NIORPHOLOGY OF SPORE DEVELOPMENT 1N CLOSTRIDIUM

Transcription

1 NIORPHOLOGY OF SPORE DEVELOPMENT 1N CLOSTRIDIUM PECTINOVORUJI PHILIP C. FITZ-JAMES Department of Bacteriology and Immunology and Department of Biochemistry, University of Western Ontario, London, Ontario, Canada Received for publication January 17, 1962 ABSTRACT FITZ-JAMES, PHILIP C. (University of Western Ontario, London, Ont., Canada). Morphology of spore development in Clostridium pectinovorum. J. Bacteriol. 84: The process of spore formation in Clostridium pectinovorum was followed by phase-contrast microscopy and by thin-section electron microscopy employing a polyester plastic for embedding. The development of the forespore membrane was found to be similar to that already described for the genus Bacillus, being, in addition, accompanied by considerable cell enlargement. The cortex, as in the bacilli, was found between the apposed layers of the double forespore membrane. The spore coat was laid down in the narrow zone of cytoplasm peripheral to the outer forespore membrane. As these layers formed, striking changes occurred in the fine structure of the spore nuclear material, mesosomes and ribosomes, reflecting the marked alterations in physical environment known to occur in a developing spore. The recent demonstration of membrane involvement in the process of sporulation in the genus Bacillus (Fitz-James, Abstr. 8th Intern. Congr. Microbiol., in press) has prompted a similar study of spore formation in the genus Clostridium. This comparison was undertaken in view of the present incomplete and somewhat confusing description of the sporulation process in the clostridia. In methacrylate-embedded sections, Robinow and Murray (1959; see also, Robinow, 1960) observed a double layer of low density surrounding the spore cytoplasm, but noted that the spore coat was the first dense layer laid down. Hashimoto and Naylor (1958) had already shown that the early stages of sporulation in C. sporogenes do indeed involve membranes, but the mode of formation and final location of the membranes they described differed considerably from that since observed in bacillus cells. As it seemed unlikely that the general process of sporulation would be different in these two genera, and probable that the failure of earlier workers to demonstrate a forespore membrane developing from the plasma membrane might be a matter of technique, the process in clostridia was investigated, using methods now known to resolve membrane structures more clearly. The results indicate that the basic process of sporulation in a member of the genus Clostridium is very similar to that already described for the genus Bacillus. MATERIALS AND METHODS The organism used, C. pectinovorum, was obtained from C. F. Robinow. A medium consisting of yeast extract (0.5%), sodium acetate (0.5%), and glucose (2 %), with and without agar, was used for growth and sporulation, respectively. Sporulation could be made to occur sooner and more synchronously if the broth were diluted with an equal volume of distilled water, either initially or after 12 hr of growth. Dissolved air was removed by boiling and cooling the broth prior to inoculation. Progress of the cultures was observed in wet smears, using the Zeiss dark phase-contrast attachment. The process of sporulation was also followed in sealed cover-slip smears observed over a period of days. Loopfuls of sporulation culture were also mixed, by the method of Mason and Powelson (1956), with small drops of medium containing gelatin (16%) on a warm cover slip, mounted, and examined by dark phase-contrast microscopy. The fixation and embedding procedure used was essentially that of Kellenberger, Ryter, and Sechaud (1958). Washing with uranyl acetate was used with the fixation, and, to insure good penetration of developing spores, samples were shaken while in mixtures 1, 11, and III, and then soaked for 2 to 4 hr through three changes of mixture IV of the Kellenberger et al. (1958) procedure. Other details of electron microscopy have been de- 104

