e biosyn- hesis in A. umiga us and encoded by sidC. A emp s o sepa a e and concen a e HMMP2 by means o gel pe mea ion o ion exchange ch oma og aphy we e unsuccess ul (da a no shown), p obably due o i s suscep ibili y o deg ada ion. Howe e , pep ide mass finge p in ing o HMMP2, ob ained ollowing elec o- pho e ic sepa a ion (Fig. 4A), yielded 88 pep ides as de- ec ed by MALDI-ToF analysis, o which 68 ma ched wi h p edic ed yp ic pep ides o he SidC (Fig. 4D). Thus, a sequence co e age o 18.6% (m/z- ole ance: 1.5 Da) o HMMP2/SidC was de e mined which, when combined wi h he appea ance o his p o ein unde condi ions o i on deple ion, s ongly indica es ha HMMP2 was a leas a componen o he sidC-encoded NRPS ( heo e ical mass: 496 kDa). The p esence o SidE could no be de ec ed by MALDI-ToF analysis. Howe e , La Clai e al. [34] ecen ly p oposed he use o bio in o fluo escen ly labeled coenzyme A analogs, in associa ion wi h unc ional 40-phosphopan e heinyl ans e ase ac i i y, o label na i e NRPS and he eby acili a e pu ifica ion [34]. Such an app oach may p o e use ul o he pu ifica ion o addi ional, low abundance, pep ide syn he ases (e.g., SidE) in A. umiga us. In summa y, he i on-media ed, diffe en ial exp es- sion o wo NRPS has been demons a ed in A. umiga- us. Fu he mo e, wo p o eins, namely SidD and C, in ol ed in side opho e biosyn hesis ha e been pu ified, analyzed by SDS–PAGE/2D-PAGE and iden ified by MALDI-ToF/MALDI LIFT-ToF/ToF mass spec om- e y. This wo k u he s ou unde s anding o side o- pho e biosyn hesis in his o ganism and offe s new insigh s in i on egula ion o gene exp ession in Aspe gil- lus spp. Acknowledgmen s This wo k was financially suppo ed by he Highe Educa ion Au ho i y o I eland unde he P og amme o Resea ch in Thi d Le el Ins i u ions (HEA-PRTLI). Clai e Ne ille was a ecipien o a Daniel OÕConnell Fel- lowship om NUI Maynoo h. P elimina y sequence da a we e ob ained om The Ins i u e o Genomic Re- sea ch websi e a h p://www. ig .o g. Sequencing o Aspe gillus umiga us was unded by he Na ional Ins i- u e o Alle gy and In ec ious Disease U01 AI 48830 o Da id Denning and William Nie man, he Wellcome T us , and Fondo de In es icagiones Sani a ias. Re e ences [1] Kon oyiannis, D.P. and Bodey, G.P. (2002) In asi e aspe gillosis in 2002: an upda e. Eu . J. Clin. Mic obiol. In ec . Dis. 21, 161– 172. [2] Mabey, J.E., Ande son, M.J., Giles, P.F., Mille , C.J., A wood, T.K., Pa on, N.W., Bo nbe g-Baue , E., Robson, G.D., Oli e , S.G. and Denning, D.W. (2004) CADRE: he Cen al Aspe gillus Da a REposi o y. Nucleic Acids Res. 32, 401–405. [3] Sch e l, M., Bignell, E., K agl, C., Joechl, C., Roge s, T., A s J ., H.N., Haynes, K. and Haas, H. (2004) Side opho e biosyn- hesis bu no educ i e i on assimila ion is essen ial o Aspe - gillus umiga us i ulence. J. Exp. Med. 200, 1213–1219. 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