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4?-Phosphopantetheinyl transferase-encoding npgA is essential for siderophore biosynthesis in Aspergillus nidulans

4?-Phosphopantetheinyl transferase-encoding npgA is essential for siderophore biosynthesis in Aspergillus nidulans
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  RESEARCH ARTICLE Harald Oberegger  Æ  Martin Eisendle  Æ  Markus SchrettlStefan Graessle  Æ  Hubertus Haas 4 ¢ -Phosphopantetheinyl transferase-encoding   npgA   is essential for siderophore biosynthesis in  Aspergillus nidulans  Received: 18 April 2003/ Revised: 27 June 2003 / Accepted: 15 July 2003/Published online: 24 September 2003   Springer-Verlag 2003 Abstract  Aspergillus nidulans  produces two major sid-erophores: it excretes triacetylfusarinine C to captureiron and contains ferricrocin as an intracellular iron-storage compound. Siderophore biosynthesis involvesthe enzymatic activity of nonribosomal peptide synthe-tases (NRPS). NRPS contain 4 ¢ -phosphopantetheine asan essential prosthetic group, which is attached by 4 ¢ -phosphopantetheinyl transferases.  A. nidulans  appearsto possess at least one gene,  npgA , encoding such anenzyme. Using a strain carrying a temperature-sensitiveallele,  cfwA2 , we showed that NpgA is essential forbiosynthesis of both the peptide bond-containing ferri-crocin and the ester bond-containing triacetylfusarineneC. The  cfwA2  strain was found to be iron-starved at therestrictive temperature during iron-replete conditions,consistent with the siderophore system being the majoriron-uptake system—as we recently demonstrated.Northern analysis indicated that, in contrast to othergenes which are involved in siderophore biosynthesisand uptake, expression of   npgA  is not controlled bythe GATA-transcription factor SreA. It was shownpreviously that NpgA is required for biosynthesisof penicillin, pigment, and potentially lysine via the a -aminoadipate pathway. Supplementation with lysineplus triacetylfusarinine C restored normal growth of the cfwA2  strain at the restrictive temperature, suggestingthat the growth defect of the mutant is mainly due toimpaired biosynthesis of siderophores and lysine. Keywords  Iron  Æ  Siderophore  Æ  Lysine  Æ Phosphopantetheinyl transferase Introduction Virtually all organisms require iron for their growth,because this metal is used in many different types of cofactors, e.g. heme moieties and iron-sulfur clusters.Despite the fact that iron is the fourth most abundantelement in the earth ¢ s crust, the amount of bioavailableiron is very limited, because this metal is most com-monly found as an insoluble ferric oxyhydroxide. Thus,microorganisms need specialized iron-mobilization sys-tems (Guerinot 1994; Leong and Winkelmann 1998). Inorder to solubilize environmental iron, most microor-ganisms synthesize and excrete siderophores—lowmolecular weight, Fe(III)-specific chelators—in iron-depleted conditions. Subsequently, cells recover the ironfrom the ferric-siderophore complexes via specific up-take mechanisms (Winkelmann 2001; Haas 2003). Fur-thermore, most fungi possess intracellular siderophoresas an iron-storage compound (Matzanke 1994). Sidero-phores have often been suggested to function as viru-lence factors, because the acquisition of iron is a key stepin the infection process of any pathogen, since this metalis tightly sequestered by high-affinity iron-binding pro-teins in mammalian hosts (Weinberg 1999). Siderophorebiosynthesis and uptake also represent possible targetsfor antifungal chemotherapy, because the underlyingbiochemical pathways are absent in human cells.Various  Aspergillus  species are important pathogensof immunocompromised hosts, causing pneumonia andinvasive disseminated diseases with high mortality(Latge 1999). In contrast to  A. fumigatus , which may beregarded as the most important airborne pathogenicfungus,  A. nidulans  is a much rarer cause of humandisease but represents a filamentous model fungus(Brookman and Denning 2000).  