JOURNAL OF BACTERIOLOGY, Sept. 1978, p. 1137-1140 0021-9193/78/0135-1137$02.00/0 Copyright © 1978 American Society for Microbiology
Vol. 135, No. 3
Printed in U.S.A.
NOTES Phycobilisomes in Spheroplasts of Anacystis nidulans JILL C. COSNER
Biological Science Group, University of Connecticut, Storrs, Connecticut 06268 Received for publication 4 May 1978
Phycobilisomes in Anacystis nidulans can be seen more readily in spheroplasts than in cells with intact walls.
Phycobilisomes are high-molecular-weight aggregates of phycobiliproteins which function as photosynthetic accessory pigments (5, 6). Under the electron microscope these aggregates appear as electron dense granules attached to the stroma side of the thylakoid membranes. Phycobilisomes are believed to be ubiquitous in all phycobiliprotein containing species of red algae and blue-green bacteria (cyanobacteria). They have been elegantly demonstrated in many organisms (10, and see Literature Cited therein). However, with some organisms, e.g., Anacystis nidulans, it has been difficult to obtain good images of phycobilisomes by standard fixing and staining techniques (4, 7, 10). This paper describes images of phycobilisomes in A. nidulans spheroplasts which appear superior to those obtainable in intact cells. An axenic culture obtained from the Indiana Culture Collection (strain 625) was grown for 7 days in 500-ml cylindrical tubes containing 450 ml of Kratz and Myers medium C supplemented with disodium ethylenediaminetetracetic acid (6.7 mg/ml). The culture was incubated at 37°C under 22.5 pE m-2 s-' PAR of fluorescent light (cool white) and was continuously mixed by bubbling 95% air-5% CO2 through it. The cells were harvested by centrifugation, washed with fresh culture medium, and suspended in 0.5 M mannitol and 0.03 M sodium phosphate buffer (pH 6.8) according to the method of Biggins (2). Spheroplasts were prepared by adding lysozyme (Sigma Chemical Co.) in 0.03 M sodium phosphate buffer (pH 6.8) to give a final concentration of 0.1%, and the cell suspension was blended in a Vortex mixer. After incubation for 2 h at 37°C under 5 PE m-2 s-' PAR of fluorescent light (cool white) on a reciprocating shaker, the cells were harvested from the suspension. The cells were washed with 0.5 M mannitol and culture medium, resuspended in this solution, and incubated under the same conditions for an
additional 6 h. The cells were then prepared for electron microscopy by washing with 0.5 M mannitol in Kratz and Myers medium C and suspended in 0.5 M mannitol and 0.05 M sodium cacodylate buffer (pH 7.0) with 2 mM Ca"!. Glutaraldehyde was added to give a final concentration of 1.5%, and the cell suspension was fixed at ambient temperature for 2.5 h. The cells were washed overnight with the cacodylate buffer and post fixed with Os04 for 1 h at 24°C and then with 1% uranyl acetate in distilled water for 1.5 h at 24°C. The fixed cells were suspended in 2% agar. Small agar blocks containing spheroplasts as well as intact cells were dehydrated in ethanol and embedded in Epon 812 (Luft). Thin sections were obtained on an LKB Ultrome by using glass knives and stained with uranyl acetate (15 min) and lead citrate (15 min). The grids were viewed with a Philips 300 electron microscope. Intact cells of A. nidulans are rod shaped and are resistent to lysis in distilled water. After treatment with lysozyme as described approximately 50% of the cells assume a spherical shape (J. C. Cosner and R. F. Troxler, Biochim. Biophys. Acta, in press). These cells are osmotically fragile and will burst in distilled water, indicating the loss of cell wall rigidity. In the electron microscope it was found that the treated cells lack the LI, layer of the cell wall. This finding is in agreement with Jensen and Sicko's work with Cylindrospermum sp., a filamentous cyanobacterium (8). A large proportion of the remaining layers of the cell wall can still be seen around the cells although often they are discontinuous. Thus, these cells would not be considered true protoplasts, but they are better defined as spheroplasts. Spheroplasts are shown in Fig. 1 through 3. Phycobilisomes can be seen oni the stroma side of the photosynthetic thylakoids. Figure 1 shows the phycobilisomes in face view (cross-section). 1137
1138
J. BACTERIOL.
NOTES
FIG. 1. Thin section of a spheroplast of A. nidulans. Phycobilisomes in face view (cross section) are indicated by arrows and those in edge view (longitudinal section) are indicated by arrow heads. All bars represent 0.1 pm.
