Further Electron Microscope Characterization of Spore Appendages of Clostridium bifermentans

Transcription

1 JOURNAL OF BACTERIOLOGY, Jan. 1968, p Copyright (e 1968 American Society for Microbiology Vol. 95, No. I Prinited in U.S.A. Further Electron Microscope Characterization of Spore Appendages of Clostridium bifermentans DIANE P. YOLTON, LEODOCIA POPE, M. GLENN WILLIAMS, AND L. J. RODE Departmentt of Microbiology anid Electron Microscope Laboratory, The Unliversity of Texas, Austini, Texas Received for publication 14 September 1967 Two distinct spore appendage types of Clostridium bijermentans, a pinlike appendage and a tubular appendage, were studied by electron microscopy. The pinlike appendage is characterized by a shaft, about 100 A in diameter, which has a lobed caplike structure. The tubular appendage, 500 to 600 A in diameter, is characterized by a hirsute region consisting of small filaments or fibrils. Gross morphology and ultrastructural features of both types are described. Recently, we reported the occurrence of four distinct spore appendage types among 12 strains of Clostridium bifermentans examined (9). In that report, we characterized two of the appendage types, one a smooth tubular appendage (strain 9-SDH) and the other a featherlike appendage (strain 1A-SDH). In the present report, we characterize the remaining two appendage types, a pinlike appendage (strain U-11) and a hirsute tubular appendage (strain FDA-1). In addition, some ultrastructural features of the previously reported smooth tubular appendage of strain 9-SDH are described. MATERIALS AND METHODS The experimental approach and procedures used in the present investigation were identical with those detailed in a previous report (9), and thus only an abbreviated account of these methods is given here. Three strains of C. bifermenttanis were used. Strains U-li and FDA-i were received from Stanley M. Harmon, Food and Drug Administration, Washington, D.C., and strain 9-SDH was obtained from Mary Jane Ashby, Texas State Department of Health. Spores were produced on Brain Heart Infusion (Difco) medium containing sodium thioglycolate and 2%/to agar. Carbon replicas (replicas) were prepared according to conventional means (1l, 12), with platinum shadowing at approximately 30. Sodium phosphotungstate (PTA), 2(/C at ph 6.0, and uranyl acetate, 0.5% at ph 3.5, were employed for negative stains. Specimens were fixed in 0.5% glutaraldehyde at 4 C (13), followed by routine Kellenberger osmium fixation at 4 C (6). They were then embedded in a plastic mixture of 70c% dodecenyl succinic anhydride, 20%;' Araldite 6005, and 10%, Epon 812, with drop of DMP-30 per ml of plastic. Sections were cut on a Sorvall Porter-Blum MT-2 microtome, and were stained with uranyl acetate or lead citrate, or with both (10). 231 Electron microscopy was accomplished with Hitachi HS-7S and the RCA EMU-3G microscopes. Initial magnifications ranged from 8,300 to 60,000. Micrographs were taken on Dupont Cronar Ortho S Litho film or on Kodak projector slides, contrast grade. RESULTS Pinlike appendages of strain U-11. Free spores of this strain, viewed as replicas, are characterized by a prominent exosporium and numerous (100 or more) small appendages, visible on the spore surface as flaccid structures and projecting from the spore periphery as pinlike protrusions (Fig. 1). The individual appendages may have a length of up to 0.3,u (Fig. 2), but probably are longer. Each appendage has a cap which appears lobed, and a shaft which possesses ultrastructure (Fig. 2). In some cases, the shaft ultrastructure proximal to the cap appears to differ from that found along the remainder of the shaft (Fig. 2). Negative stains (Fig. 3 and 4) revealed that the appendage shafts have a diameter of approximately 90 A (smaller than most bacterial flagella) and are comprised of spherical units, 40 to 50 A in diameter, which may be arranged in helical fashion (Fig. 4). The repeating pattern, along the length of the shaft, has a periodicity of 45 to 55 A. No shaft lumen is apparent, and the shafts are not considered to be tubular in the usual sense. The terminal cap has a width of 300 to 350 A, and the cap lobes, which measure approximately 100 A, appear to consist of aggregates of spherical units similar to those comprising the shaft (Fig. 4). Each cap appears to have two or more lobes (Fig. 2 and 4). Pinlike appendages were not detected in sections of sporangia (Fig. 5) due to their small size and to the masking effect of cytoplasmic constit-

