JOURNAL OF BACTERIOLOGY, Mar. 1969, p. 1486-1493Copyright © 1969 American Society for Microbiology

Vol. 97, No. 3Printed in U.S.A.

Ultrastructure of Blue-Green Algae'E. GANTT AND S. F. CONTI

Radiation Biology Laboratory, Smithsonian Institution, Washington, D.C. 20560, and Microbiology Department,University of Kentucky, Lexington, Kentucky 40506

Received for publication 29 November 1968

Two freshwater blue-green algae, Tolypothrix tenuis and Fremyella diplosiphon,and an oscillatorialike marine alga, were found to possess structures on the photo-synthetic lamellae which appear to correspond to the phycobilisomes of red algae.These homologous structures are important because they contain the phycobilinswhich are accessory pigments involved in photosynthesis. As in the red algae, thephycobilisomes were attached on the outer side of each lamellae, i.e., the side fac-ing away from its own membrane pair. Although our study on Anacystis nidulanshas not thus far revealed the presence of phycobilisomes, some observations weremade on the structure of the polyhedral bodies. After negative staining, the poly-hedral bodies were seen to be composed of regularly spaced subunits arrangedin a crystalline array. Elongated segmented rods, which differed from the poly-hedral bodies, were found in the nuclear region of apparently healthy Tolypothrixcells.

Numerous studies on blue-green algae haveelucidated various aspects of their fine structure[see Echlin and Morris (5) and Pankratz andBowen (17) for extensive literature reviews]. Ithas generally been accepted that chlorophyll a islocated in the photosynthetic lamellae. However,the location of the phycobiliproteins has largelybeen ignored by morphologists using electronmicroscopy. The role of the phycobiliproteinsas accessory pigments and their involvement inenergy transfer of photosynthesis (4, 6) lendsspecial significance to their physical location.As has been shown (7-9) in the red alga Por-

phyridium cruentum, the phycobiliproteins arepresent as aggregates (phycobilisomes) on thephotosynthetic lamellae. Because of our interestin the localization of phycobiliproteins, wedeemed it necessary to examine various blue-green algae for the presence of phycobilisomes.Our success in finding them was mediated bydifficulty in obtaining consistent preservationwith known fixation procedures. It was difficultto obtain the same excellent fixation which isnecessary for the preservation of phycobilisomesin red algae and, apparently, in blue-green algae.Although our primary interest was in the exist-ence of phycobilisomes, we also made observa-tions on polyhedral and other structured bodies inthe blue-green algae examined.

1 Published with the approval of the Secretary of the Smith-sonian Institution.

MATERIALS AND METHODS

Cultures. Anacystis nidulans was obtained from J.Myers of the University of Texas. Cultures were grownin liquid medium D, as described by Kratz and Myers(14). The cultures were illuminated with light fromincandescent lamps with an incident illumination of150 ft-c.

Tolypothrix tenuis was obtained from M. Gibbs atBrandeis University, and Fremyella diplosiphon fromM. Krauss at the University of Delaware. The mediumfor these algae was that employed by Hattori and Fu-jita (11). The cultures were grown in liquid or on agarunder cool, white fluorescent light (150 ft-c) at roomtemperature.An unidentified oscillatorialike marine blue-green

alga was grown under the same conditions as T. tenuisand F. diplosiphon, except that the medium used wasan artificial seawater medium described by Jones etal. (12).

Electron microscopy. The cells were fixed in 4%glutaraldehyde in 0.1 M phosphate buffer (pH 6.8)for 1 to 2 hr. The cells were usually rinsed in the bufferprior to fixation. Occasionally, the glutaraldehyde wasadded directly to the medium. Similar results wereobtained with both procedures. Postfixation was in 1%osmium tetroxide (in 0.1 M phosphate buffer, pH 6.8)for 2 to 2.5 hr. The embedding and staining procedureswere the same as those previously described (7).

