JOURNAL OF BACTERIOLOGY, Jan. 1969, p. 367-375Copyright @ 1969 American Society for Microbiology

Vol. 97, No. 1Printed in U.S.A.

Mesosomes in Escherichia coliR. 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 ap-peared to develop, in dividing cells, as coiled infoldings of the cytoplasmic mem-brane. Mature mesosomes formed a link between the cytoplasmic membrane andthe nucleus of the cell. The arrangement of the mesosomes in dividing cells led tothe hypothesis that division of the nucleus in these cells is accomplished by twoseparate polar mesosomes. One mesosome is derived from the parent cell and ispresent at one pole of the daughter cell. The other is freshly synthesized at or nearthe newly forming pole of the daughter cell. While the old mesosome remains at-tached to the chromosome received from the parent cell, the newly synthesizedmesosome becomes attached to and initiates replication of the new chromosome. Asthe 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 formtubular structures, termed mesosomes by Fitz-James (4), have been most commonly observed ingram-positive organisms (4, 5, 14, 15). Observa-tions of such structures in gram-negative orga-nisms, particularly Escherichia coli, have beenrare, having been positively reported only byKaye and Chapman (6) and by Ryter and Jacob(14). Ryter and Jacob (14) concluded that diffi-culties in observation result from the fact that themesosomes in E. coli, unlike the mesosomes ingram-positive organisms, occur as delicate foldsof the cytoplasmic membrane that can only beseen if the section is cut in a plane exactly per-pendicular to the fine folds of the mesosome.This study shows the presence of mesosomes in

E. coli more clearly than has been hitherto re-ported. Our observations not only support theconcepts of structures of mesosomes of E. coliproposed by Ryter and Jacob (14) but also pro-vide evidence for an explanation of the mode ofdivision of the nucleus.

MATERIALS AND METHODSA strain of E. coli designated as 1 y (10) was grown

overnight at 35 C in Nutrient Broth (Difco) plus0.3% yeast extract. A 0.1-ml amount of this suspen-sion was then inoculated into 40 ml of a definedmedium (Au) devised by Robern (11) and was shakenfor 4 hr at 35 C. The cells, then in the logarithmicphase of growth, were harvested and fixed for electronmicroscopy by the method of Kellenberger, Ryter,and S6chaud (7). Agar cubes containing the fixed cellswere washed for 2 hr in 0.5% uranyl acetate in Veronal

buffer with 0.1 M Ca++ (pH 6.1), progressively de-hydrated in distilled acetone kept over a "molecularsieve" (type 4A beads, Union Carbide Corp., LindeDivision, Ontario, Canada), and embedded in Epon812 (8).

Thin sections were cut on an LKB Ultrotome,stained for 20 min at 40 C with a 3% aqueous solu-tion of uranyl acetate, washed, and further stained for10 min at room temperature with lead citrate (16).The sections were examined in a Siemens Elmiskop

IA electron microscope fitted with an anticontamina-tion device. The instrument magnification employedwas 30,000 times.

Micrographs were taken on Kodak Electron ImagePlates and developed for 3 min in HRP (Kodak)containing 0.1% Kodak Antifog S 1. The negativeswere photographically enlarged to the indicated mag-nifications. The markers on all micrographs are givenin micrometers.

RESULTSTh¢ cross-section of an E. coli cell (Fig. 1)

shows a coiled infolding of the cytoplasmic mem-brane which lies very close to an extension of thenuclear material. In Fig. 2, an extension of thenucleus appears to be almost in contact with acoiled mesosome which extends inward from thecytoplasmic membrane. This coiled fold issituated very close to the site of constriction in thewall of the dividing cell and can be seen in greaterdetail in Fig. 2b. Ellar et al. (3) found mesosomesin a similar location in dividing cells of Bacillusmegaterium. The mesosome (a) shown in Fig. 3differs from that shown in Fig. 2: it is farther fromthe site of division, less tightly coiled, and intrudesinto the nuclear material. At the pole of the cell,

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FIG. 1. Cross section of an E. coli cell. A coiled mesosome can be seen in close proximity to the cytoplasmicmembrane (arrow). An extension ofthe nucleus is close to the mesosome. X 198,000.

the nucleus is in close proximity to the cyto-plasmic membrane (b). The simple extended loopof the mesosome near the pole of the cell in Fig. 4(a) is in direct contact with the compact nucleusin the center of the cell. On the opposite side ofthe cell, near the constriction, and in a location

similar to that of the mesosome seen in Fig. 2,delicate infoldings of the cytoplasmic membranecan be observed (b) close to an extension of thenucleus. In the dividing cell in Fig. 5, a partiallycoiled mesosome can be observed (arrow). Theconfiguration of this mesosome matches the one

