FINE STRUCTURE OF THE NUCLEOID AND INTERNALMEMBRANE SYSTEMS OF STREPTOMYCES

DONALD C. STUART, JR.The Division of Laboratories and Research, New York State Department of Health, Albany, New York

Received for publication February 16, 1959

Recently, there have been significant advancesin our knowledge of the biochemical and geneticcharacteristics of the streptomycetes. However,cytologic studies do not fully support the geneticmechanisms which have been advanced, and thefamiliar complaint of inadequate cytologic in-formation has been reiterated (Bradley, 1958).The variable structure of the streptomycetes

has been intensively studied, especially fortaxonomic purposes and mutant selection. Thework of several investigators clearly proves theexistence of genetic recombinations (Sermontiand Spada-Sermonti, 1956; Braendle and Szy-balski, 1957; Bradley, 1958). Early morphologicstudies supporting a two-phase life cycle havebeen reviewed (Jones, 1954). More recent in-vestigations (McGregor, 1954; Dickenson andMacdonald, 1955) favor the theory of diploidiza-tion by hyphal fusion (Klieneberger-Nobel,1947). Nuclear fusion, mediated by anastomosisbetween hyphae arising from the same and dif-ferent spores, has been demonstrated in lightmicrographs (Gregory, 1956) and hyphal bridgeshave also been seen in electron micrographs(Baldacci et al., 1956). In no case has the con-tinuity of nuclear material between hyphae ofheterogeneous origin been demonstrated. Nucleardivision and centrosome-like terminal bodies inthe ungerminated spore have been described asresembling mitosis of certain higher organisms(McGregor, 1954). The formation of sporeswithin the hyphal wall and the structure of thespore sheath have been shown in electron micro-graphs (Vernon, 1955). More recently a studybased on the electron microscopy of Streptomycescoelicolor provided a more detailed description ofthe internal nuclear and cytoplasmic structureof the genus (Hopwood and Glauert, 1958).

Streptomyces noursei was isolated in thislaboratory (Hazen and Brown, 1950). The presentstudy was undertaken to delineate the internalstructures of this microorganism and providecomparative material for a study of the originand function of cellular inclusions and membrane

systems. This first report is concerned with thevariable structure of the nucleoid and the in-ternal membrane systems of the cells of the sub-stratum and degenerating sporophore.

METHODS

Cultures of S. noursei were maintained inglucose-tryptone broth with 0.1 per cent agarat 22 C. Within 3 to 5 days these cultures de-velop a typical mycelium of dense vegetativehyphae surmounted by a grayish-white mass ofaerial sporophores. A few square millimeters ofthis growth (5 and 21 days old) were prepared forelectron microscopy. Fixation in 2 per centosmium tetroxide (in Veronal-acetate buffer,pH 7.4) for 7 hr was followed by washing inwater and dehydration within M hr in gradedalcohol series. Small pieces of the dehydratedmycelium were embedded in a mixture of 90per cent butyl and 10 per cent methyl methac-rylate monomers plus 1 per cent Lupercocatalyst. The embedding capsules were evacuatedfor 15 min and capped in a nitrogen atmosphereprior to polymerization at 45 C. Sections were cuton a Porter-Blum ultramicrotome with a dia-mond knife and mounted on Formvar-coveredcopper grids. Electron micrographs were takenat initial magnifications of 5,000 and 10,000 inthe Siemens Elmiskop I.

RESULTS

Vegetative hyphae. The long, branching hyphaein the substrate growth are 0.3 to 0.5 , in diam-eter. Figure 1 shows the upper limit of the densevegetative mycelia. The compact filaments arebound by a low-density cementing-substance,probably of cell wall material, which reduces theclarity of cell walls and is visible as strands be-tween unapposed hyphal sections. The fila-mentous network of the nucleoid (N) and cyto-plasmic filaments (CF) are evident. Figure 1(arrows) also illustrates the septal separation ofthe denser vegetative protoplast from the mem-branous aerial segment of the same hypha.

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Figure 1.. Myeium of Stre;.ptmy e nr..a.b.orderbete.,e.;aer.ialandvegetative..g.rw ( 24

Figure 1. Mycelium of Streptomnyces noursei at border between aerial and vegetative growth (X 24,000).

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STUART [o.7

Figure 2. Sections of vegetative hyphae of Streptomyces noursei with branching hypha, septum, and"peripheral bodies" (X 28,500). Insert. Parallel section showing continuity of "peripheral body" andcytoplasmic membranes (X 28,500).

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Figure S. Sections of vegetative hyphae of Streptomyces noursei (X 28,500). Insert. Enlargementshowing components of cell wall, plasma membrane and nucleoid (X 57,000).