For negative staining, A. nidulans cells were exposedto brief sonic treatment (30 to 45 sec at an output ofabout 30 d-c amp in a Branson Sonifier (Branson In-struments, Inc., Stamford, Conn.). Then they wereimmediately applied to Formvar carbon-coated gridsand stained with 1% phosphotungstic acid (pH 7.2).

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ULTRASTRUCTURE OF BLUE-GREEN ALGAE

RESULTS

The ultrastructures of T. tenuis and F. dip-losiphon are very similar. The cells of these fila-mentous algae differ primarily in shape; T. tenuisis more elongated and F. diplosiphon more bul-bous. The overall structure of both is not unlikethat of the vegetative cell of Nostoc (15). Cells are

enclosed by the typical wall layers, as describedby Jost (13), and each filament is normally sur-rounded by a fibrous gelatinous sheath. Thesheath is quite thick in older cells, but it is thin orabsent (perhaps as a result of treatments involvedin fixation) in young cells. In both Fremyella andTolypothrix, older cells are characterized byextensive amounts of carbohydrate storage prod-ucts. These storage products, named a-granulesby Pankratz and Bowen (17), obscured most ofthe small internal detail; therefore, it was neces-

sary to concentrate on young cells in our in-vestigation.

Figure 1 represents a young vegetative cell ofF. diplosiphon, and Fig. 2, a section of T. tenuis.Within each cell, the photosynthetic lamellae are

randomly arranged and interspersed with severalnuclear areas. Interlamellar spaces and numerous

darkly staining bodies, which may be smallstructured granules or osmiophilic bodies, are

common in young cells. The interlamellar spaces

are absent in older cells of the same preparations,whereas the number of darkly staining bodies issmaller. The lamellae, each consisting of a mem-

brane pair, have small electron-dense granules(20 to 35 nm in diameter) attached to one surface(Fig. 1, 2). By analogy from our work on P.cruentum (9), these granules are regarded as

phycobilisomes. Phycobilisomes are characterizedby containing phycobiliproteins and being directlyattached to one side of the photosynthetic mem-branes, i.e., on the side facing away from its ownmembrane pair. Furthermore, they are orientedin evenly spaced rows on the lamellae. A sugges-

tion of the parallel rows can be seen in Fig. 1 and5. Phycobilisome rows on facing membranes can

be directly opposite one another (as in some areas

of Fig. 2), or they can have an alternate arrange-ment (Fig. 1, 2,4, 5).As anticipated, phycobilisomes were found in

both freshwater (Fig. 1, 2) and marine blue-greenalgae (Fig. 4, 5). The organism shown in Fig. 4and 5 was isolated from a marine aquarium. Inas-much as this organism has a filamentous naturewhose individual filaments are normally sur-rounded by a fibrous sheath, and because it hascharacteristic oscillations as observed under a

light microscope, it is regarded as oscillatorialike.From spectrophotometric readings on aqueous

extracts, it was determined that this alga has

phycocyanin as its accessory pigment, and that itlacks phycoerythrin altogether, whereas T. tenuisand F. diplosiphon have both phycobilins.When the plane of section passes perpendicular

to the rows of phycobilisomes, the individualaggregates can be most clearly seen. In the boxedarea of Fig. 4, the regular spacing on the lamellaeis obvious. As the lamellae are sectioned diag-onally, the phycobilisome shape changes from thebroad-face view to the longitudinal view. Whenthe plane of section is parallel to the rows, theyappear only as rather indistinct segmented bandsof somewhat greater electron density (Fig. 5)than the surrounding material. Phycobilisomeswere not observed in A. nidulans (Fig. 6, 7).

Polyhedral bodies are apparently ubiquitous inall blue-green algae and were certainly present inall four species we examined. Although our maininterest was in the phycobilisomes, some struc-tural characteristics of the polyhedral bodiesseemed worth noting, especially those in A.nidulans. Regardless of the species, the polyhedralbodies were always in the nucleoplasm (Fig. 1, 2,4-8), adjacent to ribosomes and to fibrils, whichare generally assumed to contain deoxyribo-nucleic acid. Although there sometimes is a de-finable interface (Fig. 9), limiting membraneswere not observed around the polyhedral bodies.The figures show the considerable variation in thesize and shape of the polyhedral bodies. Only incells of A. nidulans have we ever observed suchlong polyhedral bodies as that illustrated in Fig.7. In 3-week-old cultures, under our growth con-ditions, it was not uncommon to find that onepolyhedral body spanned the length of two cells.The polyhedral bodies appeared to be quite rigidsince they were always straight and in several cellsseemed to retard or prevent completion of celldivision.Inasmuch as these polyhedral bodies were

known to have a regular shape, as their nameimplies, it was not surprising to find that theypossessed a highly ordered structure. Upon closeexamination, one can observe a periodicity with ahelical pitch (Fig. 8). By negative staining (Fig. 9),it was possible to show that the periodicity is dueto the regular arrangement of equal subunitswhich causes the crystalline-type structure of thepolyhedral bodies. It should be pointed out thatthe periodicity was not always helical; sectionedmaterial sometimes revealed a periodicity parallelwith the long axis of the body.

In addition to the polyhedral bodies and whatappeared as small structured granules (Fig. 2, 3a),cells of T. tenuis possessed a third type of struc-tured body. In Fig. 3a, a segmented rod is seen topass through the cell. The enlargement in Fig. 3b

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FIG. 1. Section of F. diplosiphon with nuclear areas containing several polyhedral bodies (Pb). Phycobilisomesare evident as small dark-staining granules attached to the lamellae. The interlamellar spaces are characteristic ofyoung cells, as are the dark-staining spherical bodies. The arrows indicate a lamellar area where faint traces of theparallel phycobilisome rows are present. X 37,800. Bar indicates 0.5 jum.

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ULTRASTRUCTURE OF BLUE-GREEN ALGAE

FIG. 2. Section from a cell of Tolypothrix tenuis. Phycobilisomes (arrows) are present on the photosyntheticlamellae. Note the polyhedral body (Pb) and the clearly defined plasma membrane (arrow heads). The dark bodiesat the cell junction are probably small structured granules or osmiophilic bodies. A gelatinous sheath (not shownhere) normally surrounds older cells. X 78,000. Bar indicates 0.5 jsm.

reveals that the rod is composed of stacked disc-shaped units, each of which has an electron-densecenter and a less dense outer coat. In the middleregion of Fig. 2b a grazing section can be seenover the outer portion of the subunits where theelectron-dense center is absent. Although most of

these structured bodies were rod-shaped, someassumed a horseshoe shape. The nature and func-tion of this structure is unknown; its appearancesuggests an aggregation of virus particles, butthere was no evidence of virus infection or celllysis.

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FIG. 3. Sections of Tolypothrix tenuis. (a) A large structured body of unknown function traverses the cell. X41,000. Bar indicates 0.5 ,m. (b) Enlargement of the structured body seen in 3a. Note the periodicity and the elec-tron-dense center. The absence of the darker staining core in the middle of the photomicrograph represents a morelateral section of the rod-shaped body. X 155,000. Bar indicates 0.1 pm.

FIG. 4. Phycobilisomes in a marine blue-green alga. Their even spacing on the lamellae can be seen within theboxed area. X 74,000. Bar indicates 0.5 ,um.

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ULTRASTRUCrURE OF BLUE-GREEN ALGAE

FIG. 5. Tangential section of a filamentous marine blue-green alga. Two views of the phycobilisomes are presenton the lamellae. In the lamellar area on the right, the phycobilisomes are seen in cross section. Ifone traces the la-mellae toward the top left, phycobilisome rows evolve and appear as more darkly staining segmented bands. Poly-hedral bodies (Pb) are present in the center of tCze cell. X 81,000. Bar indicates 0.5 ,m.

DISCUSSIONEven though phycobilisomes have not as yet

been isolated from blue-green algae, we feltjustified in assuming that the granules attached tothe lamellae (Fig. 1, 2, 4, 5) are equivalent to thoseseen in red algae (7-9). Since both algal groupspossess phycobiliproteins as accessory pigments,and because their lamellar arrangement is essen-tially the same (grana or face-to-face fusions ofadjacent lamella are absent), it is certainly plau-

sible that they both possess phycobilisomes. Phy-cobilisomes are clearly aggregates of phycobili-proteins, but it is not known whether the entirephycobiliprotein content is present within them.

Experiments using T. tenuis and F. diplosiphonare now in progress to establish whether the shapeof the phycobilisomes in the blue-green algae isdetermined by the predominant phycobiliproteinpresent, as seems to be the case in P. cruentum andPorphyridium aerugineum [with spherical and

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FIG. 6. Ultrastructure ofa representative cell ofA. nidulans. Six densely staining polyhedral bodies are presentin the central region (nucleoplasm); they are surrounded by ribosomal particles andfine fibers, generally believed tocontain deoxyribonuclekic acid. Four lamellae are present along the cellperiphery. The carbohydrate storage productsappear black between the lamellae. Phycobilisomes are not visible. X 55,000. Bar indicates 0.5 ,m.

FIG. 7. In this dividing cell, as noted by the invagination of the lamellae and the wall layers, a very long poly-hedral body extends across both potential daughter cells. X 44,000. Bar indicates 0.5 jAm.

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ULTRASTRUCTURE OF BLUE-GREEN ALGAE

disc-shaped phycobilisomes, respectively; (10)].When the cultures are grown under red light,phycocyanin production predominates; undergreen light, phycoerythrin is produced in excess ofphycocyanin. In this way, the possible relation-ship between phycobilisome shape and relativepigment content will be studied. The limitingfactor at present is the lack of consistent excellentpreservation of the phycobilisomes. The difficultyin preservation ofphycobilisomes is thought to bethe cause for their apparent absence in A. nidulans.Although the general ultrastructure of this algahas been well illustrated by Ris and Singh (18) andAllen (1, 2), their illustrations show no evidenceof phycobilisome-type structures.

Phycobilisomes in blue-green algae have onlybeen reported once in the literature. AlthoughLefort (16) did not identify the structures asphycobilisomes until later (3), she neverthelessshowed them on the lamellae of two blue-greenendosymbionts, Glaucocystis nostochinearum andCyanophora paradoxa. Furthermore, she was un-able to find them in free-living blue-greens. Asidefrom this report, we are aware of three otherspecies in which phycobilisomes have been ob-served; Synechococcus (M. R. Edwards et al., J.Phycol., in press), Plectonema, and Nostoc (M. R.Edwards and J. Reese, personal communication).We expect that phycobilisomes are present in allalgae (Cyanophyta, Rhodophyta,and Cryptophyta)where phycobiliproteins are present as accessorypigments. The reason phycobilisomes have notbeen more commonly observed is believed tobe due to the difficulty in preserving them forelectron microscopy. Preservation of phycobili-somes in free-living blue-green algae is rather ran-dom, similar to the problem encountered in pre-serving chloroplast ribosomes. The problem is notencountered with preservation of cytoplasmicribosomes.From our observations, it is clear that the

polyhedral bodies, or "crystalline bodies" asWildon and Mercer (19) call them, are composedof small units in an orderly array. The crystallinearrangement suggests that the units are of aregular size and perhaps of the same nature. Un-fortunately, to our knowledge, the chemicalcomposition of the polyhedral bodies is notknown. Our limited experience suggests that their

FIG. 8. Clear helical periodicity is present in thelongitudinally sectioned polyhedral body. X 176,000.Bar indicates 0.1 ,um.

FIG. 9. Subunits are evident in this top view ofa pol-yhedral body stained with 2% solution of sodium phos-photungstate. Note evident periodicity. X 250,000. Barindicates 0.1 ,um.

isolation will not be a problem and that theirgeneral chemical nature could easily be de-termined.

ACKNOWLEDGMENTS

This investigation was supported by Atomic Energy Com-mission grant AT(30-1)-3913 and by National Science Foundationgrant GB-5336.We would like to thank K. M. Towe (Paleobiology Depart-

ment, Smithsonian Institution) for the use of the Philips EM-200and associated facilities.

LITERATURE CITED

1. Allen, M. M. 1968. Photosynthetic membrane system inAnacystis nidulans. J. Bacteriol. 96:836-841.

2. Allen, M. M. 1968. Ultrastructure of the cell wall and cell di-vision of unicellular blue-green algae. J. Bacteriol. 96:842-852.

3. Bourdu, R., and M. Lefort. 1967. Structure fine, observee encryode capage, des lamelles photosynthetiques des Cyano-phycees endosymbiotiques: Glaucocystis nostochinearumItzigs, et Cyanophora paradoxa. Compt. Rend. 265:37-40.

4. Duysens, L. N. M. 1951. Transfer of light energy within thepigmnent systems present in photosynthesizing cells. Nature168:548-550.

5. Echlin, P., and I. Morris. 1965. The relationship betweenblue-green algae and bacteria. Biol. Rev. Cambridge Phil.Soc. 40:143-187.

6. French, C. S., and V. K. Young. 1952. The fluorescence spec-tra of red algae and the transfer of energy from phycoerythinto phycocyanin and chlorophyll. J. Gen. Physiol. 35:873-890.

7. Gantt, E., and S. F. Conti. 1965. The ultrastructure ofPorphyridium cruentum. J. Cell Biol. 26:365-381.

8. Gantt, E., and S. F. Conti. 1966. Granules associated with thechloroplast lamellae of Porphyridium cruenttum. J. CellBiol. 29:423-434.

9. Gantt, E., and S. F. Conti. 1966. Phycobiliprotein localiza-tion in algae, p. 393-405. In Energy conversion by the photo-synthetic apparatus. Brookhaven Symp. Biol. no. 19.

10. Gantt, E., M. R. Edwards, and S. F. Conti. 1968. Ultrastruc-ture of Porphyridium aerugineum a blue-green coloredrhodophytan. J. Phycol. 4:65-71.

11. Hattori, A., and Y. Fujita. 1959. Formation of phycobilin pig-ments in a blue-green alga, Tolypothrix tenuis, induced byillumination with colored lights. J. Biochem. (Tokyo) 46:521-524.

12. Jones, R. F., H. L. Speer, and W. Kury. 1963. Studies on thegrowth of the red alga Porphyridium cruentum. Physiol.Plantarum 16:636.

13. Jost, M. 1965. Die Ultrastruktur von Oscillatoria rubescens.Arch. Mikrobiol. 50:211-245.

14. Kratz, W. A., and J. Myers. 1955. Nutrition and growth ofseveral blue-green algae. Am. J. Botany 42:282-287.

15. Leak, L. V. 1965. Electron microscopic autoradiography in-corporation of H3-thymidine in a blue-green alga, Anabaenasp. J. Ultrastruct. Res. 12:135-146.

16. Lefort, M. 1965. Sur le chromatoplasma d'une cyanophycdeendosymbiotique: Glaucocystis nostochinearum Itzigs.Compt. Rend. 261:233-236.

17. Pankratz, H. S., and C. C. Bowen. 1963. Cytology of blue-green algae. L. The cells of Symploca muscorum. Am. J.Botany 50:387-399.

18. Ris, H., and R. N. Singh. 1961. Electron microscope studieson blue-green algae. J. Biophys. Biochem. Cytol. 9:63-80.

19. Wildon, D. C., and F. V. Mercer. 1963. Ultrastructure of thevegetative cell of blue-green algae. Australian J. Biol. Sci.16:585-596.

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