Mesosomes in Escherichia Coli R

JOURNAL OF BACTERIOLOGY, Jan. 1969, p. 367-375 Vol. 97, No. 1 Copyright @ 1969 American Society for Microbiology Printed in U.S.A. Mesosomes in Escherichia coli R. D. PONTEFRACT, G. BERGERON, AND F. S. THATCHER Research Laboratories, Food and Drug Directorate, Department of National Health and Welfare, Ottawa 3, Ontario, Canada Received for publication 19 October 1968 When Escherichia coli was grown in a synthetic medium and fixed with osmium, sections of the cells revealed clearly defined mesosomes. These mesosomes appeared to develop, in dividing cells, as coiled infoldings of the cytoplasmic membrane. Mature mesosomes formed a link between the cytoplasmic membrane and the nucleus of the cell. The arrangement of the mesosomes in dividing cells led to the hypothesis that division of the nucleus in these cells is accomplished by two separate polar mesosomes. One mesosome is derived from the parent cell and is present at one pole of the daughter cell. The other is freshly synthesized at or near the newly forming pole of the daughter cell. While the old mesosome remains attached to the chromosome received from the parent cell, the newly synthesized mesosome becomes attached to and initiates replication of the new chromosome. As the cell grows and elongates, the two mesosomes, attached to their respective chro- mosomes move apart, thus effecting nuclear division.

Infoldings of the plasma membrane to form buffer with 0.1 M Ca++ (pH 6.1), progressively de- tubular structures, termed mesosomes by Fitz- hydrated in distilled acetone kept over a "molecular James (4), have been most commonly observed in sieve" (type 4A beads, Union Carbide Corp., Linde 5, Division, Ontario, Canada), and embedded in Epon gram-positive organisms (4, 14, 15). Observa- 812 (8). tions of such structures in gram-negative orga- Thin sections were cut on an LKB Ultrotome, nisms, particularly Escherichia coli, have been stained for 20 min at 40 C with a 3% aqueous solu- rare, having been positively reported only by tion of uranyl acetate, washed, and further stained for Kaye and Chapman (6) and by Ryter and Jacob 10 min at room temperature with lead citrate (16). (14). Ryter and Jacob (14) concluded that diffi- The sections were examined in a Siemens Elmiskop culties in observation result from the fact that the IA electron microscope fitted with an anticontamina- mesosomes in E. coli, unlike the mesosomes in tion device. The instrument magnification employed gram-positive organisms, occur as delicate folds was 30,000 times. that can be Micrographs were taken on Kodak Electron Image of the cytoplasmic membrane only Plates and developed for 3 min in HRP (Kodak) seen if the section is cut in a plane exactly per- containing 0.1% Kodak Antifog S 1. The negatives pendicular to the fine folds of the mesosome. were photographically enlarged to the indicated mag- This study shows the presence of mesosomes in nifications. The markers on all micrographs are given E. coli more clearly than has been hitherto re- in micrometers. ported. Our observations not only support the RESULTS concepts of structures of mesosomes of E. coli Th¢ cross-section of an E. coli cell (Fig. 1) proposed by Ryter and Jacob (14) but also pro- shows a coiled infolding of the cytoplasmic mem- vide evidence for an explanation of the mode of brane which lies very close to an extension of the division of the nucleus. nuclear material. In Fig. 2, an extension of the MATERIALS AND nucleus appears to be almost in contact with a METHODS coiled mesosome which extends inward from the A strain of E. coli designated as 1 y (10) was grown cytoplasmic membrane. This coiled fold is overnight at 35 C in Nutrient Broth (Difco) plus situated very close to the site of constriction in the 0.3% yeast extract. A 0.1-ml amount of this suspen- wall of the dividing cell and can be seen in greater sion was then inoculated into 40 ml of a defined medium (Au) devised by Robern (11) and was shaken detail in Fig. 2b. Ellar et al. (3) found mesosomes for 4 hr at 35 C. The cells, then in the logarithmic in a similar location in dividing cells of Bacillus phase of growth, were harvested and fixed for electron megaterium. The mesosome (a) shown in Fig. 3 microscopy by the method of Kellenberger, Ryter, differs from that shown in Fig. 2: it is farther from and S6chaud (7). Agar cubes containing the fixed cells the site of division, less tightly coiled, and intrudes were washed for 2 hr in 0.5% uranyl acetate in Veronal into the nuclear material. At the pole of the cell, 367 368 PONTEFRACT, BERGERON, AND THATCHER J. BACTERIOL.

