C A N . J . MICKOBIOL. VO1.. 25. 1979
Mycophenolic acid production by Peizicillium brevicompacturn in two media D. L. D ~ E R F L EC. K ,D. BAKTMAN. A N D I. M. C A M P B ~ L L I ) ~ ~ ~ ~ I I c~f'Rir~lo~yic~iil . ~ I ~ I ~ I I /St.it,~ii.c..\.U~rirc.r.(3). 75. ant1 3 mL) wcre taken ;~septicully h u m the c~lltllrci t t the st;~tedtimes fol. isotope irlcorpor;lti~n. biom;~ssnie:lsul.enlcnt. 2nd f'ol-~nicroscopicexamination. I.so10~~t~ /II(,OI./JOI.(II;(III l~.v/)t~~.itt~~'tr~,\ (III(/T / ~ ( ~ i ~ ~ , - t t i ( ~ / , ~ . ~ i . \ S u h c ~ ~ l t ~( ~ 5 0~m. eLsin a 125-mL Er1ennievc1-flask) wcre prepa~.cd13. 24. 36. 48. 66. ant1 9Oh following inoculation. These were incubateti ftrl- 3 h with [ I-l"Clacetalc (25 1tCi. 58 1tCilltlnol OEntries are the r c s r ~ l tof single measurements. Confidence levels were independently; live replicate assays of a liquid culture that had ( 1 Ci = 37 GBq), Nc\v Eriglancl Nuclear Corp.. Boston.Mi~s- aestablished n average biomass d r y weight of 0 29 8/75 m L gave a standard deviation sachusetrs). At the end of the ilicuh~~tion period. the culture of t 0.005 g. 'Initial spore density was 3.1 i: 107/rnL. (medilrnl ant1 biomass together) was supplerncnteil with internal clnirial spore density was 2.2 x lO'/mL. stand;irtls Or-pcnta(1ccanoic acitl. 48.411g: dotl-iasontanc. 101.8 .ILK). - k a s acitlifictl with concentr;ttcd HCI ( I mL).;tnd was homogenized with ethyl :~cct;ttc(75 mL) for I min in a blc11dc1-. M y c . o p h c i ? o l i c A c i t l Protlrtc~tio17it? Merlicr A ot?cl B '1-hc orgi~nic1;1ycr W;IS rern~vetl.PI.CCLI of' b ~ l v c n t .i ~ n dtl.catccl Since medium B is nominally richer than the irith crhe~.e;~l iliazorncthane for 15min. Del.ivatized extracts \YCI.C analyzetl by radiogas chron1;ltography (KGC) and ~.adiog;~s media in which Brar o t (11. (1968) a n d Audhya and Russell (1975) encountcrcd idiophadic production ch~.t)mi~tog~-;tphy - rnilss spectrc)nietry (KGC-MS) as described PI-cviously(Doel-Hcl-c.1 ill. 1978) with the exception that a finill of the ergot alkaloids and enniatin, ~.espectively, 8 1 s chromatographic tcmpcl-aturc of 300°C \\.;IS ~rsetl.The and since it suppol~s a markedly faster growth rate logal.ithn?ic clata compl.cssion 511-i~tcgy~.ccently advocatctl P. 1 ~ i . ~ \ ~ i c ~ o i ? ? p a i ~(I-elative trri17 to medium A). we of (Ilocrcflcr ant1 Can?pbell 1978) \vas adoptctl in all gas al-g~~ed that for mycophenolic acid PI-oduction to ch~.omatogr.aphicanalyses. Biotrr(c.c.\! \ l ( , o . c r i ~ . c , t ~ ~ c , ~ r ~ . \ At selcctcd time\ ; ~ l i q ~ ~(75mL.) ots of the C L I I ~ L I I . C S were rec h and n c r c tilterccl thro~rgha moved from thc F ~ I - n b i ~ fl;~sk prci\'righetl ant1 predriccl (IOOoC. 48 h) tiltel. papel- (Whatman # I ) lrsing a Huchncr f ~ ~ n n c Filter l. pilpel. plus biomass were \i':t\hetl with ?la\.;-tlistilletl water ( 2 5 0 nil.) i~nrl\\'el-c tlricil at IOO°C fol-48 h before ~.e\vcighi~lg.
