ELECTRON MICROSCOPY OF POLYRIBOSOMES WITHIN BACILLUS CEREUS

Transcription

1 -JOURNAL OF B3ACTERIOLOGY Vol. 88, No. 4, 1) October, 1964 Copyright 1964 Aitieiican Society for Microbiology Printed in U.S.A. ELECTRON MICROSCOPY OF POLYRIBOSOMES WITHIN BACILLUS CEREUS R. M. PFISTER1 AND D. G. LUNDGREN Departnment of Bacteriology and Botany, Syracuse University, Syracuse, New Y'or7k ABSTRAC1T PFISTERt, R. M. (Syracuse University, Syracuse, N.Y.), AND 1). G. LUNDGREN. Electron microscopy of polyrihosomes within Bacilluis cereus. J. Bactcriol. 88: Clusters of ribosomes (polyribosomnes) were identified in thin sections of Bacillus cereus. Cells were treated by freezing and thawing to induce partial lysis to permit a closer examination of structural detail. The polyribosonies were (at times) attached to the cytoplasmic meimbrane, and ribosome clusters contained about 10 to 55 individual ribosomes. Indiv-idual ribosomes ranged from 70 to 100 A, whereas polyribosomes were about 150 to 850 A wide. A membrane served as the fabric holding the rihosomes, which were about 50 to 100 A apart. The polyribosome concept has been established in different cellular systems (Gierer, 1963) and has been extended to bacteria. Polyribosomes have seldom been demonstrated in vivo in bacteria, due to interference from the normally dense cytol)lasm; however, they have been isolated from bacteria (Schlessinger, 1963; Staehelin et al., 1963; Schaechter, 1963). Fitz-James (1964) found festoons of ribosomes (polyribosomes) within Bacillus megateriuni l)rotop)last ghosts. The strings of ribosomes appeared to be attached to the cytoplasmic membrane. The investigation of B. megateriumn was primarily concerned with the in vitro study of polyribosomes where 126S and 155S ribosome peaks were noted, which corresponded closely to the tri- and tetrapolyribosomes of rabbit reticulocytes (W1arner, Rich, and Hall, 1962). The present investigation used bacteria that, were subjected to freezing and thawing, prior to fixation and embedding, to induce cell-wall breakage and thereby reduce cytoplasmic density. 1 Portion of a thesis submitted in partial fulfillment of the requirements for the Ph.). degree in Microbiology, Syracuse University, Syracuse, N.Y. Received for publication 14 May 1964 Ultrathin sections, standard embedding procedures, modified lpreparative techniques, and electron optics were employed to investigate polyribosomes in B. cereus. MATERIALS AND iiethods The bacteria studied were B. cereus ATCC 4342 and an organic sulfur-requiring auxotroph of B. cereus designated C-1. The mutant and its culture conditions have been described (Lundgren and Bott, 1963). B. cereus was grown in the glucose-glutamateglycine-salts culture system described by Cooney and Lundgren (1962). Cell samples were removed at various time intervals, rapidly frozen in a Dry Ice bath, and stored until lprepared for embedding. The auxotroph was grown in the minimal medium lplus cyst(e)ine or methionine in an identical culture svstem. In certain experiments, cell samples of auxotroph were repeatedly frozen and thawed to induce some cell-wall breakage (lysis), and thereby decrease cytopljasmic density. Osmium tetroxide (1 %c in eieronal buffer) was added to the frozen cultures prior to thawing at room temperature; the cells were centrifuged at 2,000 X g; and the pellet was treated with fresh osmium tetroxide for 12 hr atiroom teml)erature. Fixed cell specimens were plrelared for embedding by use of the agar block technique of Kellenberger, Ryter, and S6chaud (1958). The agar blocks were dehydrated through an ethyl alcohol seiries in the following sequence: 50 %, 25 min; 70%A, 25 min; 95%c, 25 min; 95%, 25 min; 100%, 20 min; 100%, 20 min. After dehydration, the blocks were twice treated for 20 min each with propylene oxide to remove the alcohol, and were placed in a mixture containing equal parts of propylene oxide and complete resin monomer for 1 hr. The monomer was prepared according to the method of Luft (1961) with 55 parts of mixture A and 45 parts of mixture B. Standard 00 gelatin capsules were filled with monomer, and the agar blocks were placed on 1119