2 VO. 84, 1962 SPORE DEVELOPMENT IN C. PECTINOVORUM1105 FIG. 1. Dark phase-contrast photomicrographs of a sporulating culture of Clostridium pectinovorum mounted in the original culture medium (la) and mixed with an equal volume of 16% gelatin (lb). The small dense bodies clearly seen in the gelatin mount are probably mesosomes. X3,900. scribed previously (Fitz-James, 1960). Most sections were stained with lead by the method of Dalton and Zeigel (1960), the lead acetate solution being filtered through a 0.45, Millipore filter just before use. RESULTS After 30 to 40 hr of incubation at 30 C in the undiluted broth (or for 24 hr in the diluted broth), cell division ceased. The small vegetative cells characteristic of a growing culture now lost their mobility, and, although maintaining, at first, the same cell width, grew about 2.5 times longer. The marked increase in cell size which accompanies spore formation in C. pectinovorum has already been noted (Robinow, 1960). In such long narrow cells, which develop at the end of growth, the initial spore membrane is readily apparent by dark phase-contrast microscopy (Fig. la), and even more so if gelatin is added to the mounting medium (Fig. lb). The relation of this cross wall to the developing spore was followed by continually observing a sealed slide culture (Fig. 2). The subsequent increase in cell width to 1.5 times the initial dimension could also be followed and, in spite of displacement due to slime. production and the continued cell enlargement, the walled-off end of the cell could always be found as the site of subsequent swelling and spore formation. However, regardless of mounting fluid, the density of the cell hides much of this later development from phase-contrast observations. In the electron microscope, various stages of the evolution of the forespore membrane were found to be similar to those already described for members of the genus Bacillus (Fitz-James, 1960). The segregated piece of spore chromatin so clearly depicted in stained preparations by Robinow (1960) was readily found in thin sections as fibrous nuclear bodies enclosed behind a double layer of membrane (Fig. 3). The subsequent extension of this septum of membrane into a forespore membrane was very like the process of bacilli (Fig. 4, 5, 6, and 7). It can be seen from the photomicrographs that the club-shaped swelling of the sporulating end of the cell begins as the forespore outline develops. The completely formed, double-forespore membrane becomes now, as in Bacillus species, the site of the cortex formation, this characteristic

3 AM'tual6....:.... ws u;0xf 1! <~~~~ X..: * 2to7 ar deie frmrcndiiin.b6hrelnainipoedngwtutfutediviin At. 11 h, a bt laer an both... ar ful e atl ntefnlpoorp. X3,900- FIG. 2. Series of phase-contrast photomicrographs showing spore formation in Clostridiunm pectinovorunm. The time (hr) from the start (O) of the observations on this 30-hr culture are indicated to the left of each picture. At 30 hr, some vegetative cells could still be found among the sporulating cells in final stages of division (O time); 0.5 hr later, division of these cells wa-s complete. By 2 hr, they had separated. Cells numbered 2 to 7 are derivedfrom recent divisions. By 6 hr, elongation is proceeding withoutfurther division. At 11 hr, a cross septum is prominent at one endof each cell (cells 4 and 7 are here out of focus), which by 24 hr further differentiated into a developing spore. Later stages3 of spore formation are indicated in other- cells by the arrows. The upper-arrowed cell at O time starts to turn white at llhr. The lower-arrowed cell at O timie follows a bit later, and both are fully refractile in thefinal photograph. X3,

4 PECTIAVOVORUM VOL. 84, 1962 SPORE DEVELOPMENT IN C. 107 Downloaded from FIG. 3. Thin section of a sporulating rod at stages shown in Fig. 1 showing the spore nuclear body (N) trapped behind the apparently completed forespore septum. X 100,000. FIG. 4. Slightly later stage of clostridial sporulation showing further development of the forespore membrane to the points indicated (arrows). Part of a mesosome (M) is seen adjacent to a nuclear body outside the forespore. X95,000. spore layer again appearing between the membrane layers (Fig. 8a). The developing spore coat is again first seen after some cortex formation has already occurred and is first laid down discontinuously, producing the scalloped effect (Fig. 8b) already noted in sections of methacrylateembedded cells (Robinow and Murray, 1959; see also Robinow, 1960). As the cortex formation becomes complete, these dense cell-wall plates develop into a continuous layer (Fig. 8c and 8d), although in ripe spores, multiple and apparently superfluous layers of coat material appear in the sides and end of the spore (Fig. 9 and 10). It should be recalled that a sleeve of cell wall on October 21, 2018 by guest

5 108 FITZ-JAMES J. BACTERIOL. 9 Downloaded from on October 21, 2018 by guest FIG. 5-7.