Their basal diameter agreed with that already published for phycobilisomes in intact bluegreen bacteria (3, 10). Phycobilisomes in edge view (longitudinal section) appeared as short rods approximately 10 nm in diameter. This datum agrees with previously published datum for intact cells (10). In Fig. 3 phycobilisomes can be seen in grazing view as short rods. These three views depict a disk-shaped phycobilisome, as expected for A. nidulans, since it contained only the phycobiliproteins phycocyanin and allophycocyanin. Organisms containing phycoerythrin as well as phycocyanin and allophycocyanin have granules which appear rounded in all
three views and thus are semispherical in shape
(6).
Other subcellular structures such as polyhedral bodies, plasmalemma, and DNA fibers can be seen in these spheroplasts. Continuity of the photosynthetic lammellae with the plasmalemma also can be seen as previously shown by Allen for intact cells of A. nidulans (1). These spheroplasts are metabolically active as shown by incorporation of L-[ U- 4C]leucine into trichloroacetic acid-insoluble material and purified phycocyanin, thus demonstrating the occurrence of protein synthesis (Cosner and Troxler, in press). The dimensions of the phycobilisomes
NOTES
VOL. 135, 1978
in the spheroplasts agree with previously published data for those in intact cells. It seems warranted to assume that the phycobilisomes in spheroplasts are identical to those in intact cells. With the standard techniques used to fix and stain these preparations, intact cells were generally more dense and lacked good contrast, whereas adjacent spheroplasts on the same section had better contrast. It is possible that during
-
m
.
n
*
q
1139
the preparation procedures for electron microscopy more of the soluble constituents leached out of the spheroplasts than out of intact cells, and it is this which allowed better contrast. However, leaching could not have been extensive since there was no phycocyanin in any of the supematants at the various steps of the electron microscopy preparation procedures. It is also possible that fixatives and stains as well
wG'u.
',-.
I
_
_
FIG. 2. Section of a spheroplast showing rows ofphycobilisomes in edge view (arrow heads).
FIG. 3. Section of a spheroplast showing phycobilisomes in grazing view (wavy arrows).
1140
NOTES
as other solutes can more easily enter and leave the cells when the LI, layer of the cell wall is removed. Pigott and Carr showed that spheroplasts incorporate a larger percentage of exogenous purine and pyrimidine bases than intact cells (9). They concluded that treatment with lysozyme affects the permeability barrier of the cells (9). Clearer images of phycobilisomes are seen in spheroplasts than in intact cells, suggesting that the lack of a rigid cell wall permits better visualization of the cell's internal structure. The preparation of spheroplasts for electron microscopy has the potential of allowing workers to obtain structural data on phycobilisomes in other organisms in which good definition previously has not been obtainable. This work was supported by the Biological Science Group, University of Connecticut. I am grateful to A. Wachtel and D. Wetherell for their helpful discussions and constructive comments on this manuscript.
J. BACTERIOL. LITERATURE CITED 1. Allen, M. M. 1968. Photosynthetic membrane system in Anacystis nidulans. J. Bacteriol. 96:836-840. 2. Biggins, J. 1967. Preparation of metabolically active protoplasts from the blue-green alga Phormidium luridium. Plant Physiol. 42:1442-1446. 3. Edwards, M. R., and E. Gantt. 1971. Phycobilisomes of the thermophilic blue-green alga Synechococcus lividus. J. Cell Biol. 50:896-900. 4. Evans, E. L., and M. M. Allen. 1973. Phycobilisomes in Anacystis nidulans. J. Bacteriol. 113:403-408. 5. Gantt, E., and S. F. Conti. 1966. Granules associated with the chloroplast lamellae of Porphyridium cruentum. J. Cell Biol. 29:423-434. 6. Gantt, E., and S. F. Conti. 1966. Phycobiliprotein localization in algae. Energy conversion by the photosynthetic apparatus. Brookhaven Symp. Biol. 19:393-405. 7. Gantt, E., and S. F. Conti. 1969. Ultrastructure of bluegreen algae. J. Bacteriol. 97:1486-1493. 8. Jensen, T. E., and L M. Sicko. 1971. The effect of lysozyme on cell wall morphology in a blue-green alga, Cylindrosperrnum sp. J. Gen. Microbiol. 68:71-75. 9. Pigott, G. H., and N. G. Carr. 1971. The assimilation of nucleic acid precursors in intact cells and protoplast of the blue-green alga Anacystis nidulans. Arch. Microbiol. 96:83-92. 10. Wildman, R. B., and C. C. Bowen. 1974. Phycobilisomes in blue-green algae. J. Bacteriol. 117:866-881.
Vol. 135, No. 3
Printed in U.S.A.
NOTES Phycobilisomes in Spheroplasts of Anacystis nidulans JILL C. COSNER
Biological Science Group, University of Connecticut, Storrs, Connecticut 06268 Received for publication 4 May 1978
Phycobilisomes in Anacystis nidulans can be seen more readily in spheroplasts than in cells with intact walls.
Phycobilisomes are high-molecular-weight aggregates of phycobiliproteins which function as photosynthetic accessory pigments (5, 6). Under the electron microscope these aggregates appear as electron dense granules attached to the stroma side of the thylakoid membranes. Phycobilisomes are believed to be ubiquitous in all phycobiliprotein containing species of red algae and blue-green bacteria (cyanobacteria). They have been elegantly demonstrated in many organisms (10, and see Literature Cited therein). However, with some organisms, e.g., Anacystis nidulans, it has been difficult to obtain good images of phycobilisomes by standard fixing and staining techniques (4, 7, 10). This paper describes images of phycobilisomes in A. nidulans spheroplasts which appear superior to those obtainable in intact cells. An axenic culture obtained from the Indiana Culture Collection (strain 625) was grown for 7 days in 500-ml cylindrical tubes containing 450 ml of Kratz and Myers medium C supplemented with disodium ethylenediaminetetracetic acid (6.7 mg/ml). The culture was incubated at 37°C under 22.5 pE m-2 s-' PAR of fluorescent light (cool white) and was continuously mixed by bubbling 95% air-5% CO2 through it. The cells were harvested by centrifugation, washed with fresh culture medium, and suspended in 0.5 M mannitol and 0.03 M sodium phosphate buffer (pH 6.8) according to the method of Biggins (2). Spheroplasts were prepared by adding lysozyme (Sigma Chemical Co.) in 0.03 M sodium phosphate buffer (pH 6.8) to give a final concentration of 0.1%, and the cell suspension was blended in a Vortex mixer. After incubation for 2 h at 37°C under 5 PE m-2 s-' PAR of fluorescent light (cool white) on a reciprocating shaker, the cells were harvested from the suspension. The cells were washed with 0.5 M mannitol and culture medium, resuspended in this solution, and incubated under the same conditions for an
additional 6 h. The cells were then prepared for electron microscopy by washing with 0.5 M mannitol in Kratz and Myers medium C and suspended in 0.5 M mannitol and 0.05 M sodium cacodylate buffer (pH 7.0) with 2 mM Ca"!. Glutaraldehyde was added to give a final concentration of 1.5%, and the cell suspension was fixed at ambient temperature for 2.5 h. The cells were washed overnight with the cacodylate buffer and post fixed with Os04 for 1 h at 24°C and then with 1% uranyl acetate in distilled water for 1.5 h at 24°C. The fixed cells were suspended in 2% agar. Small agar blocks containing spheroplasts as well as intact cells were dehydrated in ethanol and embedded in Epon 812 (Luft). Thin sections were obtained on an LKB Ultrome by using glass knives and stained with uranyl acetate (15 min) and lead citrate (15 min). The grids were viewed with a Philips 300 electron microscope. Intact cells of A. nidulans are rod shaped and are resistent to lysis in distilled water. After treatment with lysozyme as described approximately 50% of the cells assume a spherical shape (J. C. Cosner and R. F. Troxler, Biochim. Biophys. Acta, in press). These cells are osmotically fragile and will burst in distilled water, indicating the loss of cell wall rigidity. In the electron microscope it was found that the treated cells lack the LI, layer of the cell wall. This finding is in agreement with Jensen and Sicko's work with Cylindrospermum sp., a filamentous cyanobacterium (8). A large proportion of the remaining layers of the cell wall can still be seen around the cells although often they are discontinuous. Thus, these cells would not be considered true protoplasts, but they are better defined as spheroplasts. Spheroplasts are shown in Fig. 1 through 3. Phycobilisomes can be seen oni the stroma side of the photosynthetic thylakoids. Figure 1 shows the phycobilisomes in face view (cross-section). 1137
1138
J. BACTERIOL.
NOTES
FIG. 1. Thin section of a spheroplast of A. nidulans. Phycobilisomes in face view (cross section) are indicated by arrows and those in edge view (longitudinal section) are indicated by arrow heads. All bars represent 0.1 pm.
