fleming’s penicillin producing strain is not penicillium ... · abstract: penicillium chrysogenum...

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doi:10.5598/imafungus.2011.02.01.12 IMA FuNgus · voluMe 2 · No 1: 87–95

Fleming’s penicillin producing strain is not Penicillium chrysogenum but P. rubens

Jos Houbraken1,JensC.Frisvad2,andRobertA.Samson1

1CBS-KNAWFungalBiodiversityCentre,Uppsalalaan8,NL-3584CTUtrecht,theNetherlands;correspondingauthore-mail:[email protected],Building221,SøltoftsPlads,TechnicalUniversityofDenmark,DK-2800Kgs.Lyngby,Denmark

Abstract: Penicillium chrysogenum isacommonlyoccurringmould in indoorenvironmentsandfoods,andhasgainedmuchattentionforitsuseintheproductionoftheantibioticpenicillin.Phylogeneticanalysisof the most important penicillin producing P. chrysogenum isolates revealed thepresenceof twohighlysupported clades, and we show here that these two clades represent two species, P. chrysogenum and P. rubens. These species are phenotypically similar, but extrolite analysis shows thatP. chrysogenum producessecalonicacidDandFand/orametaboliterelatedtolumpidin,whileP. rubens does not produce thesemetabolites.Fleming’soriginalpenicillinproducingstrainandthefullgenomesequencedstrainofP. chrysogenumarere-identifiedasP. rubens.Furthermore,thewell-knownclaimthatAlexanderFlemingmisidentifiedtheoriginalpenicillinproducingstrainasP. rubrumisdiscussed.

Article info:Submitted29April2011;Accepted24May2011;Published7June2011.

Key words: Flemingindoor mycologyphylogenytaxonomy

INtroductIoN

Penicillium chrysogenum is a commonly occurring mould in indoorenvironmentssuchasdust, indoorair,anddampbuilding materials (Chang et al. 1995, Gravesen 1999,Hunter & Lea 1995). Furthermore, P. chrysogenum is frequentlyidentifiedasafoodspoilageagentandhasgainedmuch attention for its use in the production of the antibiotic penicillin(Samsonet al.2010). ThetaxonomyofthisspecieswasstudiedextensivelybyRaper&Thom(1949)andtheyaccepted four species in the “Penicillium chrysogenum series”: P. chrysogenum, P. notatum, P. meleagrinum, and P. cyaneofulvum.However,Samsonet al. (1977) regardedthis series as one broad species and did not accept Raper and Thom’s“P. chrysogenumseries”.Morerecently,otherspecieshavebeen included inPenicillium series Chrysogena, such as P. flavigenum, P. nalgiovense, and P. dipodomyis(Frisvadet al. 1987, Banke et al. 1997, Frisvad & Samson 2004),and these species are clearly different from P. chrysogenum althoughallspeciesproducepenicillin (Frisvadet al.1987,Andersen & Frisvad 1994, Banke et al. 1997, Färber &Geisen 1994). Scottet al. (2004) studied the taxonomy ofP. chrysogenum using multilocus sequence typing (MLST)dataandshowedthatitcouldbesubdividedintofourclades.However, thatstudymainly focusedon indoor isolatesanddid not link the synonyms of P. chrysogenumtothoseclades.

Penicillium chrysogenumwasdescribedbyThom(1910),but the name P. chrysogenum is predated by P. griseoroseum, P. citreoroseum, and P. brunneorubrum, which were described by Dierckx (1901). Pitt (1980) accepted P. griseoroseum and P. chrysogenum, and synonymized P. citreoroseum and P. brunneorubrum with P. griseoroseum. However, subsequentmorphological, physiological and extrolite data showedthat these specieswere conspecific withP. chrysogenum (Frisvad & Filtenborg 1989, Banke et al. 1997). Since P. chrysogenum is a widely used name, Frisvad et al.(1990) and Kozakiewicz et al. (1992) suggested topreservethenameP. chrysogenumandtorejecttheoldername P. griseoroseum with its synonyms P. citreoroseum, and P. brunneorubrum. This proposal was acceptedby the Committee for Fungi and Lichens and the name P. chrysogenum is currently listed as nomen conservandum (McNeillet al.2006).