6 VOL. 84, 1962 SPORE DEVELOPMENT IN C. PECTIXOVORUM109 remains over the spore of this organism when it is separated from the rest of the clostridial cell (Robinow, 1960). Although the cell wall appears relatively structureless on the sporulating cell, the cell wall on the ripe spore can often be resolved into four dense and light layers with an interperiod dimension of about 50 A (Fig. 11). Like the ripening and ripe spores of the genus Bacillus, spores of the clostridia often show imperfections of fixation or embedding or both when cut in thin sections. Cleft defects are particularly common in the outer region of the developing cortex (Fig. 8b, 10, 11, 12, and 13). When closely examined (Fig. 8b), this cleavage is seen to lie between the two dense lines making up the outer forespore membrane in a zone which, in light of recent studies of membrane ultrastructure, is now considered as the site of the fatty acid chains of the bimolecular leaflet of membrane (Stoeckenius, Circulation, in press). With the development of complete refractility, as with the spores of the genus Bacillus, the sections show a low affinity for osmium; ribosomes, mesosomes, and nuclear material become practically invisible (Fig. 9). Moreover, these structures, particularly the ribosomes, can be only faintly located when the sections are stained with lead (Fig. 10 and 11; compare with Fig. 8d) or if the cut sections are floated onto osmium solution. In spite of extra embedding precautions, however, there was, as is usual, considerable evidence of poor penetration of the polyester monomer into the ripe spore. Nevertheless, this procedure seemed equal, if not superior, to methods employing methaerylate (Fitz-James, 1959; Robinow, 1960). In addition to the above loss of ribosome staining, two other changes were of interest in the developing spore. The first change is in the fine structure of the chromatin bodies. During the late vegetative stage and the stages of forespore formation, the fibrous material of the nuclear bodies appears as a randomly arranged and open fibrous network which is often branched (Fig. 3, 4, 5, 7, and 14c). Then, during the stage of cortex formation, these nuclear fibers become much more orderly and appear to run parallel to the long axis of the nuclear body (Fig. 8a, 12, and 13). Branching parts can still be seen, but the over-all structure appears to be drawn out throughout the spore. In Feulgen or HCl-Giemsa preparations made at this stage, the entire nuclear body is displayed as a straight or wavy cord (Robinow, 1960). The nuclear material in that l)art of the clostridial cell outside the spore area possesses the more randomly arranged fibrous l)attern. The nuclear material of the resting spore appears in a low-density area with no definite fibrous structure despite heavy-metal "staining" (Fig. 10). In photomicrographs of uranyl-stained nuclear or deoxyribonucleic acid (DNA) fibers such as those shown in Fig. 12 and 13, the fibers, although often superiml)osed, could in some areas be seen as small wavy segments 20 to 25 A wide and of variable length (Fig. 13, insert). Such dimensions, although near the limit of resolution with the methods and equipment used, are in agreement with those recently found on isolated DNA molecules (Stoeckenius, 1961). Since the above differences in fibrous areas of spore nuclear bodies might possibly be those of a functional or duplicating (that is, open, randomly arranged fibers) and nonduplicating (parallel fibers) DNA, a study was made of the cell content of DNA in a culture of synchronously sporulating FIG. 5. Developing forespore membrane partly proliferated around the sporullating end of the cell (small arrows) and maintaining, at all times, its double structure. The uranyl acetate stains the wide central zone formed between the two apposing membranes rather deeply here and in many areas obscures the two main dense lines of the component membranes. At the site of the apposing arrows, however, these can be discerned. X 100,000. FIG. 6. Enlargement of part of the cell as the forespore membrane proliferates towards the cell end. The arrows mark the site of juncture of the forespore membrane with the plasma mnembrane on either side of the cell. Approx. X40,000. FIG. 7. Totally enclosed forespore, showing what may be the beginning of the formation of the cortex. The ribosomes in the forespore are still as heavily stained with lead as those without. The occluded iiesosomies are quite dense and now appear to be somewhat more compact than those (M) seen outside the spore. The low density masses in the sporangium are the sites of polysaccharide material. Strands of extracellular slime can be detected in the background of the original photomicrograph. X95,000.