Their basal diameter agreed with that already published for phycobilisomes in intact bluegreen bacteria (3, 10). Phycobilisomes in edge view (longitudinal section) appeared as short rods approximately 10 nm in diameter. This datum agrees with previously published datum for intact cells (10). In Fig. 3 phycobilisomes can be seen in grazing view as short rods. These three views depict a disk-shaped phycobilisome, as expected for A. nidulans, since it contained only the phycobiliproteins phycocyanin and allophycocyanin. Organisms containing phycoerythrin as well as phycocyanin and allophycocyanin have granules which appear rounded in all
three views and thus are semispherical in shape
(6).
Other subcellular structures such as polyhedral bodies, plasmalemma, and DNA fibers can be seen in these spheroplasts. Continuity of the photosynthetic lammellae with the plasmalemma also can be seen as previously shown by Allen for intact cells of A. nidulans (1). These spheroplasts are metabolically active as shown by incorporation of L-[ U- 4C]leucine into trichloroacetic acid-insoluble material and purified phycocyanin, thus demonstrating the occurrence of protein synthesis (Cosner and Troxler, in press). The dimensions of the phycobilisomes
NOTES
VOL. 135, 1978
in the spheroplasts agree with previously published data for those in intact cells. It seems warranted to assume that the phycobilisomes in spheroplasts are identical to those in intact cells. With the standard techniques used to fix and stain these preparations, intact cells were generally more dense and lacked good contrast, whereas adjacent spheroplasts on the same section had better contrast. It is possible that during
-
m
.
n
*
q
1139
the preparation procedures for electron microscopy more of the soluble constituents leached out of the spheroplasts than out of intact cells, and it is this which allowed better contrast. However, leaching could not have been extensive since there was no phycocyanin in any of the supematants at the various steps of the electron microscopy preparation procedures. It is also possible that fixatives and stains as well
wG'u.
',-.
I
_
_
FIG. 2. Section of a spheroplast showing rows ofphycobilisomes in edge view (arrow heads).
FIG. 3. Section of a spheroplast showing phycobilisomes in grazing view (wavy arrows).
1140
NOTES
as other solutes can more easily enter and leave the cells when the LI, layer of the cell wall is removed. Pigott and Carr showed that spheroplasts incorporate a larger percentage of exogenous purine and pyrimidine bases than intact cells (9). They concluded that treatment with lysozyme affects the permeability barrier of the cells (9). Clearer images of phycobilisomes are seen in spheroplasts than in intact cells, suggesting that the lack of a rigid cell wall permits better visualization of the cell's internal structure. The preparation of spheroplasts for electron microscopy has the potential of allowing workers to obtain structural data on phycobilisomes in other organisms in which good definition previously has not been obtainable. This work was supported by the Biological Science Group, University of Connecticut. I am grateful to A. Wachtel and D. Wetherell for their helpful discussions and constructive comments on this manuscript.
J. BACTERIOL. LITERATURE CITED 1. Allen, M. M. 1968. Photosynthetic membrane system in Anacystis nidulans. J. Bacteriol. 96:836-840. 2. Biggins, J. 1967. Preparation of metabolically active protoplasts from the blue-green alga Phormidium luridium. Plant Physiol. 42:1442-1446. 3. Edwards, M. R., and E. Gantt. 1971. Phycobilisomes of the thermophilic blue-green alga Synechococcus lividus. J. Cell Biol. 50:896-900. 4. Evans, E. L., and M. M. Allen. 1973. Phycobilisomes in Anacystis nidulans. J. Bacteriol. 113:403-408. 5. Gantt, E., and S. F. Conti. 1966. Granules associated with the chloroplast lamellae of Porphyridium cruentum. J. Cell Biol. 29:423-434. 6. Gantt, E., and S. F. Conti. 1966. Phycobiliprotein localization in algae. Energy conversion by the photosynthetic apparatus. Brookhaven Symp. Biol. 19:393-405. 7. Gantt, E., and S. F. Conti. 1969. Ultrastructure of bluegreen algae. J. Bacteriol. 97:1486-1493. 8. Jensen, T. E., and L M. Sicko. 1971. The effect of lysozyme on cell wall morphology in a blue-green alga, Cylindrosperrnum sp. J. Gen. Microbiol. 68:71-75. 9. Pigott, G. H., and N. G. Carr. 1971. The assimilation of nucleic acid precursors in intact cells and protoplast of the blue-green alga Anacystis nidulans. Arch. Microbiol. 96:83-92. 10. Wildman, R. B., and C. C. Bowen. 1974. Phycobilisomes in blue-green algae. J. Bacteriol. 117:866-881.
