JOURNAL OF BACTERIOLOGY, June 1968, p. 2358-2364 Vol. 95, No. 6Copyright © 1968 American Society for Microbiology Printed in U.S.A.

Composition and Ultrastructure ofStreptomyces venezuelae

S. G. BRADLEY AND DONNA RITZIDepartment of Microbiology, University of Minnesota, Minneapolis, Minniesota 55455

Received for publication 11 April 1968

Streptomyces venezuelae is a filamentous bacterium with branching vegetativehyphae embedded in the substrate and aerial hyphae bearing spores. The exterior ofthe spore is inlaid with myriads of tiny rods which can be removed with xylene.The spore wall is approximately 30 nanometers thick. Occasionally, it canbe seen that the plasma membrane and the membranous bodies within aspore are connected. The spore's germ plasm is not separated fromthe cytoplasm by a nuclear envelope. The cell walls of the vegetative hyphae,which are about 15 nanometers thick, are structurally and chemically similar tothose of gram-positive bacteria. The numerous internal membranous bodies, someof which arise from the plasma membrane of the vegetative hypha, maybe vesicular, whirled, or convoluted. Membranous bodies are usually prominent atthe hyphal apices and are associated with septum formation. The germ plasm is anelongate, contorted, centrally placed area of lower electron density than the hyphalcytoplasm. The spores differ from the vegetative hyphae, not only in fine structure,but also in the arginine and leucine contents of their total cellular proteins.

The only streptomycete whose fine structurehas been adequately examined is Streptomycesviolaceoruber. The excellent studies of Glauertand Hopwood (8, 12, 13) have established thatthe streptomycetes are typical prokaryotic orga-nisms with extensive internal membranous bodies.Although S. violaceoruber has been the object ofintensive genetic investigations (3), most of theresearch on actinophages has employed S.venezuelae as the host (4, 14). Accordingly, adetailed examination of the fine structure of S.venezuelae is needed as a prerequisite for furtherstudies on intracellular multiplication of actino-phages. Moreover, certain aspects of the strepto-mycete fine structure have not been resolved; forexample, Hagedorn (11) concluded that thestreptomycete spore is an endospore, whereasGlauert and Hopwood (10) concluded that it ismerely a hyphal segment surrounded by a thick-ened wall.

In this study, the fine structure of the hyphaeand spores has been examined; in addition, a fewdeterminations of the chemical compositions ofstreptomycete components have been made. Dur-ing sporulation, there are changes in both cellularfine structure and chemical composition.

MATERIALS AND METHODS

Fine structure studies. Stock cultures of Strepto-myces venezuelae S13 were maintained at 30 C on

oatmeal-tomato paste-agar (5). The germinatingspores and hyphal elements used for electron micro-scopic studies were obtained as follows. Spores wereharvested from 4-day-old stock cultures of S. vene-zuelae; this material was suspended in peptone-yeastextract broth (16); the suspension was homogenizedwith a tissue grinder; and the dispersed spores wereshaken at 30 C for the appropriate time. The desiredsamples were collected and fixed with 1% bufferedosmium tetroxide (15), dehydrated by transferthrough a graded ethyl alcohol series, and embeddedin Epon (17). Thin sections were prepared with anLKB Ultrotome by use of glass knives and weremounted on copper grids covered with a carbon-coated Parlodion film. The sections were stained withlead citrate (19) or with 2% (w/v) uranyl acetate andexamined with a Siemens Elmiskop I electron micro-scope at initial magnifications between 10,000 X and28,000 X. Samples for electron microscopic studieson sporogenesis were scraped from oatmeal-tomatopaste-agar cultures, fixed in osmium tetroxide, andthereafter treated in the same manner as the hyphalpreparations.

Carbon replicas of S. venezuelae spores were pre-pared by the method of Dietz and Mathews (6). Thespores to be examined were picked up on a glasscover slide by gently touching it to the surface ofsporogenous aerial growth on oatmeal-tomato paste-agar. The dried film of spores was first shadowed withchromium at an angle of 200 from the horizontal andthen coated with carbon at an angle of 90°. The coverslide was immersed in 25% (w/v) aqueous KOH for24 hr to destroy the spores per se. The replica-film was

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STREPTOMYCETE ULTRASTRUCTURE

teased from the cover slide and washed by floating it,first on 0.1 N HCI and then by several transfers todeionized water. The carbon replicas were mountedon copper grids and examined at an initial magnifi-cation of 10,000 X.

Analytical determinations. Approximately 25 g(wet weight) of S. venezuelae spores or vegetativemycelia were extracted at 50 C for 2 hr with 250 mlof acetone, followed by 250 ml of diethyl ether at30 C for 2 hr. The residue was suspended in 20 ml ofdeionized water and then exposed to ultrasonicvibrations (20,000 cycles/sec generated by an MSEultrasonic disintegrator) while resting in an ice bath.The disrupted cellular material was centrifuged in thecold at 5,000 X g for 15 min to sediment intact cellsand large cellular fragments. The residue wassuspended in 20 ml of deionized water and shakenvigorously for 10 min at 4 C. This preparation wascentrifuged in the cold at 5,000 X g for 15 min. Thetwo supernatant fractions were pooled and centri-fuged in the cold at 15,000 X g for 15 min to removeany remaining hyphal walls. The cellular proteinswere precipitated and were leached for 30 min at 4 Cwith two 50-ml changes of 5% trichloroacetic acid.This residue was extracted twice with 50 ml of 5%trichloroacetic acid for 30 min at 100 C. The trichloro-acetic acid remaining after these extractions wasremoved by washing the residue with 25 ml of acetone,followed by two washes with diethyl ether. Thisresidue was acid hydrolyzed with 6 N HCl for 22 hr at110 C. The resulting protein hydrolysate was analyzedwith a Beckman Spinco model 120 automatic aminoacid analyzer.

Vegetative mycelia from overnight, peptone-yeastextract broth cultures of S. venezuelae were collectedby centrifugation and washed three times with de-ionized water. The hyphae were ruptured by ultra-sonic treatment (20,000 cycles/sec). Whole cells wereremoved by centrifugation at 500 X g, and the hyphalwalls were collected by centrifugation at 15,000 X g.The hyphal wall preparations were subjected to 20to 25 washes with deionized water until the ab-sorbance at 260 nanometers (nm) of the supernatantfluid was less than 0.001. The final, purified wallpreparation was 5.8% nitrogen and 5.2% phosphorus;the A260/A28o was less than 1.3, and the ratio A260/A450 was less than 2 (W. J. Cooney, M.S. Thesis,Univ. of Minnesota, Minneapolis, 1964). The hyphalwalls were hydrolyzed and analyzed by the samemethods used for the total cellular proteins.

RESULTS AND DISCUSSIONCarbon replicas of S. venezuelae spores show

that the spore surface is covered with a mosaicpattern formed by ordered arrangements ofmyriads of rods approximately 10 x 100 nm.These tiny rods frequently become separated fromthe spore surface, indicating that they are super-ficially placed and loosely attached to the sporewall (Fig. 1). This surface material is not solublein water or carbon tetrachloride, but is extractedby benzene, xylene, and ethyl alcohol (Fig. 2).This layer probably accounts for the hydrophobic

properties of S. venezuelae spores. Similar mosaicshave been observed on the spore surface of anumber of streptomycetes (6).

Spores of S. venezuelae are enclosed by a cellwall about 30 nm thick. Patches of the outercomponent of the cell wall of the sporogenousaerial hypha frequently remain attached to themature spore surface. The plasma membrane isclearly evident in sections of spores, and an oc-casional membranous body can be found at-tached to the outer limiting membrane. Thecytoplasm of the uranyl acetate-stained sectionsis dense and homogeneous. The electron-trans-parent germ plasm, with its delicate, electron-dense network is concentrated in the center of thespore. A membranous body is usually found with-in the nucleoid (Fig. 3). Similar structures ingram-positive bacteria have been variously des-ignated as chondrioids, plasmalemmosomes, andmesosomes by other workers.

