Macromolecular synthesis during the germination of Streptomyces spores in a chemically defined medium

FEMSMicrobiologyLetters 7 (1980) 233-235 © Copyrigl;dFederationof EuropeanMicrobiologicalSocieties Publishedby F.IsevierlNortlt-HotlandBiomedicalPress

233

MACROMOLECULAR SYNTHESIS DURING THE GERMINATION OF STREPTOMYCES SPORES IN A CHEMICALLY DEFINED MEDIUM CARLOS HARDISSON *, JOS~ A, SALAS, JOSI~ A GUIJARRO and JUAN E. SUAREZ Departametffode Microblologia. UniversMadde Ovledo,O,'iedo, 8pahl

Received12 December1979 Accepted4 January 1980 1. Introduetion

It has previously been shown thatmacromolecular

synthesis during germination of Streptomyees anti. bioticus spores in a complex medium (glucose.aspara-

gine-yeastextract'miner,d salts)takes p!ace in a sequential order: RNA, protein and finally DNA [1]. Incorporation Of [3Hluridine into RNAoccurred after about 40 rain in~:ubatlon,~that of [3H]leu¢ine into protein after about 60 rain incubation and that of [all]thymine into DNA after about I40-160 min incubation coinciding with the outgrowth, Followingour researchon Streptomyces spore germination Veehave analysed the occurrence of free amino acids and nu~~'~.ieacid precursors in dormant spores ofS. antibiot¢ats, and have found (unpublished results) the20 most common amino acids and some precursors of nucleic acids. The presence of a pool of free amino acids and nu¢I¢i¢acid precursors in dormant spores ofS. antibioticus prompted us to study macromolecularsynthesis during germination inn minimal synthetic medium.

2. Materials and Methods

2. l. Prevaration of spore suspensions S. antibioticus ATCU 11~5~1was grown for 7to 9 days at 28'C in 250 ml flasks containing.75 ml GAE solid mediuni (consisting of (g/l)~.!K2pO,iH, O.5, MgSO4'- 7HaO,~0.5; FeSO4L:'7H2O, 0.01 ; glucose ! 0:0; aspa~agln¢;i .0; Yeast'e'xi!act",.0.5 ;iagar, 20.0). Preparation of spore suspensions was ear-

tied out as previouslydescribed [! ] except where indicated. Sometimes a rapid method of preparation of spore suspensionswas used. In fl~esecases, steril. ized glass beads (3-4 mm diameter) were gently rolled around on tile agar surface and immediatdy transferred t~a flask containing 15 ml of cold distilled water in an ice-bath.:The resultant suspension was immediately filtered through Whatman No.l filter paper, adjusted to 10~ spores/mi (Asso = 0,3) and the components of the medium were added in sterilized, small, concentrated volunies. 2.2. Germination

Freshly harvested spore suspensions (I 0 ml) were incubated in a minimal sytlteti¢ medium (consisting of (g/l); K2PO4H,0.5; MgS04..7 H:O, 0.5; FeSQ4 7 I:I20, 0.01 ;'glucose, 10.0; (NI-h)~S04, 2.0) in 10CI ml flasks at 350C and 200 rev./min in a Gallenkamp Orbital incubat0i.:Germination was determined as previously described [ 1]. 2.3. Rate o f syntheas Ol'macroraoteodes

The rates of synthesis of RNA, protein and D~A were determined by nieasudng the incorporat!Onof [3I'I]uridine Opec. act. 27 Citnlmol), [~Hl[eucine Opec: act. 50 Ci/mmol) and-[ Hlthymine (sp¢¢. act. :30 C!/nim01)ir~Pectively, (e~ichat ! t~Ci/ml)into the correspOnrd!ngmacr0moleculesas previously described [ l ] except fltat the incorporatt0n of label was stopped by adding tdchl0roacetiC:idd (i$% (w/v) final e0dedntraitdn),"aadthit th~sampl~we~ incubdiidat 900C for t5'rnin ~fdr,' I'dieringin experiments Wtthi:JH]leucine:

234 3. Results and Discussion

The germination process in tile mininml synthetic medium was similar to that observed in GAE medium with regard tothe synchronization of the process attd absorbance changes. Germination was slower: a maxi. mum of swollen spores was observed after abot~t 4 it incubation and germ tube emission took place after about 5 it incubation. Incorporation of exogenous labelled precursors (Fig. l) and pulse-labelling experiments showed that RNA and protein synthesis during germination of S, antibDticus spores in a minimal syntlletic medium was initiated at 5 rain incubation, 'Il~ese results are similar to those found by Mikuiik et a[. [21 ill 3:: gra}mticolor. Our previous results obtained ina complex medium containing yeast extract [ 1] may bedue to the dilution of the radioactive exo. genous precursors by the cold precursors present in tile complex medium;under these conditions RNA and protoin syntlmsis only was observed after about

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40 mid 60 rain incubation, res ~Lectively.Tile incorporation of tall]thymine into DNA began after about 300 rain lneubatio'i!, at tile moment of outgrowth (Fig. 1C). The rates of RNA and protein synthesis during germination ~;~erealso determined by' pulse. labelling of samples taken at different times from the start of germination. The rate of [aH|uridine incorpo. 9 increased"continuously from.the ration (Fig. ,.A) onset of incubation, reaching maximum values after about 180 rain incubation and remained constant for up to 240 min, coincident with a maximum of swollen spores. Later, tile incorporation rate decreased to reach very low values, Tile turnover of cellular RNA may explain this fatl in the incorporation rate, The pattern of [aH]leucine incorporation (Fig. 2B) 'showed two different rates of increase of incorporation: the slower rate occurred from the start of incubation to 120 rain; ff0m this moment the rate of inceease of incorporation became higher and rem,~ined higher. All analogous pattern was found in a similar experiment on complex ntedium: In this ease macr0moleeulatSynthesis'was als6 determined by chemical methods and it was again found that the total amount ofall macromolecules continuously increased once tile s'ynthesis started [ 1]. The incorporation ofexogen0us labelled precursors([3H]uridine and [3H]leucine) by dormant spores in distilled water was also determitled: We found (Fig. IA; B) that both pre*ursors were incorporated into RNAor protein', and this incorporation was confirmed.by pulse-labelling (data not shown). The spores were really in the dormant'stage, as con. firmed [6oth by their phase brightness and by' the