2 FISTER ANT) LUND)GREN J. BAs(7rERIOI,. Mc- -Mb Ps- }. ~ ~~~~~~~~~~~, EL.~~~~~~~~~~~~.4 r~~~~~~~~~~~~~~~~~~~~~~~~' I I. _, A 'Aop.- 4" "rs FIG. 1. Thin section of Bacilluis cereucs. Cell with a reduced cytoplasmlic content showing clusters of ribosomes (polyribosomes, Ps) with an interlacing retic'ulumnike (Rt) network. The cell wall (W) shows several layers which may represent a microcapsule (Mc). Cell also contains a terminal membranous body (Mb), with a mesosomle (M1), in close association with the cytoplasmlic mnembrane (Cm) (56,500 X). 4z the surface. The blocks settled to the bottom of the capsule during polymerization. Polymerization was achieved by heating the capsules first at 35 C for 12 hr, then at 45 C for 12 hr, and finally at 60 C for 12 hr. The monomer was Epon 812 (Shell Chemical Co., Division of Shell Oil Co., New York, N.Y.). Sections wer-e cut on a AMT-2 lorter-blum

3 VOL. 88, 1964 POLYRIBOSOMES AND MEMBIIANES IN B. ('EREUS 1121 microtome, transferred to carbon-coated grids, and post-stained with a saturated solution of lead hydroxide by the method of Watson (1958); generally, the staining time was increased to 45 min. Preparations were examined in either an EMU-2D electron microscope with an objective aperture of 25 A and an accelerating voltage of 50 kv or an EMU-3C at 100 kv with an aperture As- Ts _ N.. x R. 2 FIG. 2. Thin section of Bacillus cereus. Sporulating cell containing a normal cytoplasmic content with ribosomes (R) and possessing a partially formed transverse septum (Ts) and a forespore (Fs) containing 70 to 100 A bodies presumed to be ribosomes (R). Nuclear material (N) is shown between the transverse septum. A layered cytoplasmic membrane (Cm) is associated with the cell wall (W) and the transverse septum. A developing exosporium (X) is forming around the forespore associated with a mesosome (M) (59,000 X).

4 1122 PFISTER AND LUNDGREN J. BACTERIOL. 3 ~~~o' 5 JA..r FIG. 3. Thin section of Bacillus cereus C-1. The partially lysed bacterium shows a cytoplasmic membrane (Cm) which has pulled away from the cell wall (W) throughout the cell. Polyribosomnes (Ps) appear as clusters and seem to be attached to the cytoplasmic membrane. Two large poly-g-hydroxybutyrate granules surrounded by a membrane (PHBm) are within this cell (44,000 X).

5 VOL. 88, 19G4 POLYRIBOSOMES AND MEMBRANES IN B. CEREUS PHB 'HBM.SVM V.. M 11 ṛ. J*. ' 40tst^.gp OW FIG. 4. Thin section of Bacillus cereus C-1. Cell contains a large poly-,b-hydroxybutyrate granule with membrane (PHBm) and polyribosomes (Ps), some of which are attached to a layered cytoplasmic membrane (Cm) (84,000 X>). of 25,u. Exposures were made at 7,000 to 15,800 revealing some type of membranous complex at magnification, and structures were enlarged the pole of the cell which appears to be against photographically. (or attached to) the cytoplasmic membrane. Dense granules, presumed to be ribosomes, are RESULT AND DscussoNarranged in apparent clusters and connected to a Figure 1 shows an older, partially lysed B. reticulumlike network. A bacterium containing cereus cell, with reduced cytoplasmic density a normally dense cytoplasm as shown in Fig. 2

6 1124 PFISTER AND LUNDCUREN J. BACTERIOL. has these dense granules scattered throughout the cytoplasm. In this cell, there is a forespore which also has ribosomes. These 70 to 100 A particles (ribosomes) were routinely observed in thin sections fixed and stained as described above. In an attempt to study ribosomal structures in greater detail, cells were treated by freezing and thawing to rupture cell walls and to free cytoplasm. This treatment allowed observations of organelles without the interference of a dense cytoplasm. That this treatment did not lead to the production of artifacts is attested to by the Ps - PHB ( ia AZK I FIG. 5. Thin section of Bacillus cereus C-1. Cell contains polyribosomes (Ps) associated with the cytoplasmic membrane; specific polyribosomes are labeled 1, 2, and 3, and are shown enlarged in Fig. 6. The broken cell wall (bw), allowing the escape of cytoplasm and reducing the cell density, is clearly shown. In the center of the cell is a poly-f,-hydroxybutyrate granule with its membrane (PHBm) (56,500 X).

7 VOL. 88, 1954 POLYRIBOSOMES ANI) ME] MIBRANES IN B. CEREU,S 1125 general intactness of cells, except at specific locations where the cell wall was broken. The generally accepted criteria for cell normalcy are appearance and structure. Bacteria with decreased amounts of cytoplasmic material are seen in Fig. 3; the lead staining has accentuated the features of both membranes and ribosomes. The cytoplasmic membrane has moved away from the cell wall and demonstrates some elaboration into folds. An enlargement of a mutant cell is shown in Fig. 4, where the cytoplasmic membrane is seen as a J,,c; 4'~T 77 FIG. 6. Thin section of Bacillus cereus C-1. Enlargement (390,000 XK) of the arrowed area in Fig. showing the cytoplasmic membrane (Cm) pulled away from the wall (W) and polyribosomnes (Ps) attached. The three polyribosomes are labeled 1, 2, and 3, and the attachments to the cytoplasmic membrane are identified by arrows.

8 1126 PFISTER AND LUNI)GREN J. BACTERIOL. layered structure. The spherical membranous organelles (Fig. 3 and 4) are poly-0-hydroxybutyrate (PHB) granules. The membrane coat of these granules was repeatedly identified in this auxotroph after freezing and thawing treatments. The membrane was poorly discernible in normally treated wild-type B. cereus. Some controversy exists about the presence of such a membrane; however, its existence was established in both B. cereus and B. megaterium by use of a carbon PH PHBm j.,,4 4,. '..4I" IBlo 7...i,.1..^2... 1,e- i FIG. 7. Thin section of Bacillus cerents C-i. Portion of a cell showing large dense polyribosomes (Ps), and a poly-13-hydroxybutyrate granule with membrane (PHBm) (100,000 X). Two polyribosomes have been labeled A and B; enlargements of these are shown in Fig. 9.