Results Bioi~?rt.c..s P i . o l i f o i . c ~ r i o i ~(it?(/ h'lorplio/ogic.cl/ ~ ~ o I o / > i ? ;it ~ ~Mc,(/ilr ~ t r i A (ri1(1B
Dc-
follow the ergot alkaloid-enniatin patterns i t would need to be idiophasic in medium B. Since rnycophenolic acid production in strain ATCC 9056 is already known to be growth-linked in riiedium A (Nulton and Campbell 1977; Doel-fler ~t (11. 1978) a simple test for idiophasic PI-oductionin medium B is as follows: whereas mycvphenolic acid biosynthesis i n ATCC 9056 would be expected to be occurring in rnediunl A from the time (48 h) when the tissue biomass begins to increase. i t sho~lldnot occur to any significant extent in n~ediumB until growth becomes rest[-ictedaround the 90th h. The relevant data 31-e shown in Table 2. Mycophenolic acid p~~oduction. as measur.ed both by pool size increase and radioactivity inco~.po~-ation from [14C]~~cetate. is active in both media from the time when tissue proliferation begins. Clearly, therefore, rnycophenolic acid production in P. hi.rl.ic.oii?j>crc.rrri??does not conform to the pattern fo~lndin C'/rt~.ic.c~l)sbv BI-ar c t trl. (1968) and i n F r t s t ~ i ~ i r rby t ~ i Audhya and Russell (1975): rather, i t resembles the A . prrrrrr1rr.s case (Burtman and Campbell 1979).
I t is shown in Table 1 how I?iomass dry weight changed with time in the two media. In metlium B. gel-mination occurr-ed between the 34th and 35th h: by the 36th h. tissue ;\ccumul;\tion was well under way. I n niediuni A. germination was delaycd until the 36th h and significant tissue accumulatiun was not seen till the 66th h. Event~lalbiomass pl-oliferation was approximntcly sixfold more rapid in medium B than i n medium A. Correspondingly. biomass PI-oliferation was continued for at least Discussion 212 h in medium A, but was terminated i n medium The results ofthis study, taken together with our B ar-ound the 90th h. The culture in medium A sporulated around the 66th h but no sporulation previous findings (Nulton and Campbell 1977; occurred at the 212th h i n medium B. Doerflcr c t at. 1978; Bartman and Campbell 1979)
942
CAN. J . MICROBI(
TABLE2. Yicld (milligrams per 50 m L or culture alicluot) anti specific ratlioactivity (disintegrations per minute per niicrogram) of 1iiycopher1olic acid p r o d ~ ~ c e in d media A ant1 B rollowing incubation with [I-'"Clacetate Yield" ancl specific radioactivity" (in parenthesis)
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by YORK UNIV on 11/20/14 For personal use only.
Tinieartcr inoculation (11)
Acknowledgments The financial support of the National Science Foundation (PCM-78-032852) and the National Institute of Health (GM-00149) is gl-atefully acknowledged.
A H A K ~ ~ U O WYI .-.I:tnd . Z . A. L. DI.MAIN. 1978. Carbonc:~litholite regulation of cephalosporin production in Srreptottr~c.cs c~lr~brrligo.~is. Antimicl-ob. Agents Chemothe~..14: 159-164. A U I I H Y AT. . K.,and D . W . Russr.1 I . 1975. Enniatinp~.oduction by F~c.tro.i~ol~ .sr~rrrhrrc~itrr~t~~: primary secondary and 11nita1.y metabolism. J . Gen. Microhiol. 86: 327-331. B A R . I ~ ~ C. A ND.. . and 1. M. C ' A ~ I P B I . L1979. L . Naphth;~lenonc production in d.spergillrt.~porc~tlrr.~. Can. J . Microhiol. 25: 130- 137. "Each entry is the result ol' a single measurcn~cnr.Conlidcncc i n mass BHAR.S. S.. c. S. G ~ ~ h l . i W. ~ ~ A. l d T A B ~ R1968. . Pattel-nsofir~ s;rl~ieswas cl~cckedto be w ~ t l > i lnh c n n ~ i.c in a t e d-+ 10'2 1975) ,,,,(Slavhack PI 01. ~. -, r.iro crgot al kaloicl production by C'lo~:ic.ol>.\ po.spcili and thcir by making Rve replic;ile measurements in a n independent experiment; !he average value of 0.23 mg/50 mL uns :!ccompanied by a standard deviat~on association with different g r o s t h rates. Mycologia. 