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Fig; 2. Pulse-labeUingpattern ofRNA (A) and protein (B) synthefis during germination0f Strepromyces antibioticz~s spores. At th'e indicht6d;times~t n~i sampleswere taketi from the staff Of incubation; incubated With'labelled~r~cmson:f~r 3 rain and a~;ed for ra~activity;* Ea'ch point~a~ d~t6/mined in triplicate.

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Fig. 3. Effect of rtfampictn(10 ,u$/rnl)on protein synthesis duringgerminationof Strepromycesanffb~ot~eusspores in a minimalsynthetic medium•Symbols: e, control; % dfampieln and 13H] leuein¢added at the onset of incubation;•, rifalnplcinadded at tile onset of incubationand 13H]leucine added 60 rain tater. Each point was detexminedin quintuplicate.

absence of any change in th e absorbance during the thne the spores were incubated in distilled water, This seems to pro~,e that the spores have a functional system of protein and RNA synthesis; and imernal pools ofprecursoiS of both macromoiecules. We have found using thin.layer chromatography, that:the 20 iuost Common amino acids and some precursors of nucleic acids were present in the dormant Spore.'The total anaount of amino acids in the dormant sporeswas measured as 1.58 +-0.1 i ttmol/mg dry weight (unpublished results). The energy necessarY for these biosynthetic processes maybe driven from the endogenous reserves Of the dormant spore (QO2 in distilled water 10.42 ± 0.25 pl Oz/h/mg drywelght). The addition of antibiotics that inhibit synthesis of RNA (d fampiein !0 pg/ml), pr0t¢in (chloralfiphenicoll 00 pg]ml) Or.DNA(mitomYcin C .lOpg/mi) to dormant spores at zeroltim~i completely blocked the incorPOration of lobe!led pre¢tirs0zs into.the ¢0rrespondingmacromolecules !A,.B,C.. respectively)• However, a si~ifican t incorp0iatio n of.[3Hlleucine into protein(Fig. 3)took Pla~ in the Presence of rifampiein (10 vg/ml). Rifamp!cin 0nly. blocks !nitiation of RNA synthesis but not RNAehain elongation once the RNA.polyrnerabe has been attacl~ed t0,the promoter region of DNA [3] • TO exclude the pass[, bility that mRNAsYnthesis b)/d0nnant ispdres takes place during the p/epa~atio~.of theSp0re suspensions by the Standard method (duratiOn: 35+-45 min),a rapid method was employed(ab6m 3 rain). Undet these conditions and even when dormant Spores were prepaied in the i~resenceof fifampicifi ([ O.#gjml)the .~me'effect was observed. Thus, thepossibility of

mRNA synthesis during the preparation of spore suspensions can be eliminated. Similar results were found at ~ drug concentration of 200 pgjml. Impermeability of the drug can be excluded, because [~H] uridine incorporation was com#etely blocked I rain after the addition of dfampicin (data not shown). When dornmnt spores were it~cubated ill the minimal medium containing tifampicin (i 0 btg,/nli) i't~r 60 rain before addition of [~llltcu¢ine, no protein synthesis was observed (Fig. 3),thu s indicating that tnRNArof dormant spores was degraded during this time. Therefore, these results suggest tl~e presence of stable mRNA in dormant spores orS. antibioricus. Tile absence of incorporation in all analogous exl~rTmerit in a complex medium [l] may be explained by the dilution effect due to the cold external precursors. Our results aredifferent from those obtained by Mikulik et al. [2] inS. granaticolor, where rifampicin immediately inhibits lcucine incorporation into protein. Although not marly methodological details are given by these authors we think that the procedure they. followed to obtain spore suspensions may initiate the germination process, explaining the absence of absorban¢¢ fall found'by them, contrary to what has been reported by several 'workers [I,4,5] in different species~ofStreptotlo,ce~(this could also explain the degradation of the mRNA of the donuant spores. Further experiments Will be carried out in order to characterize theprotein(O encoded by this stable mRNA.

Acknowledgements

The authors wish to thank Dr~ K,.F, Chater, for critical reading of the manuscript: 3.A.S; was die recipient for a predoctoral fe!lowshit~ from the Ministery.of Education of Spain.

References [ t ] Hardisson. C., Manzanal, M•B, Salasi J.A. and Su~rez,

[2] ~ k U ~ r K ~.Janda;:I;;MaSKOVa,lit', Stastna~'J~and Jba[ 3] Hartm~nn, G., Honikel, K.O,, Knuml. F.I and Nuesch. Y. ([967) Bioehim~BlOphys. Acta 145¢843~844• [4] Attw¢ll, ~R.W. and Cross, T. (1973) hi The Actirmmy-

e~t~lesiC~~clc~sticslndp~=t=H~potta,~c*

(Syke.s, G. and Sktnn,:r. F.A.~ Eds.), pp•.t 97-207, Academic PxesS,New York.,

l$l ttirtch, C.F~a~d Ensign.J.C. (1976) J,BacteriaL'126, 13--23.