7 4A R. W., 0. n rx JF X. W." W ".ft ltrll3w. A T', Bc FIG. 8. Cortex and spore coat formation in Clostridium pectinovorum stained with lead. (a) The formation of the cortex (C) between the inner (IM) and outer (OM) forespore membranes. The cytoplasmic membrane (CM) is seen under the cell wall. In the narrow zone of cytoplasm between the cytoplasmic and outer forespore membrane, small areas of spore coat (SpC) can be seen. Part of a nuclear (N) body is seen. By this time, the intraspore mesosomes (M) are barely detectable. X142,000. (b) A later stage, showing more advanced spore coat formation. The outer membrane (OM) is imperfectly preserved and shows a cleft separating its two dense lines. The inner membrane (IM) and mesosomes (M) are both faintly seen. The typical scallops of spore coat stand out. X142,000. (c) A better preserved section similar to that in Fig. 8b but stained more heavily with lead, showing more sharply the cortex (C) forming between the outer and inner forespore membranes, as well as further growth of the markedly dense spore coat. X 142,000. (d) The spore coat is now continuous, but refractility is as yet incomplete. The cortex (C) is poorly differentiated, but the ribosomes in the spore still stain with lead. X110, A WA-A M.. A

8 9 FIG. 9. Section of a fully refractile clostridial spore. Not lead stained. The residual cell wall, itself doublelayered here, (CW, arrow) is seen overlying what appear to be multilayers of plasma membrane. The spore coat is quite thin in this part of the spore section. The cortex, underlying cytoplasm, and inner membrane layer show the low density usual in such osmium-fixed and uranyl-soaked ripe spore sections. X95,000. FIG. 10 and 11. Sections of fully refractile clostridial spores. Lead staining has faintly revealed some of the internal structures. Ribosomes are faint (compare with Fig. 8d). However, the site of the remnant outer membrane (OM), the cortex, and the inner membrane (IM), although not highly contrasted, are readily detectable. In Fig. 10, the spore coat has, in places, doubled and appears to be accompanied by a similar proliferation of membranelike lamellae. In Fig. 11, these wall or membrane proliferations fill the neck region of the sporangial remnant. In places, the remnant cell wall (CW) is resolved into layers. Within the spore, the site of the inner layer (IM) of the forespore double membrane can be seen and a faint profile of an attached mesosome (M) was detectable in the original photomicrograph. The lower and absent parts of the section are torn. X95,

9 FIG. 12. Cortex being formed between the double layers of the forespore membrane. As is often found at this stage, preservation of this region is poor. The cytoplasmic granules (ribosomes) stain well and the more parallel array of the nuclear fibers is clearly seen. The occluided mesosomes (M) become less prominent. A few specks of spore coat can be seen. X95,000. FIG. 13. Despite the poor preservation of the area around the spore, this photomicrograph shows good preservation of the spore nuclear body and part of a mesosome (M). Scallops of spore coat are developing around the periphery (X95,000). Although largely superimposed the fibers of the nuclear body at higher magnification (insert; X175,000 marker 0.1 At) often shown wavelike profiles (arrows). 112

10 VOL. 84, 1962 SPORE DEVELOPMENT IN C. PECTINOVORUM 113 E$.> E R i Kfi -14a. FIG. 14. Appearance of mesosomes within the completed forespores of Clostridium pectinovorum. All "stained" with lead after osmium-uranyl fixation. The close membranous type of structure seen in completed forespores (14a) is replaced by more densly staining and possibly more compact structures as the cortex is formed (14b and c). In 14b part of a peri-sporal mesosome (M) is seen. In 14c the nuclear fibers have still the miore disorganized array. X95,000. C. pectinovorum. It was found that, after the end of growth, as with many Bacillus cultures (Young and Fitz-James, 1959), synthesis of DNA ceased, and its level remained constant throughout spore formation. Hence, the marked change in fibrous structure does not reflect a change in the state of DNA replication. Another structural change of interest during sporulation, and one which coincided in part with the above change in nuclear structure, was that observed in the intraspore membranous organelles or mesosomes. The mesosomes seen in the early forespore of C. pectinovorum, like those accompanying the spore chromatin in bacilli (Fitz-James, 1960), were initially (Fig. 3) very similar in structure to the perisporal mesosomes (Fig. 4, 7, and 14b) and those seen during cell division in this species. With the completion of forespore formation, they appeared much more compact (Fig. 14a) and were often found as highly compressed electron-dense (presumably osmiophilic) masses (Fig. 14b and 14c). As the cortex was formed they became much less prominent, yet still appeared to be made up of closely compressed lamellae (Fig. 8a, 8b, 12, and 13). In the ripe spore they were almost invisible despite lead staining (Fig. 11). DISCUSSION From this and preceding studies (Fitz-James, 1960; Young and Fitz-James, 1962), it becomes obvious that the process of spore formation is a unique type of membrane-dependent, internal cell division generally similar in all species. A peculiar infolding of membrane leads to the formation of a double membrane structure, the