60: n f + 0 009 m ~ . - . - - .- - - ...-. 806-826. Dlnilial spore density was 3.1 :: 107/rnL. 'Initial spore density was 2.5 'i IO'/mL. Bu'Loc-K.J . 1). 1961. 1ntermedi:lry met;rholism and crntibiotic 'Decreasing specific activitics from this point o n reflect tile increasing size synthesis. Adv. Appl. hliuobiol. 3: 293-342. of a preexisting unlabelled pool of mycophenolic acid. -1967. E s w y s in hiosynthesih and microhiill devclopment. Chap. 3. John Wiley EL Sons. Inc. New Yol-k. pp. and those of other groups (Brarcr nl. 1968; Audhyn 42-67. and Russell 1975) lead to the conclusion that in ---- 1975. Seconclary mct;tholism in fungi and its rel;~tionl i q ~ ~ ishaken d or stirred culture there need be no ships to growth and clevelopment. Irr The filamentous fungi. Vol. I . chap. 3. f~clirc~tl h~ J . E. Smith and D. K. Bcrry. E. uniq~leassociation of seconda~-ymetabolism with AI-nolds.Loncion. pp. 33-58. the postvegetative growth phase offungal growth. BU'LOCK.J . D.. H , ~ R I I L T O M.N A. . H U L M I .A. . J . PO\VI.I.L.H. We. therefore. join other investigntol-s (e.g., AuM. S ~ I A L I . I : VI).. S H L P I ~ I : R Iand > , G . N. S ~ I I T H1965. . dhya and Russell 1975; Bu'Lock 1975; AhnronoMck~bolic development and secondary biosynthesis in P~~trir~il/irr~~r rrrric.oc,. Can. .I. Micl.ohiol. 11: 765-778. witz and Demain 1978) in calling for a reappraisal of what the fungal idiophnse and tl-ophophase DI:RI;\IN.A. L. 1974. How d o ;~ntibiot~c-proclucingnlic~.ooriivoitl suicidc? Ann. N.Y. Acitd. Sci. 233: 601-6 12. are and how these phases I-elate to the onset and D Igkinisms T K O YR. . W.. S. N. FKI.I:II.and D. J . FINNLL..1973. Remaintenance of fungal secondary metabolism. I21tionships between the hiosynthesis of virus-like pn~.ticles One area that wasrants attention in S L I C ;I~ reapand mycophenolic acid in Pcrric.illir~rrrstolorr~firrr~rr and P. h r c ~ ~ ~ i c ~ o t t r p oCra~nt.~J~. ~ Microbiol. ~r. 19: 1459- 1462. praisal is the appropriateness of submerged liquid I I .and I . M. C A M P U ~ ~ 1978. L I . . Logarithmic d a t ; ~ cultu~-e techniques in exploring the physiology and D ~ ~ K F LD.~ L.. compl-ession strategy for gas chromatoglxphy. Anal. Chem. molecular- biology of fungi that do not grow nor50: 1709-171 1 . mally in an agitated. aquatic environment. In na- D o ( - H F L ~ D. R . L.. C . P. N U L T ~ NC., D. B , ~ R I - M A F. N, J. and I. M. CAAIPBI:LL. 1978. Spore gel-n~ination. t ~ i t i r Go-r-~-LII:B. ture. 01-ganismssuch as P. b t ~ r ~ i c ~ o t n p n c ~ colcolony development. ant1 secondary mct21bolism in Pcrrt~ic.ilonize gl-ains and decaying vegetable mattel- in the lirirrr brc~~.ic~o~trpeic~riitti: ;I ~.i~diogi~s chromiltogr;~phitnd morsoil. growing on a stationary solid. moist support. phological study. Can. .I. Microbiol. 24: 1490-1501. The fungus grows vegetatively into the substratum KOFFI.I~R. H . . R . L. E ~ I E H S OD. NP . ~~Lhl,i~,anH d R. BURHIS. . and out from that substratum aerially in the PI-ocess 1945. Chemical changes in subrnelged penicillin fel-mentations. J . Bacterial. 50: 517-548. of conidiation. Vegetative and aerial hyphae oc. S . , i111dL. C. V I N I N C1970. . Metz~bolismofchloramcupy different nutritional environments and serve M A L I KV. phenicol by the producing organism. Can. J . Microbiol. 16: different biological ends. When such an organism is 173- 179. grown in the label-atory in shaken liquid culture. the MU-IH. W. L.. i ~ n d C. H . NASH. 1975. Biosynthesis of mycophenolic acid: purification and charecleriz:~tion of S distinction between vegetative and aerial hyphae adenosyl-L-methionine: demethylmycophenolic acid 0disappears. There can be no guarantee that fungi methyltransfelxse. Antimicrob. Agents Chemother. 8: will react uniformly or predictably to such a trans321-327. fer. We are currently exploring the possibility that NULTON.C. P.. and I . M ,C A M P B E L L1977. . Mycophenolic acid the variations seen in fungi regarding so-called is produced during balanced growth of Petricilliirt~~ l>~.c,cic'ot~~ptrc~trrtn.Can. J . Micl-obiol.23: 20-27. growth-linked or idiophasic secondary metabolism and E. W. in batch-mode shaken culture is attributable to dif- N U L T O NC. . P.. J . D. NAWORAL.I. M. CA~VIPBLLL. G R ~ ~ I . Z I V G1976. E R . A combined radiogas c h r o m : ~ t o g ~ ~ p h / ferences in response to the abnormality of culturing mass spectrometer detects intermediates in mycophenolic a nonaquatic organism in a stirred or shaken liquid acid biosynthesis. Anal. Biochem. 75: 2 19-233. S L A Y B A CJ. K ,R. B.. I . M. C.\MPBELL. and E. FAKISH.1975. A environment. Medium Bc (rich)
Medium A" (mini~iial)
-
~
~
~
protocol based on gas chromatog~~phy-flow counting for. munito~.ing intermei1i:u.y met:~bolism. Anal. Biochem. 69: 140- 154. S ~ I I I IJ .I ,E.. ;~ntlD. R . BI.I~. FI.IIII~.~'
Appl-ouve le 3 mai 1979 KII..BCRTUS. G.. et J . P1101.~.1979. Observation d'un sol forestier (rendzine) en microscopie electronique. C;ln. J . Microbiol. 25: 943-946. L'observation en microscopie electronique B balayage perrnet de mettre en evidence I:\ structure en agl-egats ilu sol forestier ktudie, ainsi que la presence de fil;~rnentsfongiques h I'exte~.ieur de ceux-ci. Les eaux de lavage de ces sols contiennent essentiellement des bacteries isolees, alors que les coupes dans les agrigats montrent frequemment des :~ssociationsd e bacteries entourees d e e adherent des feuillets d'argiles. mucilage e t B la s u ~ f a c duquel Ces observations conlirment I'existcnce d c deux types d e biotopes dans c e sol: I'un situe d a n s les espaces inter-agrkgats. le second i I'interieur m6me des agregats. KILBCKI-US. G . , ilnd J . PKOTH.1979. Observ:~tiond'un sol forestier (rendzine) en microscopie electronique. Can. J . Microbiol. 25: 943-946. Scanning elect~.onmicroscopy was used to evidence the aggregated stl-ucture of a forest soil as well as the presence of fungal hyphae external to soil aggregates. The supernatant of soil suspension in water mainly contained isolated bacteria. while ultrathin sections of aggregates frequently revealed groups of bacteria surrounded by a sheath of mucilage with adhering clay minelxls on the outside. These results conlirm the existence of two particular biotopes in the soil studied: oneis located spaces. inside aggregates, and the other. in the inter--agg~-egate [Translated by the joul-nal]
De nombreux chercheut-s ont suggere que les germes telluriques etaient lies ;I des matikres organiques ou minerales ou incluses dans celle-ci (Stevenson 1958; Jones et Griffiths 1964; Casida 1969; Zvyagintsev cr trl. 1969). Ces hypotheses ont ete confirmees par I'observation du sol en microscopie electronique B transmission (Balkwill ct 01. 1975; Bae et Casida 1973; Kilbertus cJt rrl. 1977): les feuillets d'al-giles adherent souvent A la surface des bacteries, des colonies du procaryotes ou de filaments de champignons. De plus, I'observation de debris vegktaux (Kilbertus et Reisinger 1975; Olah cJt (11. 1978) a montre que les microol-ganismes se developpaient generalement A I'intet-ieurdes parois ou dans les cellules et qu'il etait par consequent impossible de les dissocier de la matiere en decomposition. Dans le cas particulier du sol, Balkwill ct rrl. (1977) en utilisant differents traitements physiques (broyage, sonication) ainsi que des agents dispersants, n'ont reussi qu'i isoler 60% des germes de ce milieu. Cela prouve que la quantifica'Demandes de reimpression: G . Kilbertus.
tion des microorganismes du sol dans ces biotopes est extremement delicate. Nous avons tente d'appot-ternotl-e contribution ;I ces problemes en experimentant sur un sol forestier (rendzine) dont les caracteristiques pedologiques ont ete publiees par Vannier (1970). Apses avoir agite durant 10 min, 10 g de sol dans 100 mL d'eau, nous avons passe ['ensemble sur un tamis (mailles de 50 pm) et lave le tout ;I I'aide de I000 mL d'eau. Les eaux de lavage ont ete centi-ifugees ;I 5000g durant 5 min, le culot A recueilli. Ensuite. nous avons obsei-ve ( a ) les particules grossikres en microscopie electronique a balayage apres deshydratation B I'air et metallisation B !'or-palladium; (6) les particules grossieres et le culot A en microscopie electronique k transmission aplZs fixation selon Rytei- et Kellenberger (1958) et a p ~ e savoir contraste les coupes selon les techniques preconisees par Reynolds ( 1963) ou Thiery (1967). Le sol est constitu6 paldes agl-egats dont la taille moyenne est comprise entre 50 et 100 pm (Fig. I). Des filaments fongiques relient Iichement ces for-
0008-4 166/79/080943-04$0 1.00/0 @ 1979 National Rebearch Council of CunadalConseil national de r-echerches du Canada
Mycophenolic acid production by Peizicillium brevicompacturn in two media D. L. D ~ E R F L EC. K ,D. BAKTMAN. A N D I. M. C A M P B ~ L L I ) ~ ~ ~ ~ I I c~f'Rir~lo~yic~iil . ~ I ~ I ~ I I /St.it,~ii.c..\.U~rirc.r.(3). 75. ant1 3 mL) wcre taken ;~septicully h u m the c~lltllrci t t the st;~tedtimes fol. isotope irlcorpor;lti~n. biom;~ssnie:lsul.enlcnt. 2nd f'ol-~nicroscopicexamination. I.so10~~t~ /II(,OI./JOI.(II;(III l~.v/)t~~.itt~~'tr~,\ (III(/T / ~ ( ~ i ~ ~ , - t t i ( ~ / , ~ . ~ i . \ S u h c ~ ~ l t ~( ~ 5 0~m. eLsin a 125-mL Er1ennievc1-flask) wcre prepa~.cd13. 24. 36. 48. 66. ant1 9Oh following inoculation. These were incubateti ftrl- 3 h with [ I-l"Clacetalc (25 1tCi. 58 1tCilltlnol OEntries are the r c s r ~ l tof single measurements. Confidence levels were independently; live replicate assays of a liquid culture that had ( 1 Ci = 37 GBq), Nc\v Eriglancl Nuclear Corp.. Boston.Mi~s- aestablished n average biomass d r y weight of 0 29 8/75 m L gave a standard deviation sachusetrs). At the end of the ilicuh~~tion period. the culture of t 0.005 g. 'Initial spore density was 3.1 i: 107/rnL. (medilrnl ant1 biomass together) was supplerncnteil with internal clnirial spore density was 2.2 x lO'/mL. stand;irtls Or-pcnta(1ccanoic acitl. 48.411g: dotl-iasontanc. 101.8 .ILK). - k a s acitlifictl with concentr;ttcd HCI ( I mL).;tnd was homogenized with ethyl :~cct;ttc(75 mL) for I min in a blc11dc1-. M y c . o p h c i ? o l i c A c i t l Protlrtc~tio17it? Merlicr A ot?cl B '1-hc orgi~nic1;1ycr W;IS rern~vetl.PI.CCLI of' b ~ l v c n t .i ~ n dtl.catccl Since medium B is nominally richer than the irith crhe~.e;~l iliazorncthane for 15min. Del.ivatized extracts \YCI.C analyzetl by radiogas chron1;ltography (KGC) and ~.adiog;~s media in which Brar o t (11. (1968) a n d Audhya and Russell (1975) encountcrcd idiophadic production ch~.t)mi~tog~-;tphy - rnilss spectrc)nietry (KGC-MS) as described PI-cviously(Doel-Hcl-c.1 ill. 1978) with the exception that a finill of the ergot alkaloids and enniatin, ~.espectively, 8 1 s chromatographic tcmpcl-aturc of 300°C \\.;IS ~rsetl.The and since it suppol~s a markedly faster growth rate logal.ithn?ic clata compl.cssion 511-i~tcgy~.ccently advocatctl P. 1 ~ i . ~ \ ~ i c ~ o i ? ? p a i ~(I-elative trri17 to medium A). we of (Ilocrcflcr ant1 Can?pbell 1978) \vas adoptctl in all gas al-g~~ed that for mycophenolic acid PI-oduction to ch~.omatogr.aphicanalyses. Biotrr(c.c.\! \ l ( , o . c r i ~ . c , t ~ ~ c , ~ r ~ . \ At selcctcd time\ ; ~ l i q ~ ~(75mL.) ots of the C L I I ~ L I I . C S were rec h and n c r c tilterccl thro~rgha moved from thc F ~ I - n b i ~ fl;~sk prci\'righetl ant1 predriccl (IOOoC. 48 h) tiltel. papel- (Whatman # I ) lrsing a Huchncr f ~ ~ n n c Filter l. pilpel. plus biomass were \i':t\hetl with ?la\.;-tlistilletl water ( 2 5 0 nil.) i~nrl\\'el-c tlricil at IOO°C fol-48 h before ~.e\vcighi~lg.