The aim of this study was to find out whether the twomost common clades in P. chrysogenum sensu lato (Scottet al.2004)representoneortwospecies.Towardsthisaim,wehavestudiedthemostimportantstrainsinthehistoryofpenicillin production by combining extrolite and moleculardata.Furthermore,thehistoryofthetaxonomyofFleming’spenicillin-producerstrainisdiscussed.

Houbraken et al.ARTICLE

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MAterIAl ANd MetHods

strainsAn overview of Penicillium chrysogenum strains used in this study is given in Table 1. Three point positioninoculations of these strains were made in order to study their macromorphology. The strains were inoculated onCzapekyeastextractagar(CYA),yeastextractsucroseagar(YES), creatine agar (CREA) andmalt extract agar (MEA)(Oxoid).AllmediawerepreparedasdescribedbySamsonet al.(2010).Colonycharacteristicswererecordedafter7dofincubationat25°C.MicroscopicmountsweremadefromMEAorOAandmicroscopicalfeatureswerestudiedbylightmicroscopy(OlympusBH2andZeissAxioskop2Plus).

Molecular analysisDNAextractionwasperformedusingtheUltracleanMicrobialDNA isolation Kit (MoBio, Solana Beach, USA) accordingthe manufacturer’s instructions. Parts of the β-tubulin andcalmodulingeneswereamplifiedandsequencedaccordingto the method described by Houbraken et al. (2007).ApartoftheRPB2locuswasamplifiedusingtheprimerpairRPB2-5F_Pc 5’- GATGACCGTGACCACTTCGG-3’ and RPB2-7CR_Pc 7CR 5’- CCCATGGCTTGTTTGCCCAT-3’. Thealignmentswerecombinedandmaximumlikelihoodanalysiswas performed using RAxML v. 7.2.8. Each dataset wastreatedasaseparatepartition.TheGTRsubstitutionmatrixwasused forall threepartitionsand individualperpartition

branch length optimization was carried out (Stamatakis et al. 2008).Maximumparsimonyanalysiswaspreformedasa second measure of branch support. This analysis wasruninMEGA5andbootstrapvaluesweredeterminedusing500 replicates. Penicillium griseofulvum CBS 185.27 wasselectedasanoutgroup.NewlygeneratedsequencesweredepositedinGenbankunderJF909924–JF909977.

extrolite analysisStrainsweregrownfor7–14dat25°ContheagarmediaYES, CYA and MEA prior to extrolite extraction. Extroliteswere extracted and analyzed by HPLC-DAD as describedby Frisvad &Thrane (1987) andmodified by Smedsgaard(1997). Identification was performed by comparison of theUVspectraandretentiontimesoftheextroliteswiththoseofpurifiedandchemicallycharacterizedcompoundspresentinthecollectionattheBiocentrum-DTU(Lyngby).

results

Phylogenetic analysisThestructureofthepartitionsofthealignmentwasasfollows:the calmodulin partition was 506 basepairs long and had90 distinct alignment patterns; the RPB2 dataset included981 characters, and 91 of those had a distinct alignmentpattern and the tubulin gene partition was 434 basepairslong,62were found tohaveadistinctalignmentpattern. In

table 1.OverviewofPenicillium chrysogenum and P. rubensstrainsstudied.

No. other collection no. Name substrate, locality remarks

NRRL820 DTO100G5=IBT4395 =IBT6067=IMI92220

P. chrysogenum Unrecordedsource Ex-lectotypeofP. griseoroseum

CBS306.48T NRRL807=IBT5233 =IMI24314

P. chrysogenum Cheese,Storrs,Connecticut,USA Ex-lectotypeofP. chrysogenum

DTO87I2 P. chrysogenum Fungalgrowthonceilingofarchive,Utrecht,theNetherlands

DTO102B4 IBT26889=C238 P. chrysogenum Housedust,Wallaceburg,ON,Canada

Representativeofgroup2inthestudy of Scottet al.(2004)