11 114 FITZ-JAMES J. BACTERIOL. forespore. When this is complete, there follows the formation of two final spore layers, the cortex and the spore coat. The cortex, developing between the layers of the forespore, binds the spore protoplast and can be considered as a specialized type of cell wall. Indeed, structural and chemical studies of normal and cortexless mutants of B. cereus reveal that a-e-diamino-pimelic acid (DAP) containing spore peptide is bound in the cortex to other antigenically unique components (Fitz-James, in press). Similarly, Murrell (personal communication) has found that cortical structure disappears as DAP is extracted from isolated spore coats by lysozyme digestion. The spore coat in both the clostridia and the bacilli is formed peripheral to the outer forespore membrane in what is often a narrow zone of cytoplasm between the outer forespore membrane and the plasma membrane. Either of these two membrane layers or membranous organelles (perisporal mesosomes) connected to the plasma membrane appear, morphologically, to be intimately associated with this spore-coat formation. With the completion of these syntheses, the spore (now white in dark phase-contrast) undergoes a final ripening process and, with full refractility, takes on the characteristics associated with the term "resting spore." ACKNOWLEDGMENTS This study was supported by a term grant from the Medical Research Council of Canada. The author is grateful to Doryth Loewy for her careful technical assistance. LITERATURE CITED DALTON, A. J., AND R. F. ZEIGEL A simplified method of staining thin sections of biological material with lead hydroxide for electron microscopy. J. Biophys. Biochem. Cytol. 7: FITZ-JAMES, P. C Cytological comparison of spores of different strains of Bacillus megaterium. J. Bacteriol. 78: FITZ-JAMES, P. C Participation of the cytoplasmic membrane in the growth and spore formation of bacilli. J. Biophys. Biochem. Cytol. 8: HASHIMOTO, T., AND H. B. NAYLOR Studies of the fine structure of microorganisms. II. Electron microscopic studies on sporulation of Clostridium sporogenes. J. Bacteriol. 75: KELLENBERGER, E., A. RYTER, AND J. SfCHAUD Electron microscope studv of DNAcontaining plasms. II. Vegetative and mature phage DNA as compared with normal bacterial nucleoids in different physiological states. J. Biophys. Biochem. Cytol. 4: MASON, D. J., AND D. M. POWELSON Nuclear division as observed in live bacteria by a new technique. J. Bacteriol. 71: ROBINOW, C. F., AND R. G. E. MURRAY Cytological observations on the process of sporulation in Clostridium pectinovorunm. Can. Soc. Microbiol. 9th Ann. Meeting. Abstracts, p. 7. ROBINOW, C. F Morphology of bacterial spores, their development and germination, p In I. C. Gunsalus and R. Y. Stanier [ed.], The bacteria. Academic Press, Inc., New York. STOECKENIUS, W Electron microscopy of DNA molecules stained with heavy metal salts. J. Biophys. Biochem. Cytol. 11: YOUNG, I. E., AND P. C. FITZ-JAMES Chemical and morphological studies of bacterial spore formation. II. Formation of spores in Bacillus cereus. J. Biophys. Biochem. Cytol. 6: YOUNG, I. E., AND P. C. FITZ-JAMES Chemical and morphological studies of bacterial spore formation. IV. The development of refractility. J. Cell Biol. 12: