Synchronous Growth D

JOURNAL OF BACTERIOLOGY, Oct. 1967, p. 1189-1205 Vol. 94, No. 4 Copyright © 1967 American Society for Microbiology Printed in U.S.A. Fine Structure of Bacillus megaterium During Synchronous Growth D. J. ELLAR,1 D. G. LUNDGREN, AND R. A. SLEPECKY Department ofBacteriology and Botany, Biological Research Laboratories, Syracuse University, Syracuse, New York 13210 Received for publication 17 July 1967 A fine-structure study of synchronously dividing Bacillus megaterium revealed the sequence of events involved in the division of the cell. First, a mesosome develops as a concentric fold of the plasma membrane at the site of septum formation. The mesosome contains membrane-bound vesicular structures, 300 to 500 A in diameter, plus a large membrane-bound structure, 2,000 A in diameter. These larger vesicles are peculiar to mesosomes in this stage of division and are not observed in the mesosomes involved in spore septum formation. The transverse septum originates within the mesosome and remains enclosed during its subsequent growth across the cell. An intimate association is observed between mesosome vesicles, mesosome membrane, and the growing edge of the transverse septum. Prior to completion of the septum, the membranes bounding the mesosome fuse, and further wall thickening occurs within the structure formed by this fusion. At this time, the septum only equals the parent cell wall in thickness. The doubling in thickness of the septum, which is re- quired for the production of two normal daughter cell walls, occurs during a second phase of wall thickening, which is characterized by the appearance of a constriction at the base of the septum. As the constriction widens, the wall in this region thickens, forming the typical rounded poles of the daughter cells. Capsular synthesis at the poles occurs during this second phase of wall thickening. Throughout the division process, the nuclear material appears to be associated at one end with a mesosome at or near the pole of the cell and at the other end to the mesosome involved in septum formation. This association frequently takes the form of a stalk- like extension of the mesosome penetrating into the chromatin fibrils. Electron microscopy of the division process in MATERIALS AND METHODS gram-positive bacteria has shown that the growth The organism used was B. megaterium ATCC 19213 of the cross wall is preceded by the inward pro- grown on a defined sucrose salts medium (SS) pre- liferation of the plasma membrane (13, 18, 30, pared according to Slepecky and Foster (37). For 36, 39). Several workers have observed "periph- these studies, the medium was supplemented by the eral bodies" or mesosomes in the vicinity of the addition of 0.02%o Trypticase (BBL). developing cross wall (5, 11, 20, 23), and it has Culture technique. Details of the culture procedures been suggested that they function in some manner and the modified filtration method of Maruyama and in the orderly synthesis of cell wall material. Yanagita (25) which were used to obtain division Evidence to date, however, is not adequate to synchrony have been previously reported (19). In the the the present experiments, when the culture reached late log determine relationship between mesosome, phase (166 Klett units, measured with a no. 54 filter), the infolding plasma membrane, and the new the cells were collected on filters and aseptically pooled cross wall, nor is it possible to determine the into a sterile 3-liter Fernbach flask and placed on a precise sequence of changes in fine structure which reciprocating shaker at 30 C. The entire filtration proc- occur when the cell divides. The use of a synchro- ess took less than 5 min and was performed at room nously dividing culture of Bacillus megaterium temperature. offers a more useful method for studying both Electroni microscopy. Samples (10 ml) of the culture the nature and time of appearance of these mor- were removed from the Fernbach flask at intervals of phological changes during cell division. 1 hr and were fixed immediately with osmium by the agar block method of Kellenberger, Ryter, and 1 Present address: Department of Microbiology, Schaud (24). The procedures for postfixation and New York University School of Medicine, New York, embedding were the same as those previously de- N.Y. 10016. scribed for B. cereus (32). Sections were cut on an 1189 1190 ELLAR, LUNDGREN, AND SLEPECKY J. BACTERIOL. MT-2 Porter-Blum microtome, transferred to carbon- the mesosome contains no additional visible ma- coated copper grids, and poststained with lead hy- terial. The new cross wall begins to appear at the droxide (42). Preparations were examined in a Phillips base of the mesosome, projecting into the cyto- 200 microscope at 80 kv with a 20-,u objective aperture. plasm at right angles to the old cell wall (Fig. 3, Micrographs were made on Kodak (3.25 by 4 inches) 4, the same time, the space at the Projector slide plates (contrast) and enlarged photo- 5, 18, 19). At graphically. Final magnification of the micrographs is base of the mesosome narrows considerably, so included in the figure legends. that the only connection between the contents of the mesosome and the extracytoplasmic region RESULTS is through the 30 to 60 A space which separates In the growth system, after inoculation, the the plasma membrane from the cell wall (Fig. 4). cells divided synchronously for 4 hr. At this time, This stage and the subsequent events leading to sporulation began and was complete by the 10th the completion of the new cross wall are charac- hr after inoculation. Details of growth kinetics terized by an intimate association between are published elsewhere (19). Cells sectioned mesosome vesicles, mesosome membrane, and the during the period of synchronous division were growing edge of the transverse septum. Figure 4 observed to be in various stages of cross wall shows this association to be such that the meso- formation and to possess a varying number of some membrane is folded through 900 to cover mesosomes. the growing edge. At its base, the newly develop- Structure of the mesosome. In B. megaterium, ing transverse septum measures approximately all mesosomes have the same basic structure and 300 to 400 A, which is the same thickness as that originate as an invagination of the plasma mem- of the parent cell wall. At the growing edge, how- brane which subsequently expands into the cyto- ever, the septum is generally only half this thick- plasm. Although they vary in size, all mesosomes ness (Fig. 3, 4). Before the transverse septum is appear to contain vesicular structures 300 to 500 complete, the extension of the plasma membrane A in diameter (Fig. 1-6) which are themselves over the surface of the mesosome fuses, and the bounded by a membrane similar in appearance to final stage of this wall synthesis occurs within the the plasma membrane. In this study, including structure formed by this fusion (Fig. 6). The some of the serial sections, concentric membrane exact point at which this fusion occurs has not lamellae were only rarely seen in the mesosomes; been determined, but it probably occurs early in it would appear, therefore, that the structures development when the new wall has progressed seen are in fact true vesicles. half the distance to the cell center. An exact Cell division and the role of the mesosome. At determination of the time of fusion requires the outset of cell division, a mesosome develops further careful analysis of serial sections, since as a concentric fold of the plasma membrane at what appear to be separate mesosomes are fre- the site of future septum formation (Fig. 1, 2, 5). quently revealed in serial sections to be fused. In cross section this therefore frequently appears After fusion, it is interesting to observe that the as two separate structures. At this point in de- resulting structure is bipartite, with each half dis- velopment, the membranes at the base of the tributed to opposite sides of the developing mesosome are separated by a considerable dis- septum (Fig. 6). The growing region of the trans- tance (2,000 A), and the structure consequently verse septum is half the thickness of the com- appears to be in open connection with the space pleted wall (Fig. 6), and the region of synthesis is between the cell wall and the plasma membrane in intimate contact with the vescular contents of (Fig. 1, 2). As the mesosome enlarges, a second the mesosomes. distinct structure is observed within the mesosome Even after the new transverse septum was con- in addition to the 300 to 500 A membrane-bound tinuous across the cell, it remained only 300 to vesicles. This is a much larger membrane-bound 400 A thick. If two normal daughter cells are to vesicle, approximately 2,000 A in diameter, and is be formed, this new wall must double in thickness filled with granular material (Fig. 1, 2, 5, 6). The at some later stage. Since this organism is sur- chemical composition of this material has not rounded by a thick capsule (Fig. 20, 21), this too been determined. Except for these two structures, must be formed at a later time. An examination FIG. 1. Section ofa cell at 2 hr demoizstratinig the cell wall (cw); plasma membranze (pm); poly-3-hlydroxybuty- rate granule (PHB); nuclear material (ni) arranged in the form ofaxially disposed filaments or fibrils. A layer of densely stained ribosomes surrountds tlhe niucleus anid extentds to the plasma membrane in the form ofstrands (R). Thie mesosome (M) at the site ofseptum formation conitainis 300 to 500 A vesicular structures (vs) together with a 2,000 A vesicle (VS).

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