9 VOL. 88, 1964 POLYRIBOSOMES AND MEMBRANES IN B. CEREUS 1127 replica technique and isolated "native" PHB granules (Lundgren, Pfister, and Merrick, 1964). Boatman (1964) demonstrated the PHB membrane in thin sections of Rhodospirillum rubrum. Ribosomes seen in cells partially cleared of cytoplasm appear to be arranged in clusters. Indeed, in cells with even less cytoplasm (Fig. 5), many grapelike clusters of ribosomes are seen, and some are attached to the cytoplasmic membrane. An enlargement of the polyribosomes labeled 1, 2, and 3 in Fig. 5 is seen in Fig. 6. The attachments to the cytoplasmic membrane are marked by arrows. Clusters of ribosomes are commonly referred to as polyribosomes, polysomes, or ergosomes, and have been isolated from both Escherichia coli and B. megaterium by Staehelin et al. (1963) and Schaechter (1963), respectively. Fitz-James (1964) showed electron micrographs of ghosts of B. megaterium containing festoons of ribosomes (polyribosomes) which appeared to be associated with the cytoplasmic membrane. To learn more about the arrangements of ribosomes within a cluster, polyribosomes in bacteria were examined with electron optics at 100 kv. Figure 7 shows the electron-scattering ribosomes arranged in a cluster on a backing material of a lighter density (presumed to be a membrane about 20 to 30 A thick). An alternative suggestion is that the backing substance may be messenger ribonucleic acid (RNA) holding ribosomes together, as postulated by Warner, Knopf, and Rich (1963). The individual ribosomes in B. cereus ranged from 70 to 100 A, and a polyribosome contained about 10 to 55 ribosomes with a diameter ranging from 150 to 850 A. The ribosomes in the polyribosome were separated by about 50 to 100 A. Cluster size would be influenced by the molecular length of messenger RNA, by the structure of the supporting membrane, or by both. Treatment of thin sections with 100,ug of ribonuclease in 1 ml of tris(hydroxymethyl)aminomethane magnesium buffer for 30 min at 37 C destroyed the cluster, leaving an empty area in the bacterium. Enlargements of individual polyribosomes are shown in Fig. 8 and W*- M14 tt-j *< s r,^^1~'y Ss: :i...::' > ; " AA Ai Pi FIG. 8. Thin section of Bacillus cereus C-1. Electron micrograph of a section of partially cleared cytoplasm showing ribosomes (R) on a membranous network (M) in a polyribosomne (Ps) arrangement (320,000 X). The three polyribosomes indicated at the end of the lines (1, 2, and 3) are identified quantitatively as: (1) contains about 53 ribosomes and measures 665 by 500 A with the connecting "membrane" about 25 A thick; (2) contains about 28 ribosomes and measures 600 by 335 A; (3) contains about 53 ribosomes and measures 635 by 500 A. At the arrows, the connecting "membrane" joins to another polyribosome. The number of ribosomes given are only those for the plane of this particular section and not for the entire three-dimensional complex.

10 1128 PFISTER ANI) LUNDGREN J. BACTERIOL. 0-f..L. vl. X FIG. 9. Thin section of Bacilluts cereus C-1 showing two polyribosomtes. The ribosomnes (R) in each cluster (A and B) appear to be arranged on a 25 A thick "membrane" (M) network in some linear order, and were separated by 50 to t00 A (960,000 X). The A cluster contains about 25 ribosomes and, at its u'idest points, measures 635 A. Cluster B contains about 30 ribosomes and is 835 A wide at the farthest points. 9, with specific details given in their legends. In both figures, the electron-scattering ibosomes are distinguishable from the interlacing nembranous network. The association of protein synthesis with ribosomes (or polyribosomes) and a membrane fraction was suggested or inferred (Spiegelman, 1958; Nisman, 1959; Godson, Hunter, and Butler, 1961; Suit, 1962). Schlessinger (1963) showed that 25% of the ribosomes in crude cell lysates of B. megaterium occur in clusters of four or more, and that these clusters are bound to cell membranes. Our micrographs suggest some linear order of arrangement of the individual ribosomes within the polyribosome, but it is possible that the ribosomes are not in such clusters in situ, but are more linearly arranged, as suggested in the micrographs of Conti and Gettner (1962). Mention of ribosome arrangements was not made by these authors, but their micrographs do show linear arrangements of ribosomes and, at times, show an attachment to the plasma membrane. Zubay (1962) reported on the structure and function of ribosomes involved in protein synthesis in E. coli. Electron micrographs of ribosomes in situ were compared with 1OOS and 70S ribosomes,

