Cell Division in a Species of Erwinia

Transcription

1 JOURNAL OF BACTERIOLOGY, Oct., 1965 Vol. 90, No. 4 Copyright 1965 American Society for Microbiology Printed in U.S.A. Cell Division in a Species of Erwinia IX. Electron Microscopy of Normally Dividing Cells E. A. GRULA AND GERALI) L. SMITH Department of Microbiology, Oklahoma State University, Stillwater, Oklahoma Received for publication 17 May 1965 ABSTRACT GLIULA, E. A. (Oklahoma State University, Stillwater), AND GERAD) L. SMIITH. Cell division in a species of Erwinia. IX. Electroni microscopy of normally dividing cells. J. Bacteriol. 90: Cells of an Erwinia species (an Enlterobacteriaceae) divide by conicomitant inivaginiation of the cell wall and membranie. It is concluded that the process is iniitiated and( sustainied b)y the cell membranie. The D isomer of serine inhibits cell division in a species of Erwinia (Grula, 1960). This study, utilizing electron microscopy of thin-sectioned bacteria, was initiate(d to p)rovide infoormation relative to the morphological processes op)erating in normally grown cells. Of primary iml)ortance was the question: which structure, wall or memiibrane, initiates the cell division process' The role of the cell wall and cell miiembrane during division in Enterobacteriaceae is not clear. Conti and Gettner (1962) observed invagination of both structures during division in Escherichia coli, and concluded that cell div-ision was the result of the centripetal growth of the cell wall. Their conclusion can be contrasted with that of Cota-Robles (1963), vho presented evidence showing that membrane invagination could occur in the absence of cell-wall invag-ination in E. (oli B. MATEItIALS AND MIETHODS Cells were grown for 16 hr with shaking at 25 C in a defined medium (Grula, 1960) with manniose as the carbon-energy source. After growth, cells were prefixed directly in the growth medium by use of a final concenitrationi of 0.15%/" osmium tetroxide for 1 hr at 25 C. After centrifugatiotn, the cells were fixed for an additional 12 hr in 2 ml of 1%,c osmium tetroxide in \eronial buffer (ph 6.1) under RK+ conditions (Ryter and Kellenberger, 1958) at 25 C. Cells were then washed three times in Veronal buffer and postfixed in 0.5% uranyl acetate in Veronial buffer (ph 3.5) for 4 hr at 25 C (Scchreil. 1964). The cells were again washed three times in Veronal buffer atid embedded in agar blocks. The agar blocks were dehydrated through an ethyl alcohol series (25 to 100%), treated with three changes of propylene oxide (10-miin treatments), and placed in an equal mixture of propylene oxidecomplete Epon 812 resin monomer for 2 hr. Another equal part of complete resin monomer was thein added, mixed, aind allowed to inifiltrate overiiight. The Epon 812 monomer was prepared aecordinig to the method of Luft (1961) aind was co)mpounded by use of 4.5 parts of mixture A to 5.5 parts of mixture B. Gelatin capsules (size 00) were filled with fresh monomer, aiid the blocks were placed oin the surface anid allowed to settle to the bottom of the capsules. The blocks were then polymerized for 36 hr at 60 C. Sections were cut oni a Porter-Blum MT-2 microtome and transferred to uncoated 400-mesh copper grids. They were then stainied in a saturated solution of lead acetate in absolute methanol for 15 min at room temperature, and were washed in three changes of absolute methanol and three changes of distilled water. Preparations were examined by use of an RCA EMU-3G electron microscope with an acceleratinig voltage of 100 kv. RESULTS ANI) DISCUSSION Our electron micrographs (Fig. 1-6) document the normal division process in Erwinia species. The cell wall and membrane invaginate together. The cell membrane does not invaginate alone; leaving the cell wall at the periphery of the cell as shown by Chapman (1959) with an unidentified bacterium. A judgment cannot be absolute, since the cell wall and membrane invaginate together; however, we favor the conclusion that the membrane initiates and sustains the plrocess. Plertinent facts leading to this conclusion can be summarized as follows. (i) Excellent division activity occurs in these cells in the presence of division inhibitors under osmotically controlled conditions or in the presence of pantoyl lactone, even though (lamage system to the cell-wall mucopelptide-synthesizing is not repaired (Grula and Grula, 1964). (ii) Cells of the type shown in Fig. 7 have been observed in some of our preparations. The large internal vacuolar area seen in this cell is bounded 1054

2 VOL. 90, 1965 ELECTRON MICROSCOPY OF CELL DIVISION 1055 ::. '...: ::...::..: f..... I 2 a t.~.ss..'mi..t FIG Normal division sequence in Erwinia sp. to demonstrate concomitant invagination of the cell wall and cell membrane. X 44,600.

3 1056 GRULA AND SMITH J. BACTERIOL. M FIG Continuation of the division sequence shown in Fig by a cell membrane that has completed an invagination in the absence of association with the cell wall. This photograph is similar to some presented by Cota-Robles (1963). (iii) As shown in Fig. 8, thin sectioning of an autolyzed cell reveals that the invaginated cell membrane maintains its position even though the cell wall has been badly torn in the division area. If the cell wall exerts

4 VOL. 90, 1965 ELECTRON MICROSCOPY OF CELL DIVISION nmmmij Downloaded from on October 13, 2018 by guest 9 :!!''' -itet-'"'sa tm;ss,a::,t FIG. 7. Completed invagination of the cell membrane in the absence of complete cell-wall invagination. X 46,500. FIG. 8. Division activity observed in a partially autolyzed cell. Membrane invagination is clearly evident and the membrane maintains its invaginated position even though the cell wall has been partially torn away. X 51,800. FIG. 9. Initiation of cell division prior to completion of nuclear division. X 46,500.

5 1058 GRULA AND SMITH J. BACTERIOL. pressure, to push the membrane ahead of it, then the cell membrane probably would not maintain the invaginated appearance shown in this picture. (iv) The observations of Martin (1964) are also important in assessing the role of the cell wall and membrane in the division process. He reported that protoplasts of Proteus mirabilis (completelv devoid of cell wall) could grow and divide indefinitely. Figure 9 is included to demonstrate that nuclear division need not be completed prior to initiation of cell division (also evident in Fig. 3). Apparently, at least two types of division occur in bacteria, and both are initiated by the cell membrane. One type involves invagination of the cell membrane, leaving the cell wall at the periphery of the cell. The completed septum is synthesized after the invaginated membrane lays down new cell-wall material and, in this way, extends the cell wall into the division area (Chapman, 1959). The reason for failure of the wall to directly follow the membrane in this type division is not known, but it may be due to the bulky and rigid nature of the structure, or there may be a lack of cohesiveness between the two structures. The other type of division, documented in this study, establishes that, under normal conditions, invagination of the cell wall and membrane occurs simultaneously. ACKNOWLEDGMENTS We wish to acknowledge technical aid by Kenneth Richter during the early phases of this study. This investigation was supported by research grant GB-1532 from the National Science Foundation, by Public Health Service research grant Al from the National Institute of Allergy and Infectious Diseases, and by Public Health Service research career program award GM-13,968 from the Division of General Medical Sciences. LITERATURE CITED CHAPMAN, G. B Electron microscope observations on the behavior of the bacterial cytoplasmic membrane during cellular division. J. Biophys. Biochem. Cytol. 6: CONTI, S. F., AND M. E. GETTNER Electron microscopy of cellular division in Escherichia coli. J. Bacteriol. 83: COTA-ROBLES, E. H Electron microscopy of plasmolysis in Escherichia coli. J. Bacteriol. 85: GRULA, E. A Cell division in a species of Erwinia. Inhibition of division by D-amino acids. J. Bacteriol. 80: GRULA, E. A., AND M. M. GRULA Cell division in a species of Erwinia. VII. Amino sugar content of dividing and nondividing cells. Biochem. Biopys. Res. Commun. 17: LUFT, J. H Improvements in epoxy resin embedding methods. J. Biophys. Biochem. Cytol. 9: MARTIN, H. H Composition of the mucopolymer in cell walls of the unstable and stable L-form of Proteus mirabilis. J. Gen. Microbiol. 36: RYTER, A., AND E. KELLENBERGER etude au microscope 6lectronique de plasmas contenant de l'acide desoxyribon-nucleique. I. Les nucl6oides des bact6ries en croissance active. Z. Naturforsch. 13: SCHREIL, W. H Studies on the fixation of artificial and bacterial DNA plasms for the electron microscopy of thin sections. J. Cell Biol. 22:1-20.