A. nidulans  producestwo major siderophores: it excretes triacetylfusarinine C(TAFC) and contains intracellular ferricrocin (FC)(Charlang et al. 1981; Oberegger et al. 2001). Subsequentto chelating Fe(III), the siderophore-Fe(III) complex istaken up by MirB, a transporter belonging to the major Curr Genet (2003) 44: 211–215DOI 10.1007/s00294-003-0434-zCommunicated by U. Ku ¨ckH. Oberegger ( & )  Æ  M. Eisendle  Æ  M. SchrettlS. Graessle  Æ  H. HaasDepartment of Molecular Biology, University of Innsbruck,Peter-Mayr-Strasse 4b, 6020 Innsbruck, AustriaE-mail: hubertus.haas@uibk.ac.atTel.: +43-512-5073605Fax: +43-512-5072866  facilitator protein superfamily (Haas et al. 2003). Afteruptake, TAFC is cleaved by an esterase, the cleavageproducts are excreted, and Fe(III) is transferred to FC(Oberegger et al. 2001; Eisendle et al. 2003). TAFC is acyclic ‘‘tripeptide’’ consisting of three  N  2 -acetyl- N  5 - cis -anhydromevalonyl- N  5 -hydroxyornithine residues linkedby ester bonds; and FC is a cyclic ‘‘hexapeptide’’ withthe structure Gly-Ser-Gly-( N  5 -acetyl- N  5 -hydroxyorni-thine) 3  (Winkelmann 1993). Based on biochemical andgenetic studies of several microorganisms, biosynthesisof FC and TAFC is assumed to proceed according to theenzymatic steps depicted in Fig. 1 (Plattner and Diek-mann 1994). Recently, we found that siderophore bio-synthesis is essential for viability of   A. nidulans  (Eisendleet al. 2003). In this study, two genes involved in sid-erophore biosynthesis are characterized:  sidA  encoding L -ornithine  N  5 -monooxygenase, the first committed stepin siderophore biosynthesis, and  sidC  , which encodes anonribosomal peptide synthetase (NRPS) involved insynthesis of ferricrocin. NRPSs are large multifunctionalenzymes with a modular construction able to assemblecompounds from a remarkable range of proteinogenicand nonproteinogenic precursors (Kleinkauf and VonDohren 1996; Weber and Marahiel 2001). Each modulecontains an adenylation domain, a condensationdomain, and a peptidyl carrier domain. Like the acylcarrier domains in fatty acid and polyketide synthases,the peptidyl carrier domain requires attachment of a4 ¢ -phosphopantetheine (Ppant) group by a dedicatedPPTase. It was anticipated that most organismsemploying more than one Ppant-dependent pathwayalso have more than one 4 ¢ -phosphopantetheinyl trans-ferase (PPTase; Walsh et al. 1997). In contrast, it wasshown that  Pseudomonas aeruginosa  possesses merely asingle PPTase, which is required for both fatty acid andsiderophore synthesis (Finking et al. 2002). Primarily, itwas suggested that  Neurospora crassa ,  A. fumigatus , and A. nidulans  possess only a single PPTase (Keszenman-Pereyra et al. 2003). The encoding  A. nidulans  gene, npgA , was isolated by complementation of a null-pig-ment mutant (Han and Han 1993; Chung et al. 1996;Kim et al. 2001). Subsequently,  npgA  was found tocomplement a  Saccharomyces cerevisiae  strain defectivein  lys5 , which encodes a PPTase required for activationof the  a -aminoadipate semialdehyde reductase  Lys2 (Ehmann et al. 1999; Mootz et al. 2002).  Lys2  andconsequently  Lys5  are essential for lysine biosynthesis inthis yeast. Utilizing a strain harboring a temperature-sensitive allele of   npgA ,  cfwA2  (Aguirre et al. 1993), itwas shown that NpgA is also required for synthesis of penicillin, which involves a NRPS step (Keszenman-Pereyra et al. 2003). Recently, a second putative PPTase-encoding gene was identified in the genome sequences of various fungi, including  A. nidulans . However, its func-tion remains unclear so far (D. Keszenman-Pereyra andG. Turner, personal communication).Here, we show that NpgA is essential for siderophorebiosynthesis and that the growth defect of the  cfwA2 strain is mainly due to impairment of the biosynthesis of siderophores and lysine. Materials and methods Strains, vectors, growth media, and general molecular techniquesThe  A. nidulans  strains WGTRAN ( argB2::argB bgA0, biA1 ),SRKO1 ( sreA::argB bgA0, biA1 ), and AJC12:36 (  pabaA1, yA2,cfwA2 ) are designated in the text as  wt ,  D sreA, and  cfwA2 ,respectively. Construction of WGTRAN and SRKO1 was de-scribed by Haas et al. (1999) and Haas et al. (2003); and  cfwA2  wasisolated by Aguirre et al. (1993).Fungal strains were grown at 25   C, 37   C, or 42   C, as indi-cated, in  Aspergillus  minimal medium (AMM) according to Pon-tecorvo et al. (1953) containing 1% glucose as carbon source,20 mM glutamine as nitrogen source, 10  l M FeSO 4 , 20  l g/l biotin,and 4 mg  p -aminobenzoic acid/l. For low-iron media ( ) Fe-AMM),iron was omitted. Standard molecular techniques were performedas described by Sambrook et al. (1989).Identification, quantification, and purification of siderophoresTAFC and FC were isolated from  A. nidulans  as described byOberegger et al. (2001). Crude identification of extracellular sid-erophore production was performed using the chrome azurol Sliquid assay (Payne 1994). Characterization and quantificationof extracellular and cellular siderophores were performed by Fig. 1A, B  Siderophores of   Aspergillus nidulans .  A  Chemicalstructure of ferricrocin (FC) and triacetylfusarinine C (TAFC),adapted from Winkelmann (1993) with permission of the publisher. B  Biosynthetic pathway for FC and TAFC, according to Plattnerand Diekmann (1994)212  reversed-phase HPLC chromatography according to Konetschny-Rapp et al. (1988), as described by Oberegger et al. (2001).Extracellular siderophore production was normalized to the dryweight of the mycelia; and the intracellular siderophore content wasnormalized to the protein content of the cellular extract.Northern analysisGenerally, 15  l g of total RNA were electrophoresed on 1.2%agarose-2.2 M formaldehyde gels and blotted onto Hybond Nmembranes (Amersham). The hybridization probes used in thisstudy were generated by PCR, using oligonucleotides 5 ¢ -AGCCCGGTGTGAAAAGAG-3 ¢  and 5 ¢ -AACAGGAGGAGGATTGCGCC-3 ¢  for  mirA , 5 ¢ -ACACCCGCCCTCTAACCG-3 ¢  and 5 ¢ -CACACCCCAGTCGCACAG-3 ¢  for  npgA , and 5 ¢ -CGGTGATGAGGCACAGT-3 ¢  and 5 ¢ -CGGACGTCGACATCACA-3 ¢  for  c -actin-encoding  acnA  (Fidel et al. 1988). Results and discussion NpgA function is essential for biosynthesis of bothferricrocin and triacetylfusarinine CAn  A. nidulans  strain harboring the temperature-sensi-tive  npgA  allele  cfwA2  did not grow at the restrictivetemperature of 42   C using liquid or solid AMM.Therefore,  cfwA2  was grown at 37   C, a temperature atwhich  cfwA2  displayed about 30% of the  wt  growth rate.In order to analyze the influence of NpgA on sidero-phore biosynthesis,  cfwA2  and  wt  were grown for 24 hduring iron-depleted conditions. Subsequently, the pro-duction of extracellular and intracellular siderophoreswas quantified as described by Oberegger et al. (2001).At the permissive temperature of 25   C,  cfwA2  showedabout 90% of the intracellular and extracellular sidero-phore production of   wt . In contrast, at 37   C, the pro-duction of TAFC and FC by  cfwA2  decreased to 3%and 2%, compared with  wt  (Fig. 2). These datademonstrate that NpgA is essential for the biosynthesisof both siderophores, ferricrocin and TAFC. As shownin Fig. 1, FC synthesis involves the NRPS SidC—whichrequires activation by a Ppant. In contrast to FC, themodified ornithine residues of TAFC are linked by esterbonds and the enzymatic steps involved are unclear sofar. On the one hand, the requirement of NpgA forTAFC synthesis is therefore surprising. On the otherhand, it has been shown that some NRPS can also formester bonds—an example is  Escherichia coli   EntF, whichis involved in the synthesis of the catecholate-type sid-erophore enterobactin (Crosa and Walsh 2002). Defi-ciency in the TAFC biosynthesis of   cfwA2  at therestrictive temperature suggests that TAFC synthesisinvolves a Ppant-dependent enzymatic step—most likelyan NRPS. cfwA2  is iron-starved during iron-replete conditionsIn  A. nidulans , the siderophore system is the major iron-uptake system (Eisendle et al. 2003). Impairment of siderophore biosynthesis, e.g. deletion of the sidero-phore-biosynthesis gene  sidA  causes iron starvationduring iron-replete conditions. Expression of genes in-volved in siderophore biosynthesis and uptake is re-pressed by iron; and this control is mediated in part bythe transcriptional repressor SREA (Haas et al. 1999;Oberegger et al. 2001, 2002a). Northern analysis of  mirA , which encodes a siderophore transporter upregu-lated under iron depletion (Oberegger et al. 2001; Haaset al. 2003), displayed that, in contrast to  wt ,  cfwA2  isiron-starved during iron-replete conditions at 37   C(Fig. 3A). Therefore, with respect to iron homeostasis, cfwA2  grown at 37   C shows a similar phenotype to thatof strains with defects in siderophore-biosynthesis genes.Northern analysis indicated that, in contrast to  mirA and numerous other genes which are involved in sid-erophore biosynthesis and uptake (Oberegger et al.2002a), expression of   npgA  is only slightly regulated byiron availability.  npgA  transcript levels are about 2-foldupregulated during iron-depleted conditions, comparedwith iron-replete conditions (Fig. 3B).  D sreA displaysthe same  npgA  expression pattern as  wt , indicating thisregulation is SreA-independent (Fig. 3B). In this respect,it is interesting to note that we previously showed thatan iron-regulatory mechanism exists in  A. nidulans which does not involve SreA (Oberegger et al. 2002b).The growth defect of   cfwA2  is due to impairedbiosynthesis of siderophores and lysineLike  S. cerevisiae ,  A. nidulans  synthesizes lysine via the a -aminoadipate pathway (Arst et al. 1973; Weidner et al1997; Busch et al. 2003). This pathway includes thePpant-dependent  a -aminoadipate semialdehyde reduc-tase. Consequently,  cfwA2  is supposed to be auxotro-phic for lysine at the restrictive temperature; but Fig. 2  Reversed-phase HPLC analysis of intracellular and extra-cellular siderophore production of   A. nidulans wt  and  cfwA2  at37   C and 25   C. The fungal strains were grown for 24 h in liquidlow-iron  Aspergillus  minimal medium (AMM); and the productionof extracellular (TAFC) and intracellular (FC) siderophore wasanalyzed, as described in the Materials and methods.  Columns represent the siderophore production normalized to  wt  at therespective temperature. Samples were prepared in triplicate, andSD did not exceed 10%213  supplementation with lysine did not repair the growthdefect (Fig. 4). In  A. nidulans , siderophore biosynthesisis essential for viability; and the growth defect of mu-tants defective in siderophore synthesis can be cured bysupplementation with TAFC or FC (Eisendle et al.2003). However, supplementation with TAFC did notcure the growth defect of   cfwA2 . In contrast, supple-mentation with TAFC plus lysine led to a  cfwA2  growthrate similar to that of   wt  but did not compensate thepigmentation defect (Fig. 4). Replacement of TAFCwith FC or performing the experiment at 42   C yieldedthe same results (data not shown). These data demon-strate that the growth defect of   cfwA2  at the restrictivetemperature is mainly due to impaired biosynthesis of siderophores and lysine.Inallexperimentscomparing wt and cfwA2 ,ithastobeconsidered that these  A. nidulans  strains are not isogenic.Nevertheless, this fact seems not to interfere with theinterpretation of the databecause:(1) cfwA2 behaves like wt atthepermissivetemperatureof25   C,withrespecttosiderophore biosynthesis and regulation of expression of  mirA  and (2) the alleles  yA2  and  pabaA1 , present in cfwA2 ,donotinterferewiththeregulationofsiderophorebiosynthesis in other genetic backgrounds at a growthtemperature of 37   C (H. Haas, unpublished data).In conclusion, NpgA seems to be a broad-rangePPTase required for the modification of at least five dif-ferent enzymes involved in both primary and secondarymetabolism and, obviously, these functions cannot becomplemented by a putative second PPTase. It isrequired for: (1) pigment synthesis, where the most likelyimpaired enzyme is thepolyketide synthase WA, whichisessentialforpigmentsynthesis(MayorgaandTimberlake1992), (2) lysine biosynthesis, with the enzyme mostprobably affected being the  a -aminoadipate semialde-hyde reductase, (3) biosynthesis of the siderophoresTAFC and FC (in the latter case the putative enzymecompromised is the NRPS SidC), and (4) penicillin bio-synthesis, where the impaired enzyme is probably theNRPS  d -( L - a -aminoadipyl)- L -cysteinyl- D -valine synthe-tase. Additionally, the  cfwA2  allele might negativelyaffect another enzyme involved in penicillin biosynthesis:isopenicillin  N   synthase (IpnA). IpnA is a nonhemeiron(II)-dependent oxygenase (Schofield et al. 1997). The cfwA2  strainis iron-starved, due to impairedsiderophorebiosynthesis. Consequently, the iron-dependent IpnAmight be inactive due to lack of iron. Moreover, wepreviously showed that various genes encoding proteinsin need of iron-containing cofactors—like aconitase Fig. 3A, B  Expression of   mirA  and  npgA  in  A. nidulans . A  Expression of   mirA  in  wt  ( left ) and  cfwA2  ( center ) at 37   C( top panel  ) and 25   C ( bottom panel  ).  B  Expression of   npgA  in  wt ( left ) and  D sreA ( right ). Fungal strains were grown for 15 h in low-iron AMM ( top panel  ) or AMM with iron ( middle panel  ). TotalRNA was isolated from the harvested mycelia and subjected toNorthern analysis. Hybridization with  acnA  served as a control forthe loading and quality of RNA ( at right in  A ;  bottom panel in  B  ) Fig. 4  Growth phenotypes of   A. nidulans wt  and  cfwA2  at 37   Cand 25   C. Aliquots of 10 6 conidia of the respective fungal strainswere point-inoculated on AMM supplemented  as indicated   with10  l M TAFC and/or 10 mM lysine ( Lys ) and incubated for 48 h.Please note that  cfwA2  has yellow conidia instead of the green  wt conidia, due to carrying the  ya2  allele (O ¢ Hara and Timberlake1989)214  ( acoA ), homoaconitase ( lysF  ), and cytochrome  c ( cycA )—are transcriptionally downregulated in responseto iron depletion (Oberegger et al. 2002b); and the samemight apply to  ipnA . Acknowledgements  We thank Dr. Geoffrey Turner for providingAJC12:36. We also thank Dr. Paul Illmer and Gerlinde Ha ¨ningerfor their help in HPLC analysis of siderophores. This work wassupported in part by Austrian Science Foundation grant FWF-P15959-B07 (to H.H.) and Austrian National Bank (OENB)grant 8750 (to H.H.). References Aguirre J, Ortiz R, Clutterbuck J, Tapia R, Cardenas M (1993) vegA  and  cfwA  define two new developmental genes in  Asper- gillus nidulans . 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