CBS355.48 NRRL821=IBT4344 =DTO98D4=IMI39759

P. chrysogenum Branches of Hyssopus,Norway Ex-typeofP. notatum

CBS197.46 NRRL832 P. rubens Mustcontaminant,Belgium Thestrainfirstusedforproducing penicillin in submergedculture(Raper&Thom1949:368–370)

CBS205.57 NRRL824=IBT30142 =IMI015378

P. rubens Culture contaminant in bacterial culture,UK

Fleming’soriginalpenicillinproducing strain

DTO95E9 IBT30661 P. rubens Cap of beer bottle, Belgium

NRRL792T DTO98E8=IBT30129 P. rubens Unrecordedsource Ex-lectotypeofP. rubens

CBS307.48 NRRL1951=IBT5857 =IMI40233

P. rubens MouldycantaloupePeoria,Illinois,USA

“Wisconsinstrain”,parentofmost high yielding penicillin producing strains

Wisconsin54-1255 P. rubens MouldycantaloupePeoria,Illinois,USA

Fullgenomesequencedstrain

Penicillium rubens, the original penicillin-producer ARTIC

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themaximumparsimonyanalysis,118siteswereparsimonyinformative(3150MPtrees, length=400,consistency index0.69,retentionindex0.83).Thepercentageofreplicatetreesinwhichtheassociatedtaxaclusteredtogetherinthebootstraptest is shownnext to the branches (Felsenstein 1985).Thebest-scoringmaximumlikelihoodtreeisshowninFig.1.

Phylogeneticanalysisof thecombinedpartialβ-tubulin,calmodulinandRPB2datasetsrevealedtwohighlysupportedclades in Penicillium chrysogenum sensu lato.Onecladeis

centeredaroundtheex-typestrainofP. chrysogenum(CBS306.48).Alsotheex-typestrainsofP. notatum(CBS355.48),P. griseoroseum (NRRL820)and several other strainsareaccomodated in this clade. The other clade is centeredaroundtheex-typestrainofP. rubens(NRRL792).Fleming’soriginalpenicillinproducingstrain(CBS205.57),thewild‐type Wisconsin strain isolated from a mouldy cantaloupe (CBS307.48 = NRRL 1951), the full genome sequenced strainderivedfromNRRL1951(Wisconsin54‐1255)(vandenBerg

0.01

P. rubens CBS 205.57, Fleming’s penicillin producer

P. rubens CBS 307.48, Wisconsin strain wild-type

P. rubens Wisconsin 54-1255, full genome sequenced strain

P. rubens CBS 197.46, first strain used for submerged penicillin production

P. rubens, NRRL 792T, ex-lectotype

P. rubens DTO 95E9

100/99

P. chrysogenum CBS 355.48, type of P. notatum

P. chrysogenum DTO 87I2

P. chrysogenum DTO 102B4

P. chrysogenum NRRL 820, ex-lectotype of P. griseoroseum

P. chrysogenum CBS 306.48T, ex-lectotype 73/-

100/93

100/99

P. flavigenum CBS 419. 89T

P. confertum CBS 171.87T

P. mononematosum CBS 172.87T

100 99

100/99

P. dipodomyis CBS 110412T

P. nalgiovense CBS 352.48T

P. persicinum CBS 111235T

P. aethiopicum CBS 484.84T

P. griseofulvum CBS 185.27T

100/99

Figure 1

Fig. 1.BestscoringMLtreeofcombinedpartialβ-tubulin,calmodulinandRPB2datasets.Thebootstrapvaluesof themaximumlikelihoodanalysis(ML,firstnumber)andmaximumparsimonyanalysis(MP,secondnumber)areshownatthenodes.Thebootstrapvaluesabove70%areshownandbranchessupportedlessthan70%ineithertheMPorMLanalysis areindicatedwithahyphen.



et al.2008),thestrainfirstfoundtoproducesatisfactoryyieldsofpenicillinwhengrownsubmerged (CBS197.46=NRRL832), are accommodated in this clade. These two cladesevidently represent two different species, P. chrysogenum and P. rubens.