Results Bioi~?rt.c..s P i . o l i f o i . c ~ r i o i ~(it?(/ h'lorplio/ogic.cl/ ~ ~ o I o / > i ? ;it ~ ~Mc,(/ilr ~ t r i A (ri1(1B
Dc-
follow the ergot alkaloid-enniatin patterns i t would need to be idiophasic in medium B. Since rnycophenolic acid production in strain ATCC 9056 is already known to be growth-linked in riiedium A (Nulton and Campbell 1977; Doel-fler ~t (11. 1978) a simple test for idiophasic PI-oductionin medium B is as follows: whereas mycvphenolic acid biosynthesis i n ATCC 9056 would be expected to be occurring in rnediunl A from the time (48 h) when the tissue biomass begins to increase. i t sho~lldnot occur to any significant extent in n~ediumB until growth becomes rest[-ictedaround the 90th h. The relevant data 31-e shown in Table 2. Mycophenolic acid p~~oduction. as measur.ed both by pool size increase and radioactivity inco~.po~-ation from [14C]~~cetate. is active in both media from the time when tissue proliferation begins. Clearly, therefore, rnycophenolic acid production in P. hi.rl.ic.oii?j>crc.rrri??does not conform to the pattern fo~lndin C'/rt~.ic.c~l)sbv BI-ar c t trl. (1968) and i n F r t s t ~ i ~ i r rby t ~ i Audhya and Russell (1975): rather, i t resembles the A . prrrrrr1rr.s case (Burtman and Campbell 1979).
I t is shown in Table 1 how I?iomass dry weight changed with time in the two media. In metlium B. gel-mination occurr-ed between the 34th and 35th h: by the 36th h. tissue ;\ccumul;\tion was well under way. I n niediuni A. germination was delaycd until the 36th h and significant tissue accumulatiun was not seen till the 66th h. Event~lalbiomass pl-oliferation was approximntcly sixfold more rapid in medium B than i n medium A. Correspondingly. biomass PI-oliferation was continued for at least Discussion 212 h in medium A, but was terminated i n medium The results ofthis study, taken together with our B ar-ound the 90th h. The culture in medium A sporulated around the 66th h but no sporulation previous findings (Nulton and Campbell 1977; occurred at the 212th h i n medium B. Doerflcr c t at. 1978; Bartman and Campbell 1979)
942
CAN. J . MICROBI(
TABLE2. Yicld (milligrams per 50 m L or culture alicluot) anti specific ratlioactivity (disintegrations per minute per niicrogram) of 1iiycopher1olic acid p r o d ~ ~ c e in d media A ant1 B rollowing incubation with [I-'"Clacetate Yield" ancl specific radioactivity" (in parenthesis)
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by YORK UNIV on 11/20/14 For personal use only.
Tinieartcr inoculation (11)
Acknowledgments The financial support of the National Science Foundation (PCM-78-032852) and the National Institute of Health (GM-00149) is gl-atefully acknowledged.
A H A K ~ ~ U O WYI .-.I:tnd . Z . A. L. DI.MAIN. 1978. Carbonc:~litholite regulation of cephalosporin production in Srreptottr~c.cs c~lr~brrligo.~is. Antimicl-ob. Agents Chemothe~..14: 159-164. A U I I H Y AT. . K.,and D . W . Russr.1 I . 1975. Enniatinp~.oduction by F~c.tro.i~ol~ .sr~rrrhrrc~itrr~t~~: primary secondary and 11nita1.y metabolism. J . Gen. Microhiol. 86: 327-331. B A R . I ~ ~ C. A ND.. . and 1. M. C ' A ~ I P B I . L1979. L . Naphth;~lenonc production in d.spergillrt.~porc~tlrr.~. Can. J . Microhiol. 25: 130- 137. "Each entry is the result ol' a single measurcn~cnr.Conlidcncc i n mass BHAR.S. S.. c. S. G ~ ~ h l . i W. ~ ~ A. l d T A B ~ R1968. . Pattel-nsofir~ s;rl~ieswas cl~cckedto be w ~ t l > i lnh c n n ~ i.c in a t e d-+ 10'2 1975) ,,,,(Slavhack PI 01. ~. -, r.iro crgot al kaloicl production by C'lo~:ic.ol>.\ po.spcili and thcir by making Rve replic;ile measurements in a n independent experiment; !he average value of 0.23 mg/50 mL uns :!ccompanied by a standard deviat~on association with different g r o s t h rates. Mycologia. 60: n f + 0 009 m ~ . - . - - .- - - ...-. 806-826. Dlnilial spore density was 3.1 :: 107/rnL. 'Initial spore density was 2.5 'i IO'/mL. Bu'Loc-K.J . 1). 1961. 1ntermedi:lry met;rholism and crntibiotic 'Decreasing specific activitics from this point o n reflect tile increasing size synthesis. Adv. Appl. hliuobiol. 3: 293-342. of a preexisting unlabelled pool of mycophenolic acid. -1967. E s w y s in hiosynthesih and microhiill devclopment. Chap. 3. John Wiley EL Sons. Inc. New Yol-k. pp. and those of other groups (Brarcr nl. 1968; Audhyn 42-67. and Russell 1975) lead to the conclusion that in ---- 1975. Seconclary mct;tholism in fungi and its rel;~tionl i q ~ ~ ishaken d or stirred culture there need be no ships to growth and clevelopment. Irr The filamentous fungi. Vol. I . chap. 3. f~clirc~tl h~ J . E. Smith and D. K. Bcrry. E. uniq~leassociation of seconda~-ymetabolism with AI-nolds.Loncion. pp. 33-58. the postvegetative growth phase offungal growth. BU'LOCK.J . D.. H , ~ R I I L T O M.N A. . H U L M I .A. . J . PO\VI.I.L.H. We. therefore. join other investigntol-s (e.g., AuM. S ~ I A L I . I : VI).. S H L P I ~ I : R Iand > , G . N. S ~ I I T H1965. . dhya and Russell 1975; Bu'Lock 1975; AhnronoMck~bolic development and secondary biosynthesis in P~~trir~il/irr~~r rrrric.oc,. Can. .I. Micl.ohiol. 11: 765-778. witz and Demain 1978) in calling for a reappraisal of what the fungal idiophnse and tl-ophophase DI:RI;\IN.A. L. 1974. How d o ;~ntibiot~c-proclucingnlic~.ooriivoitl suicidc? Ann. N.Y. Acitd. Sci. 233: 601-6 12. are and how these phases I-elate to the onset and D Igkinisms T K O YR. . W.. S. N. FKI.I:II.and D. J . FINNLL..1973. Remaintenance of fungal secondary metabolism. I21tionships between the hiosynthesis of virus-like pn~.ticles One area that wasrants attention in S L I C ;I~ reapand mycophenolic acid in Pcrric.illir~rrrstolorr~firrr~rr and P. h r c ~ ~ ~ i c ~ o t t r p oCra~nt.~J~. ~ Microbiol. ~r. 19: 1459- 1462. praisal is the appropriateness of submerged liquid I I .and I . M. C A M P U ~ ~ 1978. L I . . Logarithmic d a t ; ~ cultu~-e techniques in exploring the physiology and D ~ ~ K F LD.~ L.. compl-ession strategy for gas chromatoglxphy. Anal. Chem. molecular- biology of fungi that do not grow nor50: 1709-171 1 . mally in an agitated. aquatic environment. In na- D o ( - H F L ~ D. R . L.. C . P. N U L T ~ NC., D. B , ~ R I - M A F. N, J. and I. M. CAAIPBI:LL. 1978. Spore gel-n~ination. t ~ i t i r Go-r-~-LII:B. ture. 01-ganismssuch as P. b t ~ r ~ i c ~ o t n p n c ~ colcolony development. ant1 secondary mct21bolism in Pcrrt~ic.ilonize gl-ains and decaying vegetable mattel- in the lirirrr brc~~.ic~o~trpeic~riitti: ;I ~.i~diogi~s chromiltogr;~phitnd morsoil. growing on a stationary solid. moist support. phological study. Can. .I. Microbiol. 24: 1490-1501. The fungus grows vegetatively into the substratum KOFFI.I~R. H . . R . L. E ~ I E H S OD. NP . ~~Lhl,i~,anH d R. BURHIS. . and out from that substratum aerially in the PI-ocess 1945. Chemical changes in subrnelged penicillin fel-mentations. J . Bacterial. 50: 517-548. of conidiation. Vegetative and aerial hyphae oc. S . , i111dL. C. V I N I N C1970. . Metz~bolismofchloramcupy different nutritional environments and serve M A L I KV. phenicol by the producing organism. Can. J . Microbiol. 16: different biological ends. When such an organism is 173- 179. grown in the label-atory in shaken liquid culture. the MU-IH. W. L.. i ~ n d C. H . NASH. 1975. Biosynthesis of mycophenolic acid: purification and charecleriz:~tion of S distinction between vegetative and aerial hyphae adenosyl-L-methionine: demethylmycophenolic acid 0disappears. There can be no guarantee that fungi methyltransfelxse. Antimicrob. Agents Chemother. 8: will react uniformly or predictably to such a trans321-327. fer. We are currently exploring the possibility that NULTON.C. P.. and I . M ,C A M P B E L L1977. . Mycophenolic acid the variations seen in fungi regarding so-called is produced during balanced growth of Petricilliirt~~ l>~.c,cic'ot~~ptrc~trrtn.Can. J . Micl-obiol.23: 20-27. growth-linked or idiophasic secondary metabolism and E. W. in batch-mode shaken culture is attributable to dif- N U L T O NC. . P.. J . D. NAWORAL.I. M. CA~VIPBLLL. G R ~ ~ I . Z I V G1976. E R . A combined radiogas c h r o m : ~ t o g ~ ~ p h / ferences in response to the abnormality of culturing mass spectrometer detects intermediates in mycophenolic a nonaquatic organism in a stirred or shaken liquid acid biosynthesis. Anal. Biochem. 75: 2 19-233. S L A Y B A CJ. K ,R. B.. I . M. C.\MPBELL. and E. FAKISH.1975. A environment. Medium Bc (rich)
Medium A" (mini~iial)
-
~
~
~
protocol based on gas chromatog~~phy-flow counting for. munito~.ing intermei1i:u.y met:~bolism. Anal. Biochem. 69: 140- 154. S ~ I I I IJ .I ,E.. ;~ntlD. R . BI.I~. FI.IIII~.~'
Appl-ouve le 3 mai 1979 KII..BCRTUS. G.. et J . P1101.~.1979. Observation d'un sol forestier (rendzine) en microscopie electronique. C;ln. J . Microbiol. 25: 943-946. L'observation en microscopie electronique B balayage perrnet de mettre en evidence I:\ structure en agl-egats ilu sol forestier ktudie, ainsi que la presence de fil;~rnentsfongiques h I'exte~.ieur de ceux-ci. Les eaux de lavage de ces sols contiennent essentiellement des bacteries isolees, alors que les coupes dans les agrigats montrent frequemment des :~ssociationsd e bacteries entourees d e e adherent des feuillets d'argiles. mucilage e t B la s u ~ f a c duquel Ces observations conlirment I'existcnce d c deux types d e biotopes dans c e sol: I'un situe d a n s les espaces inter-agrkgats. le second i I'interieur m6me des agregats. KILBCKI-US. G . , ilnd J . PKOTH.1979. Observ:~tiond'un sol forestier (rendzine) en microscopie electronique. Can. J . Microbiol. 25: 943-946. Scanning elect~.onmicroscopy was used to evidence the aggregated stl-ucture of a forest soil as well as the presence of fungal hyphae external to soil aggregates. The supernatant of soil suspension in water mainly contained isolated bacteria. while ultrathin sections of aggregates frequently revealed groups of bacteria surrounded by a sheath of mucilage with adhering clay minelxls on the outside. These results conlirm the existence of two particular biotopes in the soil studied: oneis located spaces. inside aggregates, and the other. in the inter--agg~-egate [Translated by the joul-nal]
De nombreux chercheut-s ont suggere que les germes telluriques etaient lies ;I des matikres organiques ou minerales ou incluses dans celle-ci (Stevenson 1958; Jones et Griffiths 1964; Casida 1969; Zvyagintsev cr trl. 1969). Ces hypotheses ont ete confirmees par I'observation du sol en microscopie electronique B transmission (Balkwill ct 01. 1975; Bae et Casida 1973; Kilbertus cJt rrl. 1977): les feuillets d'al-giles adherent souvent A la surface des bacteries, des colonies du procaryotes ou de filaments de champignons. De plus, I'observation de debris vegktaux (Kilbertus et Reisinger 1975; Olah cJt (11. 1978) a montre que les microol-ganismes se developpaient generalement A I'intet-ieurdes parois ou dans les cellules et qu'il etait par consequent impossible de les dissocier de la matiere en decomposition. Dans le cas particulier du sol, Balkwill ct rrl. (1977) en utilisant differents traitements physiques (broyage, sonication) ainsi que des agents dispersants, n'ont reussi qu'i isoler 60% des germes de ce milieu. Cela prouve que la quantifica'Demandes de reimpression: G . Kilbertus.
tion des microorganismes du sol dans ces biotopes est extremement delicate. Nous avons tente d'appot-ternotl-e contribution ;I ces problemes en experimentant sur un sol forestier (rendzine) dont les caracteristiques pedologiques ont ete publiees par Vannier (1970). Apses avoir agite durant 10 min, 10 g de sol dans 100 mL d'eau, nous avons passe ['ensemble sur un tamis (mailles de 50 pm) et lave le tout ;I I'aide de I000 mL d'eau. Les eaux de lavage ont ete centi-ifugees ;I 5000g durant 5 min, le culot A recueilli. Ensuite. nous avons obsei-ve ( a ) les particules grossikres en microscopie electronique a balayage apres deshydratation B I'air et metallisation B !'or-palladium; (6) les particules grossieres et le culot A en microscopie electronique k transmission aplZs fixation selon Rytei- et Kellenberger (1958) et a p ~ e savoir contraste les coupes selon les techniques preconisees par Reynolds ( 1963) ou Thiery (1967). Le sol est constitu6 paldes agl-egats dont la taille moyenne est comprise entre 50 et 100 pm (Fig. I). Des filaments fongiques relient Iichement ces for-
0008-4 166/79/080943-04$0 1.00/0 @ 1979 National Rebearch Council of CunadalConseil national de r-echerches du Canada