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FIG. 2. (a) The mesosome shown in this micrograph ofa dividing cell can be seen to be a tightly coiled extensionof 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 tolead directly into the nucleus.The lower nucleus of the cell in Fig. 6 is

attached to the cytoplasmic membrane by meansof a tenuous, twisted mesosome. This structure,which resembles the proposed connection betweennuclear and cytoplasmic membrane as drawn byRyter and Jacob (14), can be seen in greater detailin Fig. 7, which is a photographic enlargement of

the mesosome shown in Fig. 6. In Fig. 8, thereappear to be two mesosomes present: one (a) is adelicate extended infolding of cytoplasmic mem-brane in contact with the nucleus, similar to thatshown in Fig. 6; the other (b) more closely re-sembles the membranous infolding seen in Fig.3 and 5. Figure 9 shows a higher magnification ofa portion of the same cell containing the twomesosomes. A comparison of their different

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FIG. 3. The mesosome (a) in this dividing cell is less tightly coiled than the one shown in Fig. 2. It appears tofit in a "pocket" in the nucleus and is in contact with the nucleus. At the pole of the cell, the nucleus lies close tothe cytoplasmic membrane and appears associated with membranous elements (b) >X 114,000.

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FIG. 4. There are two mesosomes present in this dividing cell. One is a simple loop extending from the cytoplas-mic membrane to contact the nucleus (a); the other is near the division constriction of the cell (b) and is near anextension of the nucleus. X 109,000.

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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 micro-graph the connection between mesosome and nucleus is quite clear. The tip of the mesosome appears embeddedin the matrix of the nucleus. X 116,000.

shapes can be more easily made, though a con-nection cannot be seen between the coiled meso-some and the nucleus.

DISCUSSIONOther workers (14) have stated that cells of E.

coli fixed by the standard osmium technique (7)do not permit adequate demonstration of meso-somes. However, after growth of cells in oursynthetic medium, the standard fixation gaveclear, precise structural detail; particularly goodpreservation of the nuclear material was achieved,which made the tenuous contact between nucleusand mesosome easier to see (Fig. 2-4). We suggestthat the use of glutaraldehyde-osmium (9, 14)makes difficult the observation of a connectionbetween nucleus and mesosome, because of thediffuse alveolar "foamy" appearance of the nu-cleus.Mesosomes found near the constriction of the

dividing cell usually demonstrated varying degrees

of coiling [Fig. 2, 3, 4 (b), and 5], but mesosomesobserved near the poles of the cells or in non-dividing cells usually appeared as simple directextensions from the cytoplasmic membrane intothe nuclear region [Fig. 6, 7, and 4 (a)]. An excep-tion is apparent in Fig. 8 and 9 where a partiallycoiled mesosome (b) is present on the side of thecell opposite to a relatively straight extension ofthe cytoplasmic membrane. The latter contactsthe nucleus; the former does not.

Basing their calculations on work with B.subtilis and the spheroplasts of E. coli (13, 14),Ryter and Jacob came to the conclusion that inboth types of organisms each nucleus has only onepoint of attachment with the cytoplasmic mem-brane, and that, after the chromosome has dupli-cated, the attached mesosome itself divides, eachdaughter mesosome carrying with it one newlyformed chromosome. The nuclei are thenmechanically separated by the growth of the cyto-plasmic membrane between two points of attach-

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FIG. 6. The attachment between nucleus and cytoplasmic membrane is via a twisted "umbilical cord"-like meso-some. X 84,000.

FIG. 7. This is an enlargement of the area marked in brackets in Fig. 6. The linkage of the mesosome betweencytoplasmic membrane and nucleus can be more easily seen. X 247,SO0.

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FIG. 8. There are two mesosomes present in this cell, but both are on opposite sides of the upper nucleus, anunusual 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 have developed before thecell initiated division. X 48,700.

FIG. 9. An enlargement of the area in brackets in Fig. 8. The connection of the mesosome (a) with the nucleuscan be more clearly seen. X 170,000.

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MESOSOMES IN E. COLI

ment of the mesosomes (13). However, ourelectron micrographs indicate that there are twomesosomes proximal to the nucleus (Fig. 7, 8, and4). Also, in Fig. 3, there is the suggestion that aregion of the nucleus near the pole of the dividingcell is closely associated with membranous ele-ments near the cytoplasmic membrane (b). Wetentatively suggest that the double attachment ofthe nucleus to the cytoplasmic membrane at eachpole of the cell may be a means of mechanicallyseparating the two genomes in the newly formeddaughter cell. The tightly coiled structure foundnear the division constriction of the cell is thenewly forming mesosome which attaches itself tothe new daughter strand of deoxyribonucleic acid(DNA). The daughter genome is then replicated;subsequently, as the E. coli cell grows by new syn-thesis of cell wall material, which in E. coli occursmostly in the central portion of the cell (1), thepoles of the cells move apart, carrying with themthe attached daughter nuclei, thus mechanicallyeffecting nuclear division. This proposed methodof separation of the chromosome differs some-what from Ryter and Jacob's (13) interpretation.It is suggested here that there are two separateattachment points for the mesosomes, one at thepole of the parent cell and one at the divisionpoint of the cell, which will eventually become onepole of the daughter cell. In B. megaterium, twopoints of contact between mesosomes and DNAhave been observed in dividing cells (3). Chai andLark (2) presented a model for chromosome repli-cation and segregation in which replication isinitiated when the recently synthesized comple-mentary strand of DNA becomes attached to anewly synthesized portion of the cell surface. Inthe light of our own observations, we suggest thatthe newly synthesized mesosome at or near theconstriction of the dividing cell becomes attachedto the newly synthesized strand of DNA in thedeveloping daughter cell and, after completion ofthe next round of replication, nuclear division isaccomplished as discussed above. If nuclear sep-aration proceeds in this manner, each chromo-some or newly synthesizing chromosome thenwould have only one point of attachment, via themesosome, to the cytoplasmic membrane. Thiswould be in accord with the proposal made byRyter and Jacob (14).