Phenotypic and extrolite analysisPhenotypic examination using macro- and microscopicalcharacters of strains of the Penicillium rubens and P. chrysogenum clades did not reveal clear differences.Themajorityoftheexaminedstrainsshowedsimilargrowthcharacteristics, such as good growth and sporulation on YES agar, weak or moderate growth on CREA with pooracidproductionand largeclearoryellowcolouredexudatedroplets on MEA and/or CYA (Fig. 2). Three strains haddeviatinggrowthcharacteristics:CBS355.48(ex-typecultureof P. notatum)hadsmallercoloniesonalltestedagarmedia,andNRRL792(ex-typecultureofP. rubens;Fig.3),andCBS307.48 ( = NRRL 1951, wild-type full genome-sequencedstrain)deviatedinhavingreducedsporulationonmostagarmedia.

Extroliteanalysisdifferentiated theP. chrysogenum and P. rubens clades. An overview of extrolites produced bythese species, with emphasis on the important strains in the historyofpenicillin,isshowninTable2.Theseresultsshowthat P. chrysogenumproducessecalonicacidDandFand/orametaboliterelatedtolumpidin(Larsenet al.2001),while P. rubensdoesnotproducethesemetabolites.

tAxoNoMy

Penicillium chrysogenumThom,Bull. Bur. Anim. Ind. USDA 118:58(1910);nom.cons.

Synonyms:Penicillium griseoroseumDierckx,Ann. Soc. Sci. Brux. 25:86(1901);nom.rej.Penicillium citreoroseumDierckx,Ann. Soc. Sci. Brux. 25:86(1901);nom.rej.Penicillium brunneorubrumDierckx,Ann. Soc. Sci. Brux. 25: 88(1901);nom.rej.Penicillium notatum Westling,Ark. Bot.11(1):95(1911).

Type: usA: Connecticut: Storrs, isol. ex cheese, 1904,C. Thom(IMI24314–typ.cons.).

Penicillium rubens Biourge, La Cellule 33:265(1923).

Type: Unrecorded source, P. Biourge. CBS H-20595 –lectotypus hic designatus; from Biourge (402) to Thom(4733.110)1930toNRRL(792)1949toCBS2009;culturesex-lectotype–NRRL792=IBT30129=ATCC9783=CBS129667.

Penicillium chrysogenum has, in addition to the species names mentioned above, also various other synonyms(Samson et al. 1977, Samson & Frisvad 2004). Theseinclude P. meleagrinum Biourge 1923, P. cyaneofulvum Biourge 1923, P. chlorophaeum Biourge 1923, P. roseo-citreum 1923, P. camerunense Heim 1949 nom. inval.,P. chrysogenum var. brevisterigma Forster nom. inval.1953, P. aromaticum f. microsporum Romankova 1955, P. harmonenseBaghdadi1968,P. verrucosumvar.cyclopium strain “ananas-olens” Ramírez 1982, and P. chrysogenum “mut. fulvescens” Takashima et al. ex Ramírez 1982. Amore detailed molecular or extrolite analysis is needed toproperly place these species in either P. chrysogenum, P. rubens, or other currently unrecognised species belonging to P. chrysogenum sensu lato.

table 2.ExtrolitesproducedbyPenicillium chrysogenum and P. rubens strains studied, with a focus on the important strains for the history of penicillin.

No. other collection no. Name extrolites

CBS306.48T NRRL807=IBT5233 P. chrysogenum AndrastinA,andrastinB,chrysogine,citreoisocoumarin,cf.lumpidin,meleagrin,penicillin,roquefortineC,secalonicacidD,secalonicacidF,sorbicillins

CBS355.48 NRRL821=IBT4344=DTO98D4 P. chrysogenum AndrastinB,chrysogine,cf.lumpidin,meleagrin,penicillin,roque-fortine C, secalonic acid D, sorbicillins

NRRL820 DTO100G5=IBT4395=IBT6067 P. chrysogenum AndrastinA,andrastinB,chrysogine,can-741,meleagrin,cf.lumpidin,roquefortineC,sorbicillins