Walls of dispersed hyphae are well-defined (12m,u in thickness) and consist of double lamellaeseparated by a less dense interspace (figure 3,insert). The lobulated plasma membrane (4 m,)

(figure 3, insert) is most clearly visible in crosssections of hyphae. Septa are seen infrequently.The single septum shown in figure 2 is a double-walled structure formed by the centripetal infold-

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Figures 4 to 6. Filaments of Streptomyces noursei showing organized structure of tubular nucleoid(X 28,500).Figure 7. Initial stage of protoplast division and sporogenesis (X 28,500).

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ing of the plasma membrane. Numerous longi-tudinal sections with no more than one septumindicate a minimal length of 4 , for the interseptalsegment or cell. Monopodial branching usuallyoccurs at segment poles, adjacent to the septum(figure 2).The protoplast consists of dense peripheral

cytoplasm and a relatively empty core of nucleo-plasm. The nucleo-cytoplasmic ratio varies con-siderably. The cytoplasm contains a filamentouscomponent and low-density granules (figure 3).Oblique sections through hyphae show thatcytoplasmic filaments are organized into animperfect reticulum (figure 3 and insert) whichis more dense at the nucleoplasmic border andaround vacuoles. The occurrence of vacuoles invegetative hyphae is quite variable, and twotypes are distinguishable. Isolated vacuoles(figures 1 and 2, V1), surrounded by a distinctmembrane, may contain a smaller dense periph-eral body, which is not always visible in thesection. Serial sections show the peripheral bodyas a spheroid formed by the circumflexion andcondensation of several parallel cytoplasmicmembranes (figure 2, insert). The neck of themembrane system becomes pinched off, leavingthe spheroid free within the vacuole. There issome evidence from KMnO4-fixed and sectionedmycelia that a continuity of the vacuolar mem-brane and cell wall breaks down, freeing theperipheral body from the hypha. A second typeof vacuole (figures 1 and 2, V2) frequently oc-curs in clusters (figure 1) and is also surroundedby a limiting membrane.The central core of diffuse or organized nucleo-

plasm extends throughout the length of thehyphal segment. The nuclear membrane ispoorly defined. Presumably an equivalent mem-branous structure is the local condensation ofthe cytoplasmic reticulum, which appears as aninterrupted border of dense granules and shortfilaments circumscribing the nucleoid. In crosssections, radial extensions of the core frequentlysubdivide the peripheral cytoplasm (figure 4). Invegetative hyphae the nuclear core consists of anetwork of fine filaments which merge imper-ceptibly with those of the enveloping reticularcondensation (figures 1 to 4). Within the networkare numerous, discrete, dense granules which ap-pear to be the focal point of filament distribution.Although the diffuse nucleoid structure is notshown to vary within hyphal segments of the

substratum, variations do occur in the nucleoidof the developing conidiophore.

Aerial hyphae. In 3-week-old cultures, thepredominant structure within the nuclear core ofimmature conidiophores consists of a few fila-ments and an organized nucleoid consisting ofone or more tubular structures which contain aless dense material (figures 5 to 7, arrows). Thegranules of the diffuse, vegetative nucleoid andthe tubular structures of the conidiophorenucleoid are thought to be identical. In crosssections the filaments frequently radiate fromthe dense granules and tubules (figures 4 and5). Longitudinal sections clearly delineate thetubular structures suspended within the centralcore by the filaments. The tubule may follow aspiral course through the hypha (figures 5 and6). The initial stage of spore formation is evi-denced by a girdling fissure bounded by the cen-tripetal invagination of the primitive plasmamembrane (figure 7).The greater part of the sporogenous area of

older mycelia consists of degenerate sporophoresor hyphal "ghosts" (figure 8, HG), whose inter-stices are filled with a compact mat of shorttubules (figure 8, M). The straight or smoothlycurved tubules, approximately 40 m,u in diameter,are usually oriented at random within the mat.Individual components of the mat are shownclearly as tubules (figure 8, insert), though thisunique structure is obscured in the denser por-tions of the mat. Within the hyphal "ghost" de-generate hyphal walls (figure 8, W) with innermembranes are evident. It is presumed that thesequence of sporophore maturation and degenera-tion results in the deposit of the tubular mataround immature aerial filaments, whose furtherdevelopment does not alter the mat pattern.This is evidenced by the tenuous connections be-tween the degenerate hyphal walls and the mat.The diameter of the "ghost" (0.6 to 0.8 ,u) givesan approximation of the sporophore diameter.The outline of the hyphal "ghost" is usuallybroken by intruding portions of the tubular mat(figure 8, T), which, on the other hand, forms anunbroken outline around spores (figure 8, S).