2 232 YOLTON ET AL. J. BACTERIOL. FIG Strain U-l1, free spores, replicas. FIG. 1. The spore body (B) is enclosed in a prominent exosporium (E), and the pinlike appendages are visible at the spore periphery and on the spore surface (unlabeled arrow). X 53,000. FIG. 2. Each appendage is capped by a lobed structure (C) and the shiaft (S) has ultrastructure w.',ich appzars as cross striations. X 111,000. uents and cellular inclusions. Pinlike appendages are readily detected, however, in sections of free spores (Fig. 6 and 7). In these cases, they project outward most notably from the exosporium, giving the impression that they originate in that structure (Fig. 6). The question of whether the appendages have a coat origin or exosporium origin has not been resolved, but it is certain that they do project beyond the exosporium (Fig. 6 and 7). From spatial considerations, it seems certain that the pinlike appendages are not erect in the sporangial environment. Hirsute tubular appendages of strain FDA-1. Free spores of this strain, viewed as replicas, exhibit an exosporium and prominent appendages which penetrate and extend beyond the exosporium boundary (Fig. 8). All the appendages originate on the spore coat surface FIG Strain U-li. Fig. 3 and 4, negative stains with uranyl acetate; Fig. 5-7, sections; Fig. 5, post-stained with lead citrate; Fig. 6 and 7, post-stained with uranyl acetate, then lead citrate. FIG. 3. Ultrastructural features of pinlike appendages. X 165,000. FIG. 4. Each appendage consists ofa shaft (S), made up of spherical subunits (unlabeled arrow), and a cap (C), which is lobed (L). X 420,000. FIG. 5. Typical sporangium with cellular inclusions (I) and exosporium (E). Appendages are not apparent. X 47,000. FIG. 6. Free spore with numerous appendages (A) projecting from the exosporium (E). X 43,000. FIG. 7. Like Fig. 6. X 69,000.

3 VOL. 95, 1968 SPORE APPENDAGES OF C. BIFERMENTANS 233.k

4 234 YOLTON ET AL. J. BACTERIOL. r hl:;,~,- FIG Strain FDA-I, replicas. FIG. 8. Free spore, showing spore body (B), enveloping exosporium (E), hirsute tubular appendages (A), and the origin site (0) of the appendages. X 34,000. FIG. 9. Single hirsute tubular appendage showing the naked shaft region (S) and the hirsute region (H). X 66,000. FIG. 10. Four representative views ofthe coat origin site ofthe hirsute tubular appendages. X 38,000. from a common site, usually as three clusters of parallel tubes (Fig. 8 and 10). The number of appendages per cluster is variable, ranging from 1 to 5 in the examples shown (Fig. 10). Each appendage consists of a central shaft, which is naked for a distance proximal to its coat origin, but hirsute over about half its length (Fig. 9) Ėvidence for the tubular nature of the appendage shaft, obtained with negative stains, is shown in Fig. 11. Shaft diameter varies from 450 to 600 A in such a preparation, and the tube wall may approach 100 A in thickness. The hirsute region consists of innumerable filaments or fibrils, not well resolved here, which project angularly from the tubular shaft outer surface (Fig. 12). The tubular shaft has ultrastructure (Fig. 12) which appears similar to that of the smooth tubular appendages of strain 9-SDH (see also Fig. 18 and 19). FIG Strain FDA-I. Fig. 11 and 12, negative stains with PTA; Fig , sections, lead citrate poststain. FIG. I 1. Typical cluster offour appendages which originates at the spore and penetrates the exosporium (E). The tubular nature and hirsute region of the appendages are apparent. X 75,000. FIG. 12. Enlarged view of hirsute tubular appendages. X 135,000. FIG. 13. Sporangium showing spore body (B), surrounding exosporium (E), and hirsute tubular appendages, both within and external to the exosporium (unlabeled arrows). X 62,000. FIG. 14. Longitudinal section through free spore showing the attachment of three clusters ofaopendages (unlabeled arrows) to the coat (C) within the enveloping exosporium (E). X 39,000. FIG. 15. Section through free spore showing seven hirsute tubular appendages, in cross section, enclosed within the exosporium. X 40,000. FIG. 16. Cross section of three hirsute tubular appendages. X 237,000.

5 VOL. 95, 1968 SPORE APPENDAGES OF C. BIFERMENTANS 235 (14) (in) Q6

6 236 YOLTON ET AL. J. BACTERIOL. Sections of sporangia reveal appendages enclosed within the exosporium and also within the sporangial cytoplasm external to this structure (Fig. 13). Clear evidence for a "natural" exosporium opening(s) to accommodate this penetration has not been obtained (9). Cross sections through appendages within sporangia appear as opaque tubes surrounded by zones of electron-transparent (hirsute) material (Fig. 13). Within sporangia, the contrast between sections through the naked shaft region of appendages and sections through the hirsute shaft region of appendages is apparent (Fig. 13). Supporting evidence for the coat origin of the hirsute tubular appendages has been obtained with sections of free spores (Fig. 14), and these data complement the replica data presented previously (Fig. 8 and 10). Cross sections through free appendages show substructure in the tube walls (Fig. 16). Approximately 30 of these subunits constitute the tube circumference. Smooth tubular appendages ofstrain 9-SDH. The appendages of strain 9-SDH, described in a previous report (9), project from one or both ends of the spore (Fig. 17) and lack the hirsute region of strain FDA-1. Although a description of an exosporium with ultrastructure consisting of hexagonally-arranged repeating units (1, 2) was included in that report (9), ultrastructural features of the tubular appendages were not then resolved. Further experimentation has now revealed a contrasting ultrastructure for the exosporium and the tubular appendages of strain 9-SDH (Fig. 18). Negative stain appendage preparations (Fig. 18 and 19) suggest that spherical subunits are arranged in rectangular patterns. The linear arrangement results in a beaded appearance, which is most noticeable at the appendage margin (Fig. 19). The flattened appendages in Fig. 18 and 19 measure approximately 800 A in width. DIscussIoN The pinlike appendages of strain U-11 (Fig. 1-7) are new structures, not previously reported. They bear a superficial resemblance to the tubular appendages of C. botulinum type E spores (3, 4); both are small, possess caplike structures, and are more or less randomly distributed over the entire spore surface. The pinlike appendages of C. bifermentans U-11 differ, however, in important respects. Contrasted to the appendages of C. botulinum type E spores, those of C. bifermentans U-11 have a shaft of only half the diameter, and the ultrastructure of both the shaft and cap are different. There is no obvious shaft lumen and the appendages are not considered to be tubular. In addition, the appendages of strain U-11 are not confined within the exosporium. Appendages which resemble the hirsute tubular appendages of strain FDA-1 have been reported by Hodgkiss, Ordal, and Cann (5) for a strain of C. bifermentans. We assume the two appendage types are identical although there is insufficient information at hand to permit a definite conclusion. The hirsute tubular appendages of C. bifermentans strain FDA-1 differ from the smooth tubular appendages of C. bifermentans 9-SDH (9; Fig ) in several respects: possession of a hirsute region, a larger number of appendages per spore, and a characteristic attachment site to the spore coat (Fig. 8, 10, and 14) which was not observed for strain 9-SDH (9). Smooth tubular appendages resembling those of C. bifermentans 9-SDH (9; Fig ) have been reported by Hodgkiss, Ordal, and Cann (4, 5) for "OS mutants" of C. botulinum type E spores, and also for spores of the C. bifermentans- C. sordelli group (5). In addition, a recent report by Krasil'nikov and Duda (8), as well as earlier reports by the Russian group, may include sporeappendage types similar to those described by us (9; this report) for C. bifermentans. Hodgkiss, Ordal and Cann (5), on the basis of cumulative studies (3-5), reported that a constant feature of spore appendages is their microtubular nature. We have not found this to be the case with the ribbonlike spore appendages of Clostridium sp. Ni (C. taeniosporum nov. sp.; Duda, personal communication) described by Rode, Crawford, and Williams (11). Neither the featherlike appendages of C. bifermentans 1A- SDH (9) nor the pinlike appendages of C. bifermentans U-11 (Fig. 1-7) appear to be tubular. Krasil'nikov, Duda, and Sokolov (7) consider that, in the genus Clostridium, the morphology of spores is a species-specific character. Our findings (9; this report) indicated at least five distinct morphological spore types, including one which lacks appendages, in 12 strains of C. bifermentans examined. The implications of these findings for the classification of clostridia seem apparent. As we (9, 11) and others (5) have indicated, the functional role of spore appendages remains obscure. ACKNOWLEDGMENTS We would like to thank Patsy B. Templeton for capable technical assistance, and Stanley M. Harmon and Mary Jane Ashby for the cultures used in this investigation. This investigation was supported by Public Health