DTO102B4 IBT26889=C238 P. chrysogenum AndrastinA,andrastinB,chrysogine,meleagrin,cf.lumpidin,secalonicacidD,secalonicacidF

CBS205.57 NRRL824=IBT30142 P. rubens Chrysogine,meleagrin,penicillin,roquefortineC,andrastinA,sorbicillins

NRRL792T DTO98E8=IBT30129 P. rubens Chrysogine,meleagrin,penicillin,cf.roquefortine,sorbicillins

CBS307.48 NRRL1951=IBT5857 P. rubens Chrysogine,meleagrin,penicillin,roquefortineC,roquefortineD,sorbicillins

DTO95E9 IBT30661 P. rubens Meleagrin,roquefortineC


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Fig. 2. Penicillium rubensCBS205.57(Fleming’soriginalpenicillin-producer).A–c.Colonies7dold25°C.A.CYA.B.MEA.c.YES.d–H. Condiophores.I.Conidia.Bars=10µm.



Fig. 3.Penicillium rubensNRRL792.A–c.Colonies7dold25°C.A.CYA.B.MEA.c.YES.d–H.Condiophores.I.Conidia. Bars =10µm.


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dIscussIoN

Identity of Fleming’s original penicillin-producing strainIt has long been claimed that Fleming (1929)misidentifiedthe original penicillin-producing strain as “Penicillium rubrum Biourge 1923” (Raper&Thom1949, Lax 2005).However,it was the mycologist, Charles J La Touche (1904–1981),workinginaroomonthefloorbelowthatoccupiedbyFleming,whoactuallyidentifiedthestrainasPenicillium rubrum.Ourcurrent study shows that the correct name of this strain is P. rubens.Wediscusstwopossiblescenarioshere,whichcouldhaveledLaTouchetohisidentificationofFleming’sstrainasP. rubrum, both showing that his identificationwas (rather)accurate:1. LaTouche identified Fleming’s strain asP. rubrum, but

this was a lapsus for P. rubens. The epithet rubrum means “red” and rubens “to be red”, and thus it is not unlikely that these epithets have been confused. Bothyellow and red-brown reverse colours have beenobserved inP. chrysogenum sensu lato and in the true P. rubrum (i.e.P. purpurogenum)(Pitt1980).Thesetwospecies clearly differ in micro-morphology from each otherandthemycologistLaTouchewouldhavenoticedthese differences. Pink and vinaceous drab coloursare mentioned by Raper & Thom (1949) for strains of P. meleagrinum and reddish colours are part of the names of synonyms of P. chrysogenum sensu lato, such as P. citreoroseum, P. roseocitreum, P. brunneorubrum, and P. griseoroseum.

2. In theotherscenario,LaTouchecorrectly identified thestrain as P. rubrum based on the taxonomic schemespresentatthattime.In1929,themostrecentmonographon PenicilliumwasthatofBiourge(1923),andLaTouchewould have used this monograph for identificationof Fleming’s penicillin-producing strain. This is alsosupported by that monograph being cited in Fleming’sreportoftheoriginaldiscoveryofpenicillin(Fleming1929).Penicillium rubrum is listed in Biourge’smonograph as “P. rubrum(?)Grassberger-Stoll,1905”andthisspecieswas classified in Biourge’s subsection “Les Radiés, Radiata”.ThissubsectionalsocomprisesP. chrysogenum and the species P. griseoroseum, P. cyaneofulvum, P. brunneorubrum, P. notatum, P. citreoroseum, and P. baculatum, which are currently all listed as synonyms of P. chrysogenum.ExaminationofBiourge’sdrawingsof“P. rubrum(?)Grassberger-Stoll”showsthathisdefinitionof P. rubrum is very similar to that ofP. chrysogenum and does not fit in the original concept of P. rubrum (Stoll 1904,Pitt 1980).ThiswasalsonotedbyBiourge(1923) and therefore he added the question mark tohis description of P. rubrum. Furthermore, the closeconnection between P. rubrumGrassberger-Stollsensu Biourge and P. rubenswasalsonotedbyThom (1930:250)who assigned the former species toP. rubens. Inconclusion,LaToucheusedBiourge’staxonomicschemeandidentifiedFleming’sstrainasP. rubrumGrassberger-

Stoll (asdefinedbyBiourge)and this identificationwasaccurate at that time and not incorrect as claimed in literature(Thom1945,Raper&Thom1949,Lax2005).Furthermore,Biourge (1923)wrote in his descriptionofP. rubrum that the species was a common laboratory contaminant(“…unanimaldelaboratoire!”),whichcouldhave strengthenedLaTouche in his ideaof having thecorrect identification,sinceFleming’s isolatewasalsoalaboratorycontaminant.