In a review discussing nucleus-membrane asso-ciations in bacteria, Ryter (12) pointed out that,"morphological studies of gram-negative bacteriahave not so far encouraged more than mere sup-position concerning the connection of the nucleusand membrane." We conclude that our observa-tions demonstrate that the mesosome establishes a

connecting link between the cytoplasmic mem-brane and the nucleus. Its apparent delicate andtenuous form may be a characteristic of gram-negative cells.

Ryter (12) also mentioned the difficulties indrawing conclusions about such a dynamic proc-ess as nuclear division from static micrographs.However, we feel that the sequence of eventspostulated in the present discussion on the me-chanics of nuclear division in these organisms is aworkable scheme consistent with the observationsobtained from our series of micrographs of E.coli 1'y.

LITERATURE CITED

1. Beachey, E. H., and R. M. Cole. 1966. Cell wall replicationin Escherichia coli studies by immunofluorescence andimmunoelectron microscopy. J. Bacteriol. 92:1245-1251.

2. Chai, N.-C., and K. G. Lark. 1967. Segregation of deoxy-ribonucleic acid in bacteria: association of the segregatingunit with the cell envelope. J. Bacteriol. 94:415-421.

3. Ellar, D. J., D. G. Lundgren, and R. A. Slepecky. 1967.Fine structure of Bacillus megaterium during synchronousgrowth. J. Bacteric . 94:1189-1205.

4. Fitz-James, P. C. 1960. Participation of the cytoplasmicmembrane in the growth and spore formation of bacilli.J. Biophys. Biochem. Cytol. 8:507-528.

5. Glauert, A. M., E. M. Brieger, and J. F. Allen. 1961. The finestructure of vegetative cells of Bacillus subtilis. Exptl. CellRes. 22:73-85.

6. Kaye, J. J., and G. B. Chapman. 1963. Cytological aspects ofantimicrobial antibiosis. III. Cytologically distinguishablestages in antioiotic action of collistin sulfate on Escherichiacoli. J. E cteriol. 86:536-543.

7. Kellenberger, E., A. Ryter, and J. Sechaud. 1958. Electronmicroscope studies at DNA-containing plasms. H. Vegeta-tive and mature DNA as compared with normal bacterialnucleoids in differing physiological states. J. Biophys.Biochem. Cytol. 4:671-676.

8. Luft, J. H. 1961. Improvements in epoxy resin embeddingmethods. J. Biophys. Biochem. Cytol. 9:409-414.

9. Kurkdjian, A., A. Ryter, and P. Manigault. 1966. Action dela glycine sur la structure de la paroi de diffrentes souchesd'Agrobacterium tumefaciens et d'Escherichia coll. J.Microscopie 5:605-618.

10. Pontefract, R. D., and F. S. Thatcher. 1965. A cytologicalstudy of normal and irradiation resistant Escherlchia coil.Can. J. Microbiol. 11:271-278.

11. Robern, H., and F. S. Thatcher. 1968. Nutritional require-ments of mutants of Escherichia colt resistant to gamma

irradiation. Can. J. Microbiol. 14:711-715.12. Ryter, A. 1968. Association of the nucleus and the membrane

of bacteria: a morphological study. Bacteriol. Rev. 32:39-54.

13. Ryter, A., and F. Jacob. 1964. Etude au microscope elec-tronique de la liaison entre noyau et mdsosome chez Bacillussubtilis. Ann. Inst. Pasteur 107:384-400.

14. Ryter, A., and F. Jacob. 1966. Etude morphologique de laliaison du noyau a la membrane chez E. coil et les proto-plastes de B. subtilis. Ann. Inst. Pasteur 110:801-812.

15. Van Iterson, W. I. 1961. Some features ofremarkable organellein Bacillus subtilis. J. Biophys. Biochem. Cytol. 9:183-192.

16. Venable, J. H., and J. Coggeshall. 1965. A simplified leadcitrate stain for use in electron microscopy. J. Cell Biol.25:407-408.

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