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FIG. 1. Cross section of an E. coli cell. A coiled mesosome can be seen in close proximity to the cytoplasmic membrane (arrow). An extension ofthe nucleus is close to the mesosome. X 198,000. the nucleus is in close proximity to the cyto- similar to that of the mesosome seen in Fig. 2, plasmic membrane (b). The simple extended loop delicate infoldings of the cytoplasmic membrane of the mesosome near the pole of the cell in Fig. 4 can be observed (b) close to an extension of the (a) is in direct contact with the compact nucleus nucleus. In the dividing cell in Fig. 5, a partially in the center of the cell. On the opposite side of coiled mesosome can be observed (arrow). The the cell, near the constriction, and in a location configuration of this mesosome matches the one VOL. 97, 1969 MESOSOMES IN E. COLI 369

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M 2b ___e,l FIG. 2. (a) The mesosome shown in this micrograph ofa dividing cell can be seen to be a tightly coiled extension of the cytoplasmic membrane. A lobe of the recently divided nucleus lies almost in contact with the mesosome. X 76,000. (b) Mesosomes shown in greater detail. X 152,600. shown in Fig. 3. The end of this fold appears to the mesosome shown in Fig. 6. In Fig. 8, there lead directly into the nucleus. appear to be two mesosomes present: one (a) is a The lower nucleus of the cell in Fig. 6 is delicate extended infolding of cytoplasmic mem- attached to the cytoplasmic membrane by means brane in contact with the nucleus, similar to that of a tenuous, twisted mesosome. This structure, shown in Fig. 6; the other (b) more closely re- which resembles the proposed connection between sembles the membranous infolding seen in Fig. nuclear and cytoplasmic membrane as drawn by 3 and 5. Figure 9 shows a higher magnification of Ryter and Jacob (14), can be seen in greater detail a portion of the same cell containing the two in Fig. 7, which is a photographic enlargement of mesosomes. A comparison of their different 0. 2

FIG. 3. The mesosome (a) in this dividing cell is less tightly coiled than the one shown in Fig. 2. It appears to fit in a "pocket" in the nucleus and is in contact with the nucleus. At the pole of the cell, the nucleus lies close to the cytoplasmic membrane and appears associated with membranous elements (b) >X 114,000. 370 ""', "'10. -, .r.,: ,Iir 4.- "Ns, .4 t. ...,., ,

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FIG. 4. There are two mesosomes present in this dividing cell. One is a simple loop extending from the cytoplasmic membrane to contact the nucleus (a); the other is near the division constriction of the cell (b) and is near an extension ofthe nucleus. X 109,000. 371 372 PONTEFRACT, BERGERON, AND THATCHER J. BACTERIOL.