Name changes of Fleming’s penicillin-producing strainWhenCharlesThomwasabout tofinishhismonograph in1930,hereceivedtheFlemingstrain(CBS205.57=NRRL824 = IMI 015378) that had been identified asPenicillium rubrum,andhe re-identified itasP. notatum (Thom1945).He did not realize that the Fleming isolate was identifiedby the experienced mycologist Charles La Touche sincehewrote: “Not beingamycologist, he [AlexanderFleming]undertook to identify the mold from the literature and selected the name Penicillium rubrumBiourge”(Thom1945).However, the authority name Biourge is not mentioned inFleming’sworkandThomwouldhavebeenmoreaccurateif he had named it “Penicillium rubrum Grassberger-Stoll sensu Biourge”. Subequently, Samson et al. (1977)broadened the concept of P. chrysogenum and placed P. notatuminsynonymywiththeformerspecies.Thistransferwaslatersupportedinseveralotherworks(Pitt1980,Frisvad& Filtenborg 1989, Banke et al. 1997, Samson & Frisvad2004).Fromthispointon,Fleming’spenicillin-producerwasidentifiedasP. chrysogenum.Ourcurrentstudyshowsthatthe correct name of this strain is P. rubens.

origin of the Fleming strainCharles La Touche was working with moulds from indoorair andespecially those isolated fromcobwebs.He isolatedhundredsofstrainsfrompoorerpartsofLondonandSheffield(Hare1970,Kingston2008)andalargepercentageofthemwere Penicillium chrysogenum and P. rubens. Furthermore,subsequentresearchhasshownthatthesemightbethemostcommon Penicilliuminindoorair(Scottet al.2004,Samsonet al.2010).LaTouche’shypothesiswasthatthesemouldscouldcauseasthma,andthiswaslaterconfirmedby,forexample,Wardet al.(2010).ItcouldverywellbethatoneofLaTouche’scultures from indoorair contaminatedFleming’splate, sincetheywereworking in thesamebuildingandFlemingalwayshad his windows shut (Hare 1970). Both P. rubens and P. chrysogenum produce penicillin, and in that respect it is not surprising that P. rubens was present. It is noticeable thatthehistorical importantpenicillinproducers(WisconsinstrainNRRL1951,Fleming’sstrainCBS205.57=NRRL824=IMI015378, and the strain first used for producing penicillin insubmergedconditionsCBS197.46),allprovedtobeP. rubens.

current taxonomy and implicationsHere we recognize the presence of two species in Penicillium chrysogenum sensu lato. The presence of four lineages



in P. chrysogenum was described by Scott et al. (2004).Scott’s lineages 1 and 2 areP. chrysogenum, and lineage 4 corresponds toP. rubens.The statusof lineage3 is stillunder investigation.ItwassuggestedbyScottet al.(2004)that lineage 4 (i.e. P. rubens) may have a competitiveadvantageoverother lineagestoexploithuman-associatedindoor niches and support for this hypothesis requiresinvestigationofdistributionpatternsofmembersofthisgroupin other geographic regions. The full genome sequencedstrainderivedfromNRRL1951(vandenBerg2008)isalsoa P. rubens, and this shows that there is as yet no strain of P. chrysogenum sensu strictofullgenomesequenced.

AcKNowledgeMeNts

We thank Ellen Kirstine Lyhne for technical assistance and thereviewersfortheirhelpfulsuggestions.

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fleming’s penicillin producing strain is not penicillium ... · abstract: penicillium chrysogenum...

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