7 VOL. 95, 1968 SPORE APPENDAGES OF C. BIFERMENTANS 237 FIG Strain 9-SDH. Fig. 17, replica; Fig. 18 and 19, negative stains with PTA. FIG. 17. Free spore with two smooth tubular appendages. X 13,500. FIG. 18. Contrasting ultrastructure ofthe exosporium (E) and smooth tubular appendage (A). X 152,000. FIG. 19. Enlarged view of segment of smooth tubular appendage. X 240,000. Service grant AI from the National Institute of Allergy and Infectious Diseases and by Contract Nonr 375(12) from the Office of Naval Research. LITERATURE CITED 1. ABRAM, D. 1'966. Differentiation in cells of Bacillus circulans Q 19 during spore formation, p In Electron Microscopy II. Marucen Company, Ltd., Tokyo, Japan. 2. GERHARDT, P., AND E. RIBI Ultrastructure of the exosporium enveloping spores of Bacillus cereus. J. Bacteriol. 88: HODGKISS, W., AND Z. J. ORDAL Morphology of the spore of some strains of Clostridium botulinum type E. J. Bacteriol. 91: HODGKISS, W., Z. J. ORDAL, AND D. C. CANN The comparative morphology of the spores of Clostridium botulinum type E and the spores of the "OS mutant." Can. J. Microbiol. 12:

8 238 YOLTON ET AL. J. BACTERIOL. 5. HODGKISS, W., Z. J. ORDAL, AND D. C. CANN The morphology and ultrastructure of the spore and exosporium of some Clostridiumn species. J. Gen. Microbiol. 47: KELLENBERGER, E., A. R. RYTER, AND J. SECHAUD Electron microscope study of DNAcontaining plasms. II. Vegetative and mature phage DNA as compared with normal bacterial nucleoids in different physiological states. J. Biophys. Biochem. Cytol. 4: KRASIL'NIKOV, N. A., V. I. DUDA, AND A. A. SOKOLOV Protrusions on the surface of spores of anaerobic bacteria of the genus Clostridium. Microbiology (USSR) (English Transl.) 33: KRASIL'NIKOV, N. A., AND V. I. DUDA The ultrastructure of outgrowths on the surface of spores of anaerobic bacteria. Proc. Acad. Sci. USSR, Microbiol. Sect. (English Transl.) 171: POPE, L., D. P. YOLTON, AND L. J. RODE Appendages of Clostridium bifermenitans spores. J. Bacteriol. 94: REYNOLDS, E. S The use of lead citrate at high ph as an electron-opaque stain in electron microscopy. J. Cell Biol. 17: RODE, L. J., M. A. CRAWFORD, AND M. G. WIL- LIAMS Clostridium spores with ribbonlike appendages. J. Bacteriol. 93: RODE, L. J., AND M. G. WILLIAMS Utility of sodium hypochlorite for ultrastructure study of bacterial spore integuments. J. Bacteriol. 92: SABATINI, D. D., F. MILLER, AND R. J. BARRNETr Aldehyde fixation for morphological and enzyme histochemical studies with the electron microscope. J. Histochem. Cytochem. 12:57-71.