11 N OL. 88, 1964 POLYRIBOSOMES AND MEMBRANES IN B. CEREUS 1129 and some similarity to B. cereus polyribosomes was noted. The present cytological evidence supports the idea that riibosomes are associated with membranes and are at times attached to the cytoplasmic membrane. However, an understanding of the role that these membrane entities play in protein synthesis will have to wait for further experimentation. Worthy of mention is the appearance of sectioned cell walls after repeated freezino and thawing (Fig. 7). The cell wall of normal cells (Fig. 2) appeared quite uniform and dense, whereas in treated cells the walls were less dense, and showed some fine structure not too unlike the ribosomes in size and density. We are unable to explain these structures, but they were repeatedly noted and cannot be dismissed as an artifact. They may be comparable to the subunit structures comprising the lipoprotein layer of the walls of Spirillum serpens and Mllicrococcus radiodurans. ACKNOWLEDGMENTS Thanks are extended to R. Doi, R. Slepecky, and E. Balbinder for their encouragement and comments helpful to the preparation of this manuscript. We are also grateful to WV. Cote and A. Day, New York State College of Forestry, for the loan of their electron microscope for taking micrographs at 100 kv. This wor k was sup)ported by the Atomic Energy Commission, contract no. AT-30-1 (2038). LITERATURE CITED BOATMAN, E. S Observations on the fine structure of spheroplasts of Rhodospirillo ruibrumn. J. Cell Biol. 20: CONTI, S. F., AND M. E. GETTNER Electron microscopy of cellular division in Escherichia coli. J. Bacteriol. 83: COONEY, J. J., AND D. (G. LUNDGREN Studies of asporogenic mutants of Bacilluis cereus: preliminary investigations with calcium and zinc. Can. J. Microbiol. 8: FITZ-JAMES, P. C. 19(i4. Polyribosomies in protoplasts of Bacilluis inegateriumt. Can. J. Microbiol. 10: GIERER, A Function of aggregated reticulocyte ribosomes in protein synthesis. J. Mol. Biol. 6: GODSON, (l. N., C. D. HUNTEIR, AND J. A. V. BUTLER Cellular components of Bacilluis megateriumn and their role in protein biosynthesis. Biochem. J. 81: KELLENBERGER, E., A. RYTER, ANI) J. S.CHRAUD Electron microscope study of 1)NAcontaining plasms. II. Vegetative and mature phage l)na as compared with normal bacterial nucleoids in different physiological states. J. Biophys. Biochem. Cytol. 4: LUFT, J. H Improvements in epoxy resin embedding methods. J. Biophys. Biochem. Cytol. 9: LUNDGREN, I). G., AND K. F. BOTT Growth and sporulation characteristics of an organic sulfur-requiring auxotroph of Bacillus cereuis. J. Bacteriol. 86: LUNDGREN, D). C., R. M. PFISTER, AND J. M. MERRIICK Structure of poly-betahydroxybutyric acid (PHB) granules. J. Gen. Microbiol. 34: NISMAN, B Incorporation and activation of amino acids by disrupted protoplasts of Escherichia coli. Biochim. Biophys. Acta 32: SCHAECHTER, M Bacterial polyribosomes and their participation in protein synthesis in vivo. J. Mol. Biol. 7: SCHLESSINGER, 1) Protein synthesis by polyribosomes on protoplast membranes of B. mnegaterium. J. Mol. Biol. 7: SPIEGELMAN, S Protein and nucleic acid synthesis in subeellular fractions of bacterial cells. Intern. Congr. Microbiol. 7th, Stockholm, p STAEHELIN, T., C. C. BRINTON, F. 0. WETTSTEIN, AND H. NOLL Structure and function of E. coli ergosomes. Nature 199: SUIT, J. C Ribonucleic acid in a "membrane" fraction of Escherichia coli and its relation to cell-wall synthesis. J. Bacteriol. 84: WARNERZ, J. R., P. M. KNOPF, AND A. RICH A multiple ribosomal structure in protein synthesis. Proc. Natl. Acad. Sci. U.S. 49: WARNER, J. R., A. RICH, ANI) C. E. HA LL Electron microscope studies of ribosomal clusters synthesizing hemoglobin. Science 138: WATSON, M. L Staining of tissue sections for electron microscopy with heavy metals. II. Application of solutions containing lead and barium. J. Biophys. Biochem. Cytol. 4: ZUBAY, C Physical and chemical properties of particles. Intern. Congr. Microbiol. 8th, Montreal, p