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FIG. 5. The partially coiled mesosome (a) in this cell greatly resembles the one seen in Fig. 3, but in this micrograph the connection between mesosome and nucleus is quite clear. The tip of the mesosome appears embedded in the matrix ofthe nucleus. X 116,000. shapes can be more easily made, though a con- of coiling [Fig. 2, 3, 4 (b), and 5], but mesosomes nection cannot be seen between the coiled meso- observed near the poles of the cells or in non- some and the nucleus. dividing cells usually appeared as simple direct extensions from the cytoplasmic membrane into DISCUSSION the nuclear region [Fig. 6, 7, and 4 (a)]. An excep- Other workers (14) have stated that cells of E. tion is apparent in Fig. 8 and 9 where a partially coli fixed by the standard osmium technique (7) coiled mesosome (b) is present on the side of the do not permit adequate demonstration of meso- cell opposite to a relatively straight extension of somes. However, after growth of cells in our the cytoplasmic membrane. The latter contacts synthetic medium, the standard fixation gave the nucleus; the former does not. clear, precise structural detail; particularly good Basing their calculations on work with B. preservation of the nuclear material was achieved, subtilis and the spheroplasts of E. coli (13, 14), which made the tenuous contact between nucleus Ryter and Jacob came to the conclusion that in and mesosome easier to see (Fig. 2-4). We suggest both types of organisms each nucleus has only one that the use of glutaraldehyde-osmium (9, 14) point of attachment with the cytoplasmic mem- makes difficult the observation of a connection brane, and that, after the chromosome has dupli- between nucleus and mesosome, because of the cated, the attached mesosome itself divides, each diffuse alveolar "foamy" appearance of the nu- daughter mesosome carrying with it one newly cleus. formed chromosome. The nuclei are then Mesosomes found near the constriction of the mechanically separated by the growth of the cyto- dividing cell usually demonstrated varying degrees plasmic membrane between two points of attach- VOL. 97, 1969 MESOSOMES IN E. COLI 373

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FIG. 6. The attachment between nucleus and cytoplasmic membrane is via a twisted "umbilical cord"-like mesosome. X 84,000. FIG. 7. This is an enlargement of the area marked in brackets in Fig. 6. The linkage of the mesosome between cytoplasmic membrane and nucleus can be more easily seen. X 247,SO0. M (b) I

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FIG. 8. There are two mesosomes present in this cell, but both are on opposite sides of the* upperw nucleus,:w an unusual situation. One (a) is a direct extension from the cytoplasmic membrane into the nucleus. The other (b) is a coiled mesosome which does not appear to be in contact with the nucleus. It may havel-.;1'+developed before.:..the cell initiated division. X 48,700. FIG. 9. An enlargement ofthe area in brackets in Fig. 8. The connection of the mesosome (a) with the nucleus can be more clearly seen. X 170,000. 374 VOL. 97, 1969 MESOSOMES IN E. COLI 375

ment of the mesosomes (13). However, our connecting link between the cytoplasmic mem- electron micrographs indicate that there are two brane and the nucleus. Its apparent delicate and mesosomes proximal to the nucleus (Fig. 7, 8, and tenuous form may be a characteristic of gram- 4). Also, in Fig. 3, there is the suggestion that a negative cells. region of the nucleus near the pole of the dividing Ryter (12) also mentioned the difficulties in cell is closely associated with membranous ele- drawing conclusions about such a dynamic proc- ments near the cytoplasmic membrane (b). We ess as nuclear division from static micrographs. tentatively suggest that the double attachment of However, we feel that the sequence of events the nucleus to the cytoplasmic membrane at each postulated in the present discussion on the me- pole of the cell may be a means of mechanically chanics ofnuclear division in these organisms is a separating the two genomes in the newly formed workable scheme consistent with the observations daughter cell. The tightly coiled structure found obtained from our series of micrographs of E. near the division constriction of the cell is the coli 1'y. newly forming mesosome which attaches itself to the new daughter strand of deoxyribonucleic acid LITERATURE CITED (DNA). The daughter genome is then replicated; 1. Beachey, E. H., and R. M. Cole. 1966. Cell wall replication subsequently, as the E. coli cell grows by new syn- in Escherichia coli studies by immunofluorescence and thesis of cell wall material, which in E. coli occurs immunoelectron microscopy. J. Bacteriol. 92:1245-1251. mostly in the central portion of the cell (1), the 2. Chai, N.-C., and K. G. Lark. 1967. Segregation of deoxy- poles of the cells move apart, carrying with them ribonucleic acid in bacteria: association of the segregating unit with the cell envelope. J. Bacteriol. 94:415-421. the attached daughter nuclei, thus mechanically 3. Ellar, D. J., D. G. Lundgren, and R. A. Slepecky. 1967. effecting nuclear division. This proposed method Fine structure of Bacillus megaterium during synchronous