Figures 9 to 11 show successive stages in theformation of the tubular mat from the sporo-phores. Degenerate segments of the sporophoreare readily distinguished from vegetative hyphaeby the lack of cytoplasmic substance and nuclearmaterial, thus rendering their membrane systems

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Figure 9. Membranous degenerate sporophores (X 24,000).Figure 10. Tubular forms within sporophores (X 24,000).Figure 11. Final stage of sporophore degeneration (X 28,500).Figures 12 and 13. Mature spores of Streptomyces noursei (X 28,500).

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more distinct. It is presumed that the nucleoid isconcentrated within spores at this stage. Theaerial protoplast is initially reduced to a mass ofconcentric membranes which are frequentlypaired and retain their continuity with oneanother and with the plasma membrane (figures1 and 9, arrows). The orderly orientation of themembranes undergoes successive infolding (figure9, arrow) which gives rise to a large number ofparallel tubular structures (figure 10, arrow).The final stage of hyphal degeneration is therupture of the cell wall (CW) and release of thetubular membranes (TM) (figure 11). Sporo-phore degeneration does not result in the trans-formation of all membranous components intothe uniform tubular system. The fate of the cellwall is uncertain. It may remain intact (figure11) or be absent (figure 10), though usually thetwo degenerate wall layers persist (figures 8 and9, W) as a thickened membrane of low electrondensity. The latter is also noted in figure 6.

Relatively few mature spores were visible inthe sections of aerial mycelia observed in thisstudy. The spores are approximately 0.6 , indiameter (figures 12 and 13). The double layerof the spore coat consists of an inner, less densecomponent (C), which is considerably thickerthan the outer spore wall layer (SW) and is notalways distinguishable from it. The nucleoplasmof the spore consists of a network of fine fila-ments and dense granules quite similar to thenucleoid of the young vegetative hypha. Con-traction of spore material usually leaves a clearzone of varying width between the cytoplasmand spore wall. Numerous spines project fromthe dense spore wall. The spines vary in size andshape and form a tangled mass with those ofadjacent spores. The relationship between thewalls of the sporophore and spore and the mecha-nism of spine formation have not been deter-mined.

DISCUSSION

Hyphal fusion with the formation of initialcells from which the secondary or aerial hyphaedevelop (Klieneberger-Nobel, 1947; McGregor,1954; Dickenson and Macdonald, 1955) anddirect interhyphal connections by anastomosis(Gregory, 1956) or bridges (Baldacci et al.,1956) have been observed and in some cases pre-sented as evidence of nuclear interactions. How-ever, these structural findings may not yet war-

rant the genetic interpretations attributed tothem. Exchange of nuclear material betweenfused hyphae has not been demonstrated bydirect microscopic evidence. The parallel align-ment of hyphae perhaps can be accounted for bysurface forces active during the drying of speci-mens, preparatory to light and electron micros-copy. A mucoid cementing-substance betweensubstratal hyphae is observed in figure 1 andappears adequate to cause filament binding. Thecell walls of a Streptomyces species probably con-sist of a lipozyme-sensitive mucopolysaccharide(Romano and Nickerson, 1956). In no instanceswas an indication of hyphal fusion seen in severalhundred electron micrographs, but factors arerecognized which invalidate the significance ofthis negative finding. Fusion may be more fre-quent at a developmental stage other than thoseobserved and ultrathin sectioning greatly re-duces the chance of observing an infrequent andrandomly distributed entity.Hyphal anastomosis (Gregory, 1956) is a more

likely mechanism for heterokaryon formationalthough the absence of anastomosis among thestreptomycetes has been emphasized (Erikson,1949). The occurrence and significance of theseconnections must be clarified by further cytologicstudies.There is no evidence in this study for the

existence of multinucleate cells or segments. Inall vegetative hyphae, the nuclear core is vis-ualized as a diffuse network of filaments anddiscrete granules, which are not subdivided withinthe cell. There was no evidence of sporogenesiswithin the substratum. In other segments, thenucleoid is organized into one or more long,tubular spirals supported by filaments withinthe central core. It was especially noted thatthis structure is frequently paired. Subsequentdevelopment of the conidiophore nucleoid, lead-ing to spore formation, was not observed. Thereis no completely satisfactory explanation for thedifference between these findings and the dis-crete nuclear structures observed in light micro-graphs (Gregory, 1956; Bradley, 1958). Furtherinvestigation of the sequence of nucleoid changesmay provide a structural basis for the explanationof genetic phenomena.The relatively poor delineation of the plasma

and nuclear membranes of this streptomycete,as compared with those of higher fungi, mayindicate the more primitive nature of the genusand may be of evolutional and taxonomic sig-

