ultrastructural studies ofsporulation in streptomyces · ermuth and hopwood (14) call sporulation...

JOURNAL OF BACTERIOLOGY, Sept. 1976, p. 1443-1454Copyright C 1976 American Society for Microbiology

Vol. 127, No. 3Printed in U.S.A.

Ultrastructural Studies of Sporulation in StreptomycesCARLOS HARDISSON* ArD MANUEL B. MANZANAL

Departamento de Microbiologia, Universidad de Oviedo, Oviedo, Spain

Received for publication 29 April 1976

This is the first study of sporogenesis in Streptomyces carried out on arelatively high number of species (seven) which allows us, using also previouslypublished results, to establish a general picture of this process. In the sporogene-sis of Streptomyces two basic stages can be considered: the sporulation septumsynthesis and the arthrospore maturation. Our ultrastructural study of thesporulation septum formation suggests the existence within this genus of threebasic types. Type I is distinguished because the septum is formed from thebeginning by two separate cross walls. Within this type we include Streptomyceserythraeus, Streptomyces albus, and Streptomyces aureofaciens and also includeStreptomyces venezuelae, Streptomyces griseus, and Streptomyces osteogriseus.Type II is distinguished because there is a deposit of material previous to thesynthesis of the double annulus which completes the septum. This type can bedivided into two subtypes. In the first the deposits are wedge-shaped and thedouble annulus is clearly visible, and to this group belong Streptomyces flaveo-lus, Streptomyces ambofaciens, and Streptomyces coelicolor. In the second thedeposits, which have a different shape and are very well developed, constitutealmost entirely the sporulation septum in which the double annulus is barelyvisible; Streptomyces antibioticus and also Streptomyces viridochromogenesbelong to this group. Type III, represented by Streptomyces cinnamonensis, isdistinguished because the septum is formed by a single cross wall.

The genus Streptomyces belonging to the or-der Actinomycetales shows a comparativelycomplex development cycle in which from abranched vegetative mycelium submerged inthe substrate is formed an aerial myceliumwith hyphae of greater diameter. Through thesporulation process these aerial hyphae giverise to chains of arthrospores, which placed un-der favorable conditions germinate and repeatthe cycle.The term arthrospores has been proposed by

Cross (5) to refer to the asexual spores producedby Streptomyces that are formed by the annularingrowth of specialized, regularly spaced crosswalls in existing hyphae enclosed in a fibrQussheath. The arthrospores are differentiatedfrom bacterial endospores both by the sporo-genesis process and by their properties.

In recent years great interest has been shownin the study of sporulation processes in bacte-ria, because they constitute a simple, andtherefore an attractive, model for the study of aprocess of cellular differentiation. Althoughmost of these studies are performed on speciesof two genera Bacillus and Clostridium, sev-eral works have been published on the differentprocesses of sporulation within the Actinomy-cetes, particularly in the genera Streptomycesand Thermoactinomyces (6).

In this paper we present the results of theultrastructural study of the sporulation inseven species of Streptomyces. We comparethese results with those obtained previously byother authors and make a tentative classifica-tion of the genus Streptomyces according to thepatterns of the sporulation process.

MATERIALS AND METHODSMicroorganisms. The following strains of the

Streptomyces genus were used: Streptomyces albus,CMI 52766; Streptomyces ambofaciens, ATCC 23877;Streptomyces antibioticus ATCC 11891; Streptomycesaureofaciens, ATCC 13304; Streptomyces cinnamo-nensis, ATCC 12308; Streptomyces erythraeus,ATCC 11635; Streptomyces flaveolus, ATCC 3319.

Media and culture conditions. The microorga-nisms were inoculated by streaking on GAE me-dium (glucose, 1%; asparagine, 0.1%; yeast extract,0.05%; K2HPO4, 0.05%; MgSO4 - 7H20, 0.05%; FeSO4,0.001%; and agar, 2%) contained in petri plates.After incubation at 28°C well-isolated colonieswhose stage of development was checked by directobservation in the electron microscope were selectedfor the ultrastructural study of the sporulation.

Electron microscopy. Selected whole colonies atdifferent incubation times were cut out of the me-dium on small blocks of agar and fixed by the Ryter-Kellemberger method (13). The dehydration wascarried out according to Burdett and Rogers (2).Samples were embedded in Araldite. The sectioning

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1444 HARDISSON AND MANZANAL

was carried out in a LKB III ultramicrotome andstained with 2% uranyl acetate. The Philips EM300was operated at 40 and 80 kV with instrumentalmagnifications from 3,000 to 30,000. For direct ob-servation, the surface of the colonies was touchedgently with Formvar-coated copper grids. Aerialmycelium and arthrospores adhere to the surface ofthe grids and can be observed directly in the electron

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microscope. Usually colonies with abundant aerialmycelium and only a few arthrospores give goodsamples for the study of sporulation.

RESULTSIn the sporulation process of Streptomyces

two basic stages may be considered. The first

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3FIG. 1-11. Sporulation ofS. erythraeus, S. aureofaciens, and S. albus. Abbreviations: AA, Double edge of

ingrowing annulus; FS, fibrous sheath; SS, sporulation septum. Bar equals 200 nm.FIG. 1. Longitudinal section ofa sporulating hypha ofS. erythraeus showing a partially complete sporula-

tion septum. Note the constriction of the dividing nuclear material and the invagination of the plasmamembrane.

FIG. 2. Longitudinal section of a sporulating hypha of S. erythraeus showing two complete sporulationsepta and, at the center, another in the process of being formed (arrowhead).

FIG. 3. Longitudinal section of a sporulating hypha of S. aureofaciens showing a partially completesporulation septum (arrowhead). Note the two separate cross walls and the plasma membrane invagination.

FIG. 4. Longitudinal section of an aerial hypha of S. albus at the onset of sporulation showing threeforming sporulation septa (arrows).

FIG. 5. Enlarged detail ofthe above micrograph. Note the invagination of the plasma membrane and thatthe sporulation septum is formed by two separate cross walls.

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10~~~~~~~1FIG. 6. Longitudinal section of a sporulating hypha of S. erythraeus showing a complete sporulation

septum (arrowhead). Note the two clearly separated cross walls and the typical structure of the cytoplasmicmembrane.

FIG. 7. Longitudinal section of a sporulating hypha of S. erythraeus showing several sporulation septa.Note the spiny appendages of the fibrous sheath.

FIG. 8. Longitudinal section of a sporulating hypha of S. aureofaciens showing three completed sporula-tion septa (arrowheads).

FIG. 9. Longitudinal section of a sporulating hypha of S. albus showing two completed sporulation septa.Note the two separate cross walls.

FIG. 10. Longitudinal section of a chain of arthrospores of S. albus in an advanced stage of maturation.The thickness of the wall has increased to about 30 nm. Arthrospores remain attached by the fibrous sheathand the central zone of the sporulation septum, whose corners have rounded. The arthrospore cytoplasmcontains vacuole-like spaces of low electron density that may be granules ofpoly-f3-hydroxybutyrate.

FIG. 11. Section of a mature arthrospore ofS. erythraeus. No structures may be seen within the cytoplasm.The wall has thickened to about 40 nm. Note the disintegrating fibrous sheath.

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SPORULATION IN STREPTOMYCES 1447

stage lasts from the initiation of sporogenesis tothe formation of the sporulation septum. Thesecond stage includes all the changes that takeplace from the delimitation ofthe future arthro-spore to the formation of the mature arthro-spore. Each stage has been subdivided by Hop-wood et al. (4, 14).Sporulation-septum formation. Sporulation

in Streptomyces starts with the simultaneousformation of specialized cross walls that Wild-ermuth and Hopwood (14) call sporulationsepta, which divide the preexisting aerial hy-phae. Results obtained show that according tothe synthesis of the sporulation septum theStreptomyces genus can be divided into threegroups. Type I (Fig. 1-11) is distinguished be-cause the sporulation septum is originated bythe deposit on the inner face of the hyphal wallof a material with similar electron density tothat of the wall (Fig. 2-5). This deposit has theappearance of two separate annuli or crosswalls, which grow centripetally until joining inthe center of the hyphae, forming the sporula-tion septum (Fig. 6, 8, and 9). These cross wallsremain as separate entities throughout theprocess. The cytoplasmic membrane invagin-ates as the double annulus grows (Fig. 1-5) andjoins before the cross walls do. To this typebelong S. erythraeus, S. albus, and S. aureofa-ciens.

In type II (Fig. 12-33), previous to the synthe-3is of the two annuli, material whose electrondensity is slightly less than that of the wall isdeposited at regularly spaced intervals on theinside of the hyphal wall (Fig. 13, 14, 23, 24, 27,

and 28). The cytoplasmic membrane invagin-ates as this deposit of material builds up, fromwhich are subsequently formed two annuli orcross walls which by joining centrally consti-tute the sporulation septum. Between the hy-phal wall and the deposited material the exist-ence of a boundary or separation may be seen(Fig. 14, 24, and 28), as pointed out in a pre-vious work on sporulation in Streptomyces coe-licolor (14). In some instances, such as in S.flaveolus (Fig. 13-16) and S. ambofaciens (Fig.23 and 24), the deposits have the form of awedge and the cross walls are clearly visible,whereas in S. antibioticus (Fig. 28-31) the de-posits are very large, constituting almost thewhole sporulation septum (Fig. 29 and 30), andthe cross walls that complete the septum (Fig.31) are much smaller. The abundance of meso-somes in proximity to and in contact with thedeveloping septum must be emphasized (Fig.13-18, 23, and 24), as has been previously ob-served (1, 7, 8, 11, and 14).

In type III, represented by S. cinnamonensis(Fig. 34-42), the sporulation septum is origi-nated by a single deposit of electron-dense ma-terial between the hyphal wall and the plasmamembrane which invaginates (Fig. 34 and 35).This material grows inwards until joining, asdo the invaginated membranes (Fig. 36), toform the sporulation septum (Fig. 37). Lamel-lar and vesicular mesosomes are associatedwith the sporulation septum and the nuclearregion (Fig. 36 and 37). The nuclear materialundergoes remarkable changes during theprocess assuming a compact structure as septa-

FIG. 12-26. Sporulation of S. flaveolus and S. ambofaciens. Abbreviations: WS, Deposit of septummaterial; SW, spore wall; S, storage granules; B, boundary between the hyphal wall and the septum material.Other symbols as in previous figures. Bar equals 200 nm.

FIG. 12. Longitudinal section of an aerial hypha of S. flaveolus, showing the fibrous sheath, the hyphalwall, the fibrillar nuclear region, and storage granules.

FIG. 13. Longitudinal section of an aerial hypha of S. flaveolus showing the initial stage of sporulationseptum formation. Note the deposit of septum material (arrowhead) between the hyphal wall and theinvaginating membrane in association with tubular vesicular mesosomes.

FIG. 14. Longitudinal section ofa sporulating hypha ofS. flaveolus showing the final stages.ofsporulationseptum completion. From the wedge-shaped deposited material (arrow head) two separate annuli which growinward are formed (arrowhead). Note a large vesicular mesosome in association with the forming septum anda large lamellar mesosome in association with the nuclear region.

FIG. 15. Longitudinal section of a sporulating hypha of S. flaveolus showing three complete sporulationsepta (arrowheads).

FIG. 16. Longitudinal section ofa sporulating hypha ofS. flaveolus showing a newly completed sporula-tion septum (arrowhead). Note the double cross wall between the wedge-shaped deposit material.

FIG. 17. Longitudinal section of a sporulating hypha of S. flaveolus showing the initial stage of arthro-spore maturation. Note three zones in the septum: a broad outer zone near the hyphal wall, and of similarelectron-density, that starts to become rounded; a narrower and less dense intermediate zone; and a moreelectron-dense and thicker central zone.

FIG. 18. Longitudinal section ofa sporulating hypha ofS. flaveolus showing a later stage ofarthrosporematuration. The thickness ofthe cross walls increases, and the rounding ofthe corners becomes more evident.At the same time (arrowheads) lysis in the septum zones nearest the hyphal wall occurs.

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tion proceeds (Fig. 34-37). It must be empha-sized that in this model the sporulation septumis not formed by a double cross wall as in all theother Streptomyces studied but by the deposit ofelectron-dense material which when fused atthe center of the hypha results in a single crosswall. Figure 43 shows the three types of sporu-lation septum synthesis we have found inStreptomyces.

Arthrospore maturation. Once the sporula-tion septum is formed, a series of changes oc-curs leading to the maturation of the arthro-spore, which Hopwood et al. (4, 14) divide intotwo stages. When the cellular material thatwill constitute the future arthrospore has beendelimited by the newly formed sporulation sep-tum (Fig. 6-9, 15, 16, 23, 24, 31, and 37) one canconsider the maturation process as initiated. Inthe majority of Streptomyces studied this proc-ess is initiated by the lysis of the septum zonenext to the hyphal wall, as for S. flaveolus (Fig.17 and 18), S. ambofaciens (Fig. 25), and S.cinnamonensis (Fig. 38 and 39). At the sametime, the material that will constitute the lat-eral walls of the future arthrospore is depositednear the sporulation septum (Fig. 17, 18, and25). Then the corners become more rounded(Fig. 18, 19, and 25), and the future arthrosporewalls thicken evenly until a thickness of 20 to30 nm is reached (Fig. 10, 19, and 20). In S.cinnamonensis, the electron-dense materialthat originates the sporulation septum starts tolyse in the zone next to the hyphal wall (Fig.38); the lysis proceeds (Fig. 39), and then thesynthesis of a typical arthrospore envelope, as athin layer of greater electron density, occurs(Fig. 40). Parallel to this change the cytoplas-mic structures undergo modifications. In mostcases mesosomes are not seen, and the nuclear

material is less evident (Fig. 10, 20, 25, and 40).In the cytoplasm appear abundant vacuole-likepatches with poorly defined limits and very lowelectron density (Fig. 10, 20, and 25). Thesehave been previously reported in other Strepto-mycetes (1, 8, 11, and 14) and probably corre-spond to the poly-,8-hydroxybutyrate granulesfound by Kannan and Rehacek (10) by thechemical analysis of all Streptomyces speciesthat they studied. At this stage arthrosporesremain joined only by a small central zone ofthe primitive septum and are surrounded by amore or less intact fibrous sheath (Fig. 10, 19,20, and 25). The final stage corresponds to themature arthrospore which has changed itscylindrical form for an ellipsoidal one andwhose wall has reached the thickness of40 to 50nm (Fig. 11, 21, 22, 26, 33, 41, and 42). Thearthrospores are encased in the fibrous sheath(Fig. 11, 21, 26, 41, and 42), but now no contactexists between adjacent arthrospores, except insome cases as in S. cinnamonensis in whichremnants of intersporal material remain (Fig.41). Sometimes it is possible to distinguishdisintegrating remnants of the hyphal wallbetween the arthrospore wall and the fibroussheath (Fig. 42).

DISCUSSIONAlthough sporogenesis in Streptomyces is not

as complex a process as in endospore-producingbacteria, it is accompanied by remarkable finestructural changes in the aerial hyphae, affect-ing mainly the appearance of the nuclear mate-rial and intracytoplasmic membranous sys-tems. It also involves the synthesis of the char-acteristic arthrospore coats after the comple-tion of the sporulation septa. On the other

FIG. 19. Longitudinal section of a chain of arthrospores of S. flaveolus showing an intermediate stage ofmaturation. Arthrospores remain attached by the central zone of the sporulation septum and by the fibroussheath (arrowhead).

FIG. 20. Longitudinal section ofa chain ofarthrospores ofS. flaveolus in an advanced stage ofmaturation.Note that neither nuclear material nor mesosomes can be seen. In the cytoplasm large zones of low electrondensity are observed. The wall is about 30 nm thick.

FIG. 21 and 22. Longitudinal and transverse sections (Fig. 21 and 22, respectively) ofmature arthrosporesof S. flaveolus. The wall thickness is about 40 nm. The fibrous sheath, with its characteristic appendages(arrowheads), encloses the arthrospores.

FIG. 23. Longitudinal section ofa sporulating hypha ofS. ambofaciens showing three complete sporulationsepta (arrowheads).

FIG. 24. Enlarged detail ofthe above micrograph. The existence ofa boundary between the wedge-shapeddeposit material and the hyphal wall may be seen (arrowhead). Note the double cross wall between the wedge-shaped material.

FIG. 25. Longitudinal section of a sporulating hypha of S. ambofaciens showing an early stage ofarthrospore maturation. Note the lysis in the septum zones near the hyphal wall (arrowhead) and abundantstorage granules of low electron-density.

FIG. 26. Longitudinal section ofa chain of mature arthrospores ofS. ambofaciens enclosed by the fibroussheath. The wall has reached a thickness of about 50 nm. Note that neither nuclear material nor mesosomesmay be seen.

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FIG. 27. Longitudinal section ofan aerial hypha ofS. antibioticus showing several sporulation septa in theinitial stage offormation (arrows). Note that all the septa develop simultaneously and at regular intervals.

FIG. 28-31. Successive stages of sporulation septum-completion in S. antibioticus. Note the existence of aboundary between the hyphal wall and the newly deposited septum material (Fig. 28, arrowheads). Note thatfrom the deposited material two separate annuli are formed (Fig. 29, arrows), which grow (Fig. 30, arrow-head) and finally join centrally completing the sporulation septum (Fig. 31, arrowhead).

FIG. 32. Longitudinal section ofa sporulating hypha ofS. antibioticus showing several completed sporula-tion septa. Bar equals 1 ,um.

FIG. 33. Longitudinal section of a mature arthrospore of S. antibioticus.

hand, the work of Hopwood's group (3, 9, 11)with S. coelicolor demonstrated the participa-tion of a large number of genes in sporogenesis.The ultrastructural study of the sporulation

septum formation suggests the existence withinthe genus Streptomyces of three basic types(Fig. 43). Type I includes S. erythraeus, S.

albus, and S. aureofaciens, and in our opinionmay also include Streptomyces venezuelae (1),Streptomyces griseus (15), and Streptomycesosteogriseus (16). As did Glauert and Hopwoodin 1961 (8), we essentially explain the process inthe following way. Prior to the beginning ofsporogenesis a division of the hyphal wall into

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42FIG. 34-42. Sporulation ofS. cinnamonensis. Abbreviations: IM, Remnants of intersporal material; RW,

remnants of hyphal wall material. Other symbols as in previous figures. Bar equals 200 nm.

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FIG. 43. Diagrams showing the three proposed models of sporulation septum formation in the genusStreptomyces. Model I: S. erythraeus, S. albus, and S. aureofaciens. Model II: S. flaveolus, S. ambofaciens,and S. antibioticus. Model III: S. cinnamonensis.

FIG. 34. Longitudinal section of an aerial hypha of S. cinnamonensis at the beginning of sporogenesis.Note (arrowheads) the deposit of electron-dense material between the hyphal wall and the invaginatedmembrane.

FIG. 35. Longitudinal section of an aerial mycelium spire of S. cinnamonensis showing three partiallycomplete sporulation septa (arrowheads).

FIG. 36. Longitudinal section ofa sporulating hypha ofS. cinnamonensis showing final stages ofsporula-tion septum completion.

FIG. 37. Longitudinal section ofa sporulating hypha ofS. cinnamonensis showing a complete sporulationseptum (arrowhead). Note that the septum is formed by a single, thick cross wall.

FIG. 38 and 39. Longitudinal sections ofsporulating hyphae of S. cinnamonensis showing initial stages ofarthrospore maturation. Note the lysis in the septum zones near the hyphal wall (arrows).

FIG. 40. Longitudinal section ofa sporulating hypha of S. cinnamonensis showing synthesis of the initiallayer ofthe arthrospore coat, which is seen as a thin electron-dense layer (arrowhead) completely surroundingthe future arthrospore.

FIG. 41. Longitudinal section of a chain of arthrospores of S. cinnamonensis in an advanced stage ofmaturation. The thickness of the wall is about 45 nm. Arthrospores remain attached by remnants ofintersporal material (arrowhead) and by the fibrous sheath. Neither nuclear material nor mesosomes can beseen.

FIG. 42. Section ofa mature arthrospore ofS. cinnamonensis. Note remnants of the hyphal wall (arrow-head) between the fibrous sheath and the arthrospore wall.

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Abbreviations: 0, Outer zones of similar electrondensity to the hyphal wall; I, intermediate, less elec-tron-dense zone; C, central, more electron-densezone.

layers is seen. The inner layer breaks at regu-lar intervals, and its ingrowth, accompanied byplasma membrane invagination, originates thesporulation cross walls. In our opinion, as in amore recent paper of Wildermuth and Hopwood(14), study of the published micrographs aboutsporogenesis in Streptomyces does not suggestthe participation of a possible inner layer of themycelium wall in sporulation septum forma-tion. We believe this is formed by the deposit ofnew material in the form of two separate cross

walls between the hyphal wall and the cyto-plasmic membrane that invaginates.

Williams et al. (16) describe two basic typesof cross wall formation within the genus Strep-tomyces. Type 1 refers to septum formation invegetative hyphae. Type 2 refers to septum for-mation in sporulating hyphae. They think thatsporulation septum formation in S. osteogri-seus and S. coelicolor follows a similar pattern.From previously published micrographs (16),we believe that S. osteogriseus belongs to thesame type as do S. erythraeus, S. albus, and S.aureofaciens (type I), whereas our micrographsof sporulation in S. flaveolus and S. ambo-faciens and those previously published micro-graphs of S. coelicolor (11 and 14) suggest adifferent pattern of sporulation septum forma-tion in these Streptomyces (type II).

It is worth emphasizing the remarkable simi-larity between the published micrographs ofMcVittie (11) in her study of sporulation in S.

coelicolor and our micrographs of S. flaveolusand S. ambofaciens. Like McVittie, we haveobserved that once the sporulation septum iscompleted it undergoes a series of modificationsthat mark the beginning of the maturationprocess. The first modification observed (Fig. 17and 44) is the appearance of three clearly differ-entiable zones in the sporulation septum: (i)one zone next to the hyphal wall that starts tobecome rounded and whose thickness and elec-tron density are similar to those of the hyphalwall; (ii) an intermediate, thicker, and lesselectron-dense zone; (iii) and a more electron-dense central zone.The comparative study of micrographs pub-

lished by Rancourt and Lechevalier (12) of thesporulation process in Streptomyces viridochro-mogenes and our micrographs ofS. antibioticusshows a great similarity in the sporogenesis ofboth microorganisms. The existence of a delimi-tation between the hyphal wall and the slightlyless electron-dense deposit (Fig. 28) is in con-trast to Rancourt and Lechevalier's interpreta-tion that the septum is originated,by a wallinvagination. In our opinion the cytoplasmicmembrane invaginates at regularly spaced in-tervals, and in these places newly synthesizedmaterial is deposited between the hyphal walland the invaginated membrane (Fig. 27 and28).

It is important to note that these three pro-posed types of the synthesis of the sporulationseptum do not constitute clearly distinct andwell-delimited processes; on the contrary typeII seems to represent a link between the twoextremes-types I and III.

LITERATURE CITED1. Bradley, S. G., and D. Ritzi. 1968. Composition and

ultrastructure of Streptomyces venezuelae. J. Bacte-riol. 25:2358-2364.

2. Burdett, I. D. J., and H. J. Rogers. 1970. Modificationsofthe appearance of mesosomes in sections ofBacilluslicheniformis according to the fixation procedures. J.Ultrastruct. Res. 30:354-367.

3. Chater, K. F. 1972. A morphological and genetic map-ping study of white colony mutants of Streptomycescoelicolor. J. Gen. Microbiol. 72:9-28.

4. Chater, K. F., and D. A. Hopwood. 1973. Differentia-tion in Actinomycetes, p. 143-160. In J. M. Ashworthand J. E. Smith (ed.), Microbial differentiation-23rdSymp. Soc. Gen. Microbiol. Cambridge UniversityPress, Cambridge.

5. Cross, T. 1970. The diversity of bacterial spores. J.Appl. Bacteriol. 33:95-102.

6. Cross, T., and R. W. Attwell. 1975. Actinomycetespores, p.3-14. In P. Gerhardt, R. N. Costilow, and H.L. Sadoff (ed.), Spores VI. American Society for Mi-crobiology, Washington, D.C.

7. Glauert, A. M., and D. A. Hopwood. 1960. The finestructure of Streptomyces coelicolor. I. The cytoplas-mic membrane system. J. Biophys. Biochem. Cytol.7:479-488.

8. Glauert, A. M., and D. A. Hopwood. 1961. The finestructure of Streptomyces violaceoruber (S. coelico-

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lor). III. The walls of the mycelium and spores. J.Biophys. Biochem. Cytol. 10:505-516.

9. Hopwood, D. A., H. Wildermuth, and H. M. Palmer.1970. Mutants of Streptomyces coelicolor defective insporulation. J. Gen. Microbiol. 61:397-408.

10. Kannan, L. V., and Z. Rehacek. 1970. Formation ofpoly-f3-hydroxybutyrate by Actinomycetes. Indian J.Biochem. 7:126-129.

11. McVittie, A. 1974. Ultrastructural studies on sporula-tion in wild-type and white colony mutants ofStrepto-myces coelicolor. J. Gen. Microbiol. 81:291-302.

12. Rancourt, M. W., and H. A. Lechevalier. 1964. Electronmicroscopic study of the formation of spiny conidia inspecies of Streptomyces. Can. J. Microbiol. 10:311-

316.13. Ryter, A., and E. Kellemberger. 1958. Etude au micro-

scope electronique du plasma contenant de l'acidedesoxyribonucl6ique. Z. Naturforsch. 13:597-605.

14. Wildermuth, H., and D. A. Hopwood. 1970. Septationduring sporulation in Streptomyces coelicolor. J. Gen.Microbiol. 60:51-59.

15. Williams, S. T., and G. P. Sharples. 1970. A compara-

tive study of spore formation in two Streptomycesspecies. Microbios 5:17-26.

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