Arch. Mikrobiol. 67, 328--338 (1969)

Fine Structure of Developing Polyphosphate Bodies in a Blue-Green Alga, Plectonema boryanum

THOMAS E. JENSEN*

Department of Biology, Wayne State University, Detroit, Michigan

Received April 4, 1969

Summary. Stages in the development of polyphosphate bodies in the blue- green alga, Plectonema boryanum, grown under continuous illumination in the presence of excess phosphate, are reported. During the first stage, an electron- lucent area appears near the nucleoplasm or cross walls; it gradually increases to a size approximately equal to that of the final polyphosphate body. In this area a porous structure of medium electron density develops, while simultaneously electron-dense material, interpreted as polyphosphate, is deposited in the adjacent cytoplasm. Eventually, this material seems to penetrate into the porous structure. When the polyphosphate bodies are fully formed, the surrounding cytoplasm does not contain detectable amounts of polyphosphate.

The formation of polyphosphate bodies in P. boryanum is compared with that in some bacterial species.

In an earlier report ( J~s~N, 1968) polyphosphate bodies were identified at the u]trastructural level in the blue-green alga, Nostoc pruni/orme. In this species, the bodies were found to be spherical, electron opaque, and to vary from about 0.1 to 3 ~ in diameter. While ultrastruetural details of their formation in blue-green algae have not so far been reported, several studies have shown that in bacteria they arise as the result of sequential morphological changes (KoLB~L, 1958; D~EWS, 1960 a, b; Vo]sLz et al., 1966), suggesting that similar changes might be observed in these algae.

I f grown under normal conditions, blue-green algae cells which we have investigated contain very few polyphosphate bodies, which hampers the detection of developmental changes. We have, therefore, studied the formation of these bodies in continuously illuminated cultures, supplemented with excess phosphate, which favors the formation of polyphosphate bodies (TALPASAYI, 1963). Better knowledge of their genesis may be of value in interpreting the nature of certain inclusions in blue-green algae as observed by electron microscopy.

While surveying a number of blue-green algae species, kindly sup- plied by Dr. R. C. STAIR from his collection (STAIR, 1964), for this

* Present address: Department of Biological Sciences, Lehman College, City University of New York, Bedford Park Boulevard West, Bronx, New York 10468.

Fine Structure of Polyphosphate Bodies in a Blue-Green ~Mga 329

purpose, we observed a series of s tages in Plectonema boryanum Gomont , which we in t e rp re t as a deve lopmen ta l sequence in the fo rma t ion of

po lyphospha t e bodies.

~Iaterials and Methods

P. boryanum was grown in Chu's medium l0 with soil extract (C~v, 1942). Cultures were maintained at 21 ~ under 500 ft. candles of illumination with a 12 hour alternating light and dark period. For studies on polyphosphate body formation, cultures were enriched with 0.001 M K2HPO 4 and continuously illuminated (TALPASAYI, 1963). Samples were removed for fixation after 24, 48, and 72 hours. They were fixed in buffered Os04 (1~ pH 6.2) for 3 hours at room temperature (PANKI~ATZ and BOW~N, 1963); rapidly dehydrated in a graded ethanol series, treated with propylene oxide, and embedded in Epon 812 (Lug% 1961). Sections were cut with a diamond knife on an LKB ultramicrotome and post stained with uranyl (STEMPAK and WARD, 1964) or lead salts (REYNOLDS, 1963). They were examined in a RCA EMU-3H electron microscope.

For light microscopy, material exposed to the above experimental conditions was stained for polyphosphates as described by EBEL et al. (1958) and modified by J~SEN (1968). Whole cells were photographed in bright field with a Zeiss microscope.

Results

The first ind ica t ion of po lyphospha te body fo rma t ion is the appear - ance of an e lec t ron- lucent area, be tween 150 and 200 m a in d iameter , in the cy top la sm (Figs. 1 - -2) . I t then enlarges to include an area approxi - m a t e l y equal to t h a t which the ma tu re po lyphospha te b o d y will occupy. I n P. boryanum, in i t i a t ion of the po lyphospha te bodies occurs e i ther in the cy top la sm near the nueleoplasm and thus close to po lyhedra l bodies (Fig. 1), or in the pe r iphe ry of the cell near the erosswalls. The r ibosomes which are presen t appea r to be hyd ro lyzed dur ing the forma- t ion of the e lec t ron- lucent area, as t h e y become more electron t rans- parent . I n the final s tages of deve lopment , no r ibosomes can be de tec ted in the area (Figs. 1--2) . I n Fig. 2, only fa in t images of r ibosomes near the pe r iphe ry of the e lec t ron- lucent area can be seen.

Subsequent ly , two events a p p a r e n t l y occur s imul taneous ly in the deve lopmen t of the po lyphospha te bodies. A ma te r i a l of med ium electron dens i ty is depos i ted wi th in the e lec t ron- lucent areas (Fig.3). I n most ins tances this leads to the fo rma t ion of a s t ruc ture t h a t appears porous in sections, ind ica t ing t h a t the electron t r a n s p a r e n t areas are t u b u l a r in na tu r e (Fig. 3). A t this s tage of deve lopmen t the pores are be tween 5 and 15 m a in d iameter . The porous body then enlarges, even tua l ly occupying a lmos t the ent i re e lec t ron- lucent a rea ; i ts size m a y v a r y f rom 30- -400 m~z in d iameter . A t the same t ime the porous body is developing wi th in the e lec t ron- lucent area, an e lectron-dense ma te r i a l is being depos i ted in the ad j acen t cy top la sm (F igs .4 - -7 ) . On the basis of the analysis of numerous mierographs , i t is fel t t h a t the

330 T . E . J ~ s E ~ :

Fig. i . Section of a vegetat ive cell of P. boryanum showing an electron-lucent area E which is the first indication of polyphosphate body formation. Other structures visible in this and subsequent mierographs are indicated by Pb (polyhedral bodies), T (thylakoids),

R (ribosomes), and N (nueleoplasmie areas)

Fig. 2. Port ion of a cell showing a well-developed electron-lucent area E. The ribo- somes R are faint ly visible at the edge of the area

Fig. 3. Section of the central area of a cell showing the initial stage of development of the porous body P within the electron-lucent area E. Note the porous nature of this inclusion

Fine Structure of Polyphosphate Bodies in a Blue-Green Alga 331

Fig.4. Portion of a cell showing the electron-dense polyphosphate which has apparently been deposited in the cytoplasm outside of the porous body P

332 T .E . JE~sE~:

Fig. 5. Section of a cell showing a fully developed porous body P. Note the dense poly- phosphate P P deposit at the edge of the porous body. The polyphosphate has smeared

in the direction of sectioning, which is to the right of the micrograph

Fine Structure of Polyphosphate Bodies in a Blue-Green Alga 333

Fig. 6. Portion of a cell showing a peripherally located, developing polyphosphate body. Note the dense deposit of polyphosphate P P in the cytoplasm outside the porous body P.

Direction of sectioning is toward the bottom of the mierograph

Fig. 7. Portion of a cell showing a centrally located, developing polyphosphate body. Note the numerous pores in the porous body. The polyphosphate P P is more dense in the cytoplasm outside the porous body. Direction of sectioning is toward the bottom of

the micrograph

Fig. 8. Section of a developing polyphosphate body showing a dense deposit of poly- phosphate P P at the edge of the porous body P. Direction of sectioning is toward the

bottom of the micrograph

Fig. 9. Section of a developing polyphosphate body. Note that the electron-dense poly- phosphate P P is located in the cytoplasm and in the outer portion of the porous body. The pores are of g larger diameter in this body. Direction of sectioning is toward the bottom

of the micrograph

334 T.E. J E ~ s ~ :

deposition of the electron-dense mater ial originates outside a restricted area of the body. Depending on the section angle, one could, therefore,

obta in images of either the eleetron-dense mater ial or the porous body. The electron-dense mater ia l is probably polyphosphate ; this is based

Fig. 10. Portion of a cell showing a polyphosphate body with the electron dense poly- phosphate located around its periphery. Note thaL the pores are still evident in the central

area of the body

Fig. 11. Light mierograpb of a cell stained for polyphosphates. Note that only the outer portion of ~he polyphosphate body (arrow) has stained densely. This polyphosphate body

is probably in about the same stage of development as the one shown in Fig. 13

Fig. 12. Enlargement of a small, developing polyphosphate body to show the irregular nature of lbhe pores in the porous body

Fig. 13. Portion of a cell showing a fully developed polyphosphate body in association with four polyhedral bodies Pb

Fine Structure of Polyphosphate Bodies in a Blue-Green Alga 335

on the observation that it smears out during sectioning, as previously repoted ( J ~ s E ~ , 1968) for fully developed polyphosphate bodies (Figs. 4--7).

As development proceeds, as observed in material fixed at 48 hours, electron dense polyphosphate is deposited in the porous body starting at the periphery (Figs. 8--10). Light microscope studies of similar material stained for polyphosphates also show some bodies which have stained only around the periphery (Fig. 11). The pores vary in size at this stage, being between 8 and 30 m~ in diameter (Figs. 8--10, 12). In some polyphosphate bodies the periphery of the body appears homogeneously electron-dense (Fig. 10), while in other bodies the porous structure remains visible as the electron dense polyphosphate is deposited eveny throughout the body (Fig. 12). This nniform deposition could, of course, be accounted for if the section represents the surface of a developing polyphosphate body. In the final stages of development the entire body becomes elec- tron-dense (Fig. 13). This stage, in P. boryanum, is only observed in material fixed 72 hours after the addition of excess phosphate and ex- posure to continuous light.

The developmental stages appear to be the same for polyphosphate bodies located at the periphery of cells near the crosswalls and for those located in the central area. There seems to be no morphological associa- tion between the polyphosphate body and particular cytoplasmic in- clusions in the blue-green algae. The association of polyphosphate bodies which develop in the center of the cell with polyhedi'al bodies is felt to be incidental, as no polyhedral bodies are associated with the bodies developing near the periphery of the cell.

Discussion

We have shown that polyphosphate body formation in P. boryanum proceeds by a sequential series of morphological changes which can be followed over a 72 hour period. Perhaps some polyphosphate bodies complete development in less time as has been reported for Nostoc pruni]orme ( J~sv , s, 1968). New bodies are continually being formed, thus the chances of observing later stages are increased in material exposed to the conditions favorable for development for longer periods of time. However, in P. boryanum we have not observed any of the later stages of development in the 24 hour material. The early stages of development are present in all of the material because new polyphosphate bodies are continually being formed.

During development it appears that all cytoplasmic components are excluded from the area of polyphosphate body formation. Isolation and subsequent analysis of fully developed bodies should, therefore, not be

336 T.E. JENsEN:

hampered by the presence of cytoplasmic components trapped within the developing body. I f partially formed bodies are isolated, ribosomes and probably DNA will also be present. In Myxococcus xanthus it has been reported that cytoplasmic strands served as the material onto which the polyphosphate became deposited (VoELZ et al., 1966). These authors suggested that nuclear fibers may also become trapped in the developing po]yphosphate body. They further suggest that this may be the reason for reports of DNA, RNA, protein and lipid in poly- phosphate bodies of certain bacteria. However, FuHs (1958), using cytochemical methods, has found only polyphosphate in the poly- phosphate bodies of the blue-green alga Oscillatoria amoena, thus sup- porting the proposal tha t no cytoplasmic components are trapped in the developing body.

The nature of the porous body is of interest in relation to future work on the analysis of these structures. They could serve as deposition loci for the lengthening strands of polyphosphate. However, in ear]y stages of development the polyphosphate appears to be deposited in the cyto- plasm outside of the body, though this has not yet been established with certainty, it could have preceded slightly the formation of the porous body. In early stages of polyphosphate deposition cytoplasmic elements could be acting as deposition foci, while in later stages the porous body serves this function. In the final stages, no polyphosphate can be observed in the cytoplasm; it is apparently remobilized and moved into the porous body, which itself might be composed of short chain polyphosphates. The apparent lack of staining of the central area of some porous bodies indicates that, if they contain polyphosphate, the polymer must consist of less than three phosphates because EBEL et al. (1958) reports a positive staining reaction for all polyphosphates composed of three or more phosphates. In some of the larger polyphos- phate bodies (Fig. 11), one can see, with the light microscope, a densely stained periphery and a less densely stained central portion. I t could, therefore, be that the porous body is ~composed of polyphosphate which is much less concentrated than in later stages. Preliminary results with carbon coated sections indicate that some of the pores are a type of beam artifact. I t appears that some of the pores may contain polyphos- phates which are volatilized in the beam leaving an electron transparent pore. The exact composition will have to await isolation studies of the bodies at different stages of development.

On the basis of the evidence presented here, one can obtain a number of different images of polyphosphate bodies depending on their stage of development. KSNIG and WINKLE~ (1948) and Dg]~ws (1960 a) have reported that polyphosphate bodies in bacteria may sublimate during exposure to the electron beam, leaving an electron-transparent center

Fine Structure of Polyphosphate Bodies in a Blue-Green Alga 337

and an e lectron-dense per iphery . W e have seen no ind ica t ion of such sub l imat ion to p roduce this k ind of image. I t is possible t h a t the images of po lyphospha t e bodies wi th a dense pe r iphe ry and e l ec t ron- t r anspa ren t center are po lyphospha t e bodies in the s tage of deve lopmen t shown in Fig. 10.

Vo~Lz et al. (1966) r epor t ed tha t , when Myxococcus xanthu8 is grown in cas i tone-Mg-medium conta in ing 5 • 10 -4 M phospha te as a buffer and phospha te was a d d e d dur ing the exponent ia l growth phase to a final concen t ra t ion of 5 • 10 -2 M, the po lyphospha t e was depos i ted as dense granules a round po lysacchar ide inclusions. Al though the polysacchar ide inclusions resemble, to a large degree, the e lec t ron- lucent areas which develop in P. boryanum, we do no t bel ieve t h a t t h e y represen t poly- sacehar ide deposi ts in our own mater ia l . This is based on the fact t h a t general ly, in the blue-green algae, the excess p h o t o s y n t h a t e is s to red as a polyglncoside in a discrete body of var iab le d i ame te r and length (Funs , 1963 ; GIpsY, 1964). The e lec t ron- lucent areas in no w a y resemble any of the r epor t ed polyglncoside bodies of the blue-green algae. There was also no not iceable s ta ining when lead salts were used as pos t s ta in for the sections which no rma l ly cause the polyglucoside bodies to s ta in qui te densely. The exac t na tu re of the e lec t ron- lucent area is, thus, no t known.

The technical assistance of LINDA M. SIc~o is gratefully acknowledged. This investigation was supported by NSF Grant GB 6305. Contribution No. 229 of the Department of Biology, Wayne State University.

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T~O~AS E. JEI~SEN Department of Biology Wayne State University Detroit, Michigan 48202, U.S.A.