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nificance. The membranes of the degeneratingconidiophore and the membranous precursors ofthe peripheral bodies and septa clearly demon-strate the potential of the hyphae for membraneformation. It is possible that the membranes ofthe vegetative protoplast are made less distinctby the dense cytoplasmic matrix. The plasmamembrane forms the septa and is continuouswith the membranous reticulum of the cytoplasm.This network, which appears as interruptedfilaments, is distributed throughout the proto-plast, but its density is demonstrably increasedat the borders of the nucleus, where it forms aprimitive nuclear membrane. The continuity ofprimitive endomembrane systems has beenshown in a species of Deuteromycetes (McAlearand Edwards, 1959). The membranes of thedegenerate conidiophore, which give rise to thetubular mat, were first demonstrated as anarray of narrow, pointed plates in electronmicrographs of metal-shadowed specimens (Ver-non, 1955). The significance of the tubular matand its relation to sporulation is uncertain. Someevidence was observed for the coiling of conidio-phores, which is usual among the streptomycetes.

ACKNOWLEDGMENT

I wish to express my deep appreciation to Dr.George A. Edwards and Dr. Elizabeth L. Hazenfor their helpful suggestions and interest in thisstudy.

SUMMARY

The fine structure of the substratum and aerialmycelium of a Streptomyces species, as seen inultrathin sections, was studied by means of elec-tron microscopy. Septa formed by the plasmamembrane, dense peripheral bodies formed bythe condensation of cytoplasmic membranes, anda primitive cytoplasmic reticulum which formspoorly defined membranes around the nucleusand vacuoles, were observed in the vegetativeprotoplast. Variations of the nucleoid, from afilamentous and granular structure in hyphae ofthe substratum to a spiral, tubular form in thedeveloping conidiophore, are demonstrated. Theorigin of the tubular mat of the aerial myceliumis traced to the endomembranes of the degenerat-

ing conidiophore. No evidence of hyphal fusionor multiple nuclei was found.

REFERENCES

BALDACCI, E., GILARDI, E., AND AMICI, A. M. 1956Il Ciclo di Vita degli Attinomiceti Osservatoal Microscopio elettronico. Giorn. Micro-biologia, 1, 512-520.

BRADLEY, S. G. 1958 Genetic analysis of segre-gants from heterokaryons of Streptomycescoelicolor. J. Bacteriol., 76, 464-470.

BRAENDLE, D. H. AND SZYBALSKI, W. 1957 Ge-netic interaction among Streptomycetes:heterokaryosis and synkaryosis. Proc. Natl.Acad. Sci., U. S., 43, 947-955.

DICKENSON, P. B. AND MACDONALD, K. D. 1955An electron microscope examination of theinitial cell stage in Streptomyces spp. J. Gen.Microbiol., 13, 84-90.

ERICKSON, D. 1949 The morphology, cytology,and taxonomy of the actinomycetes. Ann.Rev. Microbiol., 3, 23-54.

GREGORY, K. F. 1956 Hyphal anastomosis andcytological aspects of Streptomyces scabies.Can. J. Microbiol., 2, 649-655.

HAZEN, E. L. AND BROWN, R. 1950 Two anti-fungal agents produced by a soil actinomy-cete. Science, 112, 423.

HOPWOOD, D. A. AND GLAUERT, A. M. 1958 Theelectron microscopy of Streptomyces coelicolor.J. Gen. Microbiol. (Soc. Gen. Microbiol.Proc.), 18, vi-vii.

JONES, K. L. 1954 Variation in Streptomyces.Ann. N. Y. Acad. Sci., 60, 124-135.

KLIENEBERGER-NOBEL, E. 1947 The life cycle ofsporing Actinomyces as revealed by a study oftheir structure and septation. J. Gen.Microbiol., 1, 22-32.

McALEAR, J. H. AND EDWARDS, G. A. 1959 Con-tinuity of plasma membrane and nuclearmembrane. Exptl. Cell Research, 16, 689-692.

McGREGOR, J. F. 1954 Nuclear division and thelife cycle in a Streptomyces sp. J. Gen. Micro-biol., 11, 52-56.

ROMANO, A. H. AND NICKERSON, W. J. 1956 Thebiochemistry of the Actinomycetales. Studieson the cell wall of Streptomyces fradiae. J.Bacteriol., 72, 478-482.

SERMONTI, G. AND SPADA-SERMONTI, I. 1956Gene recombination in Streptomyces coeli-color. J. Gen. Microbiol., 15, 609-616.

VERNON, T. R. 1955 Spore formation in thegenus Streptomyces. Nature, 176, 935-936.

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