Spores placed in nutrient medium develop anextensive membranous system (Fig. 4). The cellwall of the germ tube is continuous with the innercomponent of the spore wall (Fig. 4-6). The cellwall of the germling is about 15 nm thick. Duringgermination, the ribosomes, which cannot bedistinctly seen in the spore sections, become pro-gressively well defined (Fig. 4-6). The strepto-mycete ribosomes are about 12 nm in diameter,which is comparable to values obtained for otherbacteria (22). In the vegetative hyphae, there arenumerous membranous structures along theplasma membrane (Fig. 6-9). As the vegetativehyphae grow, the nucleoid becomes elongate andtortuous, and cross walls and branches develop(Fig. 5-8). Branch formation is neither positivelynor negatively correlated with cross-wall forma-tion (Fig. 7-9). The apices of vegetative hyphaeusually contain prominent membranous bodies(Fig. 8, 9). Membranous bodies are also associ-ated with septum development in both vegetativehyphae (Fig. 7-9) and sporogenous hyphae (Fig.11, 12). These structures resembled those des-ignated as peripheral bodies, chondrioids, ormesosomes in Bacillus (22). In contrast to theobservation of Moore and Chapman (18) on anunidentified streptomycete, cross walls are rela-tively rare in S. venezuelae vegetative hyphae. Inthe stationary and death phases of growth, hyphalghosts containing membranous vesicles are com-monly seen (Fig. 10). Similar ghosts have beenobserved in degenerate sporophores of S. noursei(20) and disintegrating vegetative hyphae of S.violaceoruber (9). The plasma membrane in de-generating hyphae sometimes forms a partitionacross a filament independent of septum forma-tion (Fig. 10). This may account for our observa-tions with the light microscope in which discrete

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FIG. 1-14. Electron micrographs of sections ofStreptomyces venezuelae. The marker bars denote 500 nanome-ters. Abbreviations used throughout: CW, cell wall; FS, fibrous sheath; GP, germ plasm; MB, membranous body;PM, plasma membrane; R, ribosome; and S, septum.

FIG. 1. Carbon replica ofnormal spores. Arrow indicates free crystals.FIG. 2. Carbon replica of xylene-extracted spores.FIG. 3. Section of a normal spore, stained with uranyl acetate. Arrow indicates an attached strip of the outer

layer of the cell wall of the aerial hypha.FIG. 4. Section of a germinating spore, stained with lead citrate.FIG. 5-6. Sections ofgerminating spores, stained with uranyl acetate and lead citrate (Fig. 5) or uranyl acetate

alone (Fig. 6). Arrows indicate the points where the outer layer of the spore separates from the inner layerwhich is continuous with the cell wall of the germling.

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STREPTOMYCETE ULTRASTRUCTURE

7

FIG. 7-9. Sections of vegetative hyphae, stained with uranyl acetate.FIG. 10. Section ofa degenerate vegetative hypha, stained with uranyl acetate.

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2362 BRADLEY AND RITZI J. BACTERIOL.

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STREPTOMYCETE ULTRASTRUCTURE

number of electron-transparent foci (Fig. 11-13)which, in S. violaceoruber, were interpreted asstorage vacuoles by Glauert and Hopwood (10).The first step in sporogenesis is the simultaneousformation of many septa that eventually dividethe aerial hyphae into compartments, each ofwhich becomes a spore (Fig. 11). Initially, theplasma membrane invaginates at regular intervalsto form annular folds. At these sites, the innerlayer of the hyphal wall breaks around its entirecircumference; the free edges turn inward (Fig.11, 12). Both margins of the in-turned, delicatecell wall layer are extended centripetally at thesame rate. Each advancing cross wall is only 2.5nm thick at first (Fig. 12). It is significant that thecross walls of developing contiguous spores areseparate entities at all stages (Fig. 12, 13). Afterthe cross wall is completed, the outer and innerlayers of the hyphal wall separate laterally as thespore rounds up (Fig. 13). Ultimately, the outerlayer of the hyphal wall ruptures leaving a beltof this material firmly attached to each developingspore surface (Figs. 3, 14). The inner layer, whichnow constitutes the spore wall per se, thickens toabout 30 nm. The mature spores are held in thechains typical of streptomycetes by a thin outersheath (Fig. 14). It is significant that the outersheath, also observed by Vernon (23), Enghusen(7), and Baldacci, Gilardi, and Amici (1), isdistinct from the outer layer of the cell wall of theaerial hyphae, in contrast to the conclusionreached by Glauert and Hopwood (10) for S.violaceoruber (Fig. 13, 14). The origin and com-position of the sheath, however, is still unclear.The outer sheath is probably not the continuationof the parental cell wall of the vegetative hyphaeas proposed by Hagedorn (11) for S. griseus. Thelittle rods on the spore surface (Fig. 1) apparentlyare an integral component of this superficialfibrous layer.The total proteins of vegetative hyphae and

spores were extracted and their amino acid com-positions determined. The spore protein prepara-tion contained cell wall material, as indicated bythe presence of diaminopimelic acid in the hy-drolysate. In general, the spore protein and pro-tein from the vegetative mycelium had the sameamino acid composition; two exceptions werenoted. Both the arginine and leucine contents ofspore protein were less than that of protein fromthe vegetative mycelium (Table 1). In addition,Tewfik and Bradley (21) observed that the de-oxyribonucleic acid (DNA) from S. venezuelaespores displays a buoyant density appreciably lessthan that of the mycelial DNA. The cause for thelightness of the spore DNA has not been estab-lished, but it is presumably attributable to achange in secondary structure or to binding with

TABLE 1. Amino acid and amino sugar compositionof Streptomyces venezuelae

Amino acids andamino sugars

Alanine.Arginine ........Aspartate b......Cystine (12).....Diaminopimelic

acid...........Glutamateb.Glycine .........Histidine .......Isoleucine. ....Leucine.........Lysine.Methionine ......Phenylalanine.Proline .Serine...........Threonine .....Tyrosine ........Valine ..........Glucosamine....Muramic acid...

Amt of amino acids or amino sugar/mg oftotal cell protein from

Cell wall

,umoles

0.53

0.430.330.36

0.270.25

Vegetativemycelium

,umoles

1.260.440.660.16

1.030.910.120.320.700.280.160.290.310.310.410.200.59

Spores

j,moles1.180.280.680.09

0.160.930.960.110.280.550.210.090.310.310.280.350.170.650.100.09

a None detected.b Includes the respective amine.

some other cellular component. The cell wallsfrom vegetative hyphae were purified and ana-lyzed. The mucopeptide contained, in addition toglucosamine and muramic acid, alanine, dia-minopimelic acid, glutamate, and glycine. Theseresults agree with an earlier report (2).The fine structure and cell wall composition of

the streptomycetes are similar to those of Bacillusmegaterium and other gram-positive bacteria (22).Although membranous bodies are extensive inthis group of microbes, it has not been possible tocorrelate definitively specific physiological func-tions with particular cytological configurations.The membranous bodies at the hyphal apices andin germinating spores (Fig. 4, 8) are, generallyspeaking, convoluted, whereas those associatedwith septa are vesicular (Fig. 7, 9, 11, 12). Themembranous body associated with the germplasm seems to be made of concentric membranes(Fig. 3, 13). Conclusive correlations between finestructure and metabolic activity await the resultsof concurrent physiological, histochemical, bio-chemical, and cytological investigations.

ACKNOWLEDGMENTThis investigation was supported by Public Health

Service grant AI-06804 from the National Instituteof Allergy and Infectious Diseases.

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BRADLEY AND RITZI

LITERATURE CITED

1. Baldacci, E., E. Gilardi, and A. M. Amici. 1956.I ciclo di vita degli attinomiceti osservato almicroscopio elettronico. Gior. Microbiol.6:512-519.

2. Becker, B., M. P. Lechevalier, and H. A. Leche-valier. 1965. Chemical composition of cell-wallpreparations from strains of various form-genera of aerobic actinomycetes. Appl.Microbiol. 13:236-243.

3. Bradley, S. G. 1966. Genetics in applied micro-biology. Adv. Appl. Microbiol. 8:29-59.

4. Bradley, S. G., and D. Ritzi. 1967. Electronmicroscopic studies of actinophage multipli-cation. J. Gen. Virol. 1:285-290.

5. Bradley, S. G., and D. Ritzi. 1967. Structure ofactinophages for Streptomyces and Nocardia.Develop. Ind. Microbiol. 8:206-213.

6. Dietz, A., and J. Mathews. 1962. Taxonomy bycarbon replication. I. An examination ofStreptomyces hiygroscopicus. Appl. Microbiol.10:258-263.

7. Enghusen, H. 1955. Elektronenoptische Darstell-ungen von Streptomyceten-Sporen und-Hilllen.Arch. Microbiol. 21:329-334.

8. Glauert, A. M., and D. A. Hopwood. 1959. Amembranous component of the cytoplasm inStreptomyces coelicolor. J. Biophys. Biochem.Cytol. 6:515-516.

9. Glauert, A. M., and D. A. Hopwood. 1960.The fine structure of Streptomyces coelicolor. I.The cytoplasmic membrane system. J. Biophys.Biochem. Cytol. 7:479-488.

10. Glauert, A. M., and D. A. Hopwood. 1961.The fine structure of Streptomyces violaceoruber(S. coelicolor). III. The walls of the myceliumand spores. J. Biophys. Biochem. Cytol. 10:505-516.

11. Hagedorn, H. 1960. ElektronenmikroskopischeUntersuclhungen an Streptomyces griseus(Krainsky). Zentr. Bakteriol. Parasitenk Abt.II 113:234-253.

12. Hopwood, D. A., and A. M. Glauert. 1960.The fine structure of Streptomyces coelicolor.II. The nuclear material. J. Biophys. Biochem.Cytol. 8:267-278.

13. Hopwood, D. A., and A. M. Glauert. 1961.Electron microscope observations on the sur-face structures of Streptomyces violaceoruber.J. Gen. Microbiol. 26:325-330.

14. Jones, L. A., and S. G. Bradley. 1965. The lifecycle of an actinophage for Streptomycesvenezuelae. J. Gen Microbiol. 49:191-198.

15. Kellenberger, E., A. Ryter, and J. Sechaud. 1958.Electron microscope study of DNA-con-taining plasms. II. Vegetative and maturephage DNA as compared with normal bac-terial nucleoids in different physiologicalstates. J. Biophys. Biochem. Cytol. 4:671-678.

16. Kolstad, R. A., and S. G. Bradley. 1964. Purifi-cation of Streptomyces venezuelae phage. J.Bacteriol. 87:1157-1161.

17. Luft, J. H. 1961. Improvements in epoxy resinembedding methods. J. Biophys. Biochem.Cytol. 9:409-414.

18. Moore, R. T., and G. B. Chapman. 1959. Ob-servations of the fine structure and modes ofgrowth of a streptomycete. J. Bacteriol. 78:878-885.

19. Reynolds, E. S. 1963. The use of lead citrate athigh pH as an electron-opaque stain in electronmicroscopy. J. Cell Biol. 17:208-212.

20. Stuart, D. C., Jr. 1959. Fine structure of thenucleoid and internal membrane systems ofStreptomyces. J. Bacteriol. 78:272-281.

21. Tewfik, E. M., and S. G. Bradley. 1967. Char-acterization of deoxyribonucleic acids fromstreptomycetes and nocardiae. J. Bacteriol.94:1994-2000.

22. Van Iterson, W. 1965. Symposium on the finestructure and replication of bacteria and theirparts. II. Bacterial cytoplasm. Bacteriol. Rev.29:299-325.

23. Vernon, T. R. 1955. Spore formation in the genusStreptomyces. Nature 176:935-936.

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