of separation of the chromosome differs some- growth. J. Bacteric . 94:1189-1205. what from Ryter and Jacob's (13) interpretation. 4. Fitz-James, P. C. 1960. Participation of the cytoplasmic membrane in the growth and spore formation of bacilli. It is suggested here that there are two separate J. Biophys. Biochem. Cytol. 8:507-528. attachment points for the mesosomes, one at the 5. Glauert, A. M., E. M. Brieger, and J. F. Allen. 1961. The fine pole of the parent cell and one at the division structure of vegetative cells of Bacillus subtilis. Exptl. Cell point of the cell, which will eventually become one Res. 22:73-85. 6. Kaye, J. J., and G. B. Chapman. 1963. Cytological aspects of pole of the daughter cell. In B. megaterium, two antimicrobial antibiosis. III. Cytologically distinguishable points of contact between mesosomes and DNA stages in antioiotic action of collistin sulfate on Escherichia have been observed in dividing cells (3). Chai and coli. J. E cteriol. 86:536-543. Lark (2) presented a model for chromosome repli- 7. Kellenberger, E., A. Ryter, and J. Sechaud. 1958. Electron in which is microscope studies at DNA-containing plasms. H. Vegeta- cation and segregation replication tive and mature DNA as compared with normal bacterial initiated when the recently synthesized comple- nucleoids in differing physiological states. J. Biophys. mentary strand of DNA becomes attached to a Biochem. Cytol. 4:671-676. newly synthesized portion of the cell surface. In 8. Luft, J. H. 1961. Improvements in epoxy resin embedding methods. J. Biophys. Biochem. Cytol. 9:409-414. the light of our own observations, we suggest that 9. Kurkdjian, A., A. Ryter, and P. Manigault. 1966. Action de the newly synthesized mesosome at or near the la glycine sur la structure de la paroi de diffrentes souches constriction of the dividing cell becomes attached d'Agrobacterium tumefaciens et d'Escherichia coll. J. to the newly synthesized strand of DNA in the Microscopie 5:605-618. cell after of 10. Pontefract, R. D., and F. S. Thatcher. 1965. A cytological developing daughter and, completion study of normal and irradiation resistant Escherlchia coil. the next round of replication, nuclear division is Can. J. Microbiol. 11:271-278. accomplished as discussed above. If nuclear sep- 11. Robern, H., and F. S. Thatcher. 1968. Nutritional require- aration proceeds in this manner, each chromo- ments of mutants of Escherichia colt resistant to gamma irradiation. Can. J. Microbiol. 14:711-715. some or newly synthesizing chromosome then 12. Ryter, A. 1968. Association of the nucleus and the membrane would have only one point of attachment, via the of bacteria: a morphological study. Bacteriol. Rev. 32: mesosome, to the cytoplasmic membrane. This 39-54. would be in accord with the proposal made by 13. Ryter, A., and F. Jacob. 1964. Etude au microscope elec- tronique de la liaison entre noyau et mdsosome chez Bacillus Ryter and Jacob (14). subtilis. Ann. Inst. Pasteur 107:384-400. In a review discussing nucleus-membrane asso- 14. Ryter, A., and F. Jacob. 1966. Etude morphologique de la ciations in bacteria, Ryter (12) pointed out that, liaison du noyau a la membrane chez E. coil et les proto- "morphological studies of gram-negative bacteria plastes de B. subtilis. Ann. Inst. Pasteur 110:801-812. have not so far encouraged more than mere sup- 15. Van Iterson, W. I. 1961. Some features ofremarkable organelle the connection of the nucleus in Bacillus subtilis. J. Biophys. Biochem. Cytol. 9:183-192. position concerning 16. Venable, J. H., and J. Coggeshall. 1965. A simplified lead and membrane." We conclude that our observa- citrate stain for use in electron microscopy. J. Cell Biol. tions demonstrate that the mesosome establishes a 25:407-408.