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A unique structure in microcysts of Myxococcus xanthus
© 1968 by Academic Press Inc..
378
J. U L T R A S T R U C T U R E RESEARCH
21, 378-382 (1968)
A Unique Structure in Microcysts of Myxococcus xanthus 1 KAREN BACON2 AND F. A. EISERLING
Department of Bacteriology, University of California, Los Angeles, California 90024 Received August 8, 1967 The morphogenesis of Myxococcus xanthus from a vegetative rod to a spherical microcyst can be induced by the addition of glycerol to the vegetative growth medium. Thin sections of microcysts preserved 5 hours after induction revealed a new vesicular structure apparently between the cell envelope and microcyst capsule. This unusual structure is not seen in vegetative cells.
Myxococcus xanthus FB (2) can undergo morphogenesis f r o m a vegetative rod to a spherical, refractile microcyst. This change can be synchronously induced in liquid medium by the addition of glycerol to a vegetative culture (3). The rods progressively shorten and thicken to f o r m spheres in 120 minutes. Thereafter the cells acquire refractility and remain metabolically active for several hours before becoming resting cells. As a complement to a continuing investigation of the biochemical events occurring during microcyst formation (1, 4), we have begun to examine this process with the electron microscope. In this preliminary report we present micrographs of microcysts harvested 5 hours after glycerol induction. A vesicular structure is demonstrated which is unique to the microcyst and has not previously been described in the literature. MATERIALS A N D METHODS Vegetative cells of M. xanthus FB were grown in a casein hydrolyzate medium (2 % N - Z Case, Sheffield Chemical, Norwich, New York, and 0.1% MgSO4-7 H~O) (1). The cells were incubated in 40-ml quantities in 250-ml flasks at 30°C with shaking. Microcyst formation was induced by a modification of a procedure described by Dworkin (3). Glycerol (10 M) was added directly to exponentially growing cells (optical density at 560 m~ of 0.5 or less) to a final concentration of 0.5 M, and the induced culture was incubated as 1 This investigation was supported by National Science Foundation Grant GB-6155. Recipient of a USPHS predoctoral fellowship, Grant GM-30, 917-01. FIa. 1. Microcysts preserved 5 hours after induction. The nuclear region (NR), microcyst capsule (C), and the new vesicular structure (VS) can be seen. Note the grazing section (arrow) at the bottom of the micrograph in which the vesicular nature of the new structure is apparent.
380
KAREN BACON AND F. A. EISERLING
described above. For sectioning, microcysts were prefixed for 10 minutes in 0.1% OsO4, centrifuged, washed with Veronal buffer (pH 6.8) and resuspended in standard 1% OsO4 fixative according to the method of Ryter and Kellenberger (5). Fixation time was 14 hours followed by 2 hours in 0.5 % uranyl acetate. Cells were embedded in Vestopal W (M. Jaeger, Vesenaz, Geneva, Switzerland) and sectioned with an LKB Ultrotome using glass knives. Sections were mounted on grids coated with a Formvar film that had been stabilized with carbon. The sections were poststained with saturated uranyl acetate for 30 minutes at room temperature and examined with an Hitachi HUllA electron microscope at an accelerating voltage of 75 kV using a 50-# objective aperture.
OBSERVATIONS A survey section of a randomly chosen group of microcysts shows the characteristic features of 5-hour cysts (Fig. 1). Most cells display a distinct fibrillar nuclear area such as is typically found in Escherichia coli fixed by the same procedure (6). In addition, a localized vesicular region, generally limited to only a part of the cell circumference, and an electron dense outer layer about 250 A thick may be seen. The vesicular region and the outer layer are not present in the vegetative rod (unpublished observations and 7). A closer examination of the vesicular area revealed the following features. The vesicles are free from ribosomes and appear to lie external to the cell wall but beneath the 250 A thick outer layer (Fig. 2). In whole cells these structures are just visible with the phase contrast microscope. In such preparations they appear as one or more highly refractile regions. The individual vesicles are bounded by a triple-layered membrane structure about 50-75 A across. We describe these structures as vesicles because tubules have rarely been seen in sections through the area. A few consecutive sections about 500 A thick through several groups of cells consistently revealed vesicles and not tubules. In addition, observations of grazing sections of the cells also suggest that the structure is vesicular (Fig. 1). Although the kinetics of appearance of these vesicles has not yet been systematically determined, a few have been observed as early as 60 minutes after induction. It also appears that the microcysts m a y have an internal membrane system not visible in vegetative cells (unpublished observations). The location and appearance of the structure appears to be unique among bacteria. In the case of cyst formation in Azotobacter vinelandii peripheral bodies have been described, but they are definitely internal to the cell wall (8). Published micrographs
F~. 2. Microcysts preserved 5 hours after induction. The nuclear region (NR) and microcyst capsule (C) are indicated. Note the location of the vesicular structure (VS) between the cell envelope (E) and capsule.
UNIQUE STRUCTURE IN MYXOCOCCUS XANTHUS
381
382
KAREN BACON AND F. A. EISERLING
of microcysts of M. xanthus produced on solid medium (7) have not shown the vesicular structures we describe here, and we believe them to be a new structure. This investigation originated as part of a more general program of research on differentiation in Myxococcus xanthus in the laboratory of Dr. E. Rosenberg. We gratefully acknowledge his valuable assistance and encouragement in this project.
REFERENCES 1. BACON, K. and ROSENBERG,E., aT. Bacteriol. in press. 2. DWORKIN, M., J. Bacteriol. 84, 250 (1962). 3. DWORKIN, M. and GIBSON, S., Science 146, 243 (1964). 4. ROSENBERG, E., KATARSKI, M. and GOTTLI~, P., Y. Bacteriol. 93, 1402 (1967). 5. RYTER, A. and KELLENBERGER,E., Z. Naturforsch. 131, 597 (1958). 6. SCHREIL, W. H., J. Cell. Biol. 22, 1 (1964). 7. VOELZ, H. and DWORKIN, M., J. Bacteriol. 84, 943 (1962). 8. WYss, O., NEUMANN,M. and Socor-oFsrcv, M., J. Biophys. Biochem. Cytol. 10, 555 (1961).
378
J. U L T R A S T R U C T U R E RESEARCH
21, 378-382 (1968)
A Unique Structure in Microcysts of Myxococcus xanthus 1 KAREN BACON2 AND F. A. EISERLING
Department of Bacteriology, University of California, Los Angeles, California 90024 Received August 8, 1967 The morphogenesis of Myxococcus xanthus from a vegetative rod to a spherical microcyst can be induced by the addition of glycerol to the vegetative growth medium. Thin sections of microcysts preserved 5 hours after induction revealed a new vesicular structure apparently between the cell envelope and microcyst capsule. This unusual structure is not seen in vegetative cells.
Myxococcus xanthus FB (2) can undergo morphogenesis f r o m a vegetative rod to a spherical, refractile microcyst. This change can be synchronously induced in liquid medium by the addition of glycerol to a vegetative culture (3). The rods progressively shorten and thicken to f o r m spheres in 120 minutes. Thereafter the cells acquire refractility and remain metabolically active for several hours before becoming resting cells. As a complement to a continuing investigation of the biochemical events occurring during microcyst formation (1, 4), we have begun to examine this process with the electron microscope. In this preliminary report we present micrographs of microcysts harvested 5 hours after glycerol induction. A vesicular structure is demonstrated which is unique to the microcyst and has not previously been described in the literature. MATERIALS A N D METHODS Vegetative cells of M. xanthus FB were grown in a casein hydrolyzate medium (2 % N - Z Case, Sheffield Chemical, Norwich, New York, and 0.1% MgSO4-7 H~O) (1). The cells were incubated in 40-ml quantities in 250-ml flasks at 30°C with shaking. Microcyst formation was induced by a modification of a procedure described by Dworkin (3). Glycerol (10 M) was added directly to exponentially growing cells (optical density at 560 m~ of 0.5 or less) to a final concentration of 0.5 M, and the induced culture was incubated as 1 This investigation was supported by National Science Foundation Grant GB-6155. Recipient of a USPHS predoctoral fellowship, Grant GM-30, 917-01. FIa. 1. Microcysts preserved 5 hours after induction. The nuclear region (NR), microcyst capsule (C), and the new vesicular structure (VS) can be seen. Note the grazing section (arrow) at the bottom of the micrograph in which the vesicular nature of the new structure is apparent.
380
KAREN BACON AND F. A. EISERLING
described above. For sectioning, microcysts were prefixed for 10 minutes in 0.1% OsO4, centrifuged, washed with Veronal buffer (pH 6.8) and resuspended in standard 1% OsO4 fixative according to the method of Ryter and Kellenberger (5). Fixation time was 14 hours followed by 2 hours in 0.5 % uranyl acetate. Cells were embedded in Vestopal W (M. Jaeger, Vesenaz, Geneva, Switzerland) and sectioned with an LKB Ultrotome using glass knives. Sections were mounted on grids coated with a Formvar film that had been stabilized with carbon. The sections were poststained with saturated uranyl acetate for 30 minutes at room temperature and examined with an Hitachi HUllA electron microscope at an accelerating voltage of 75 kV using a 50-# objective aperture.
OBSERVATIONS A survey section of a randomly chosen group of microcysts shows the characteristic features of 5-hour cysts (Fig. 1). Most cells display a distinct fibrillar nuclear area such as is typically found in Escherichia coli fixed by the same procedure (6). In addition, a localized vesicular region, generally limited to only a part of the cell circumference, and an electron dense outer layer about 250 A thick may be seen. The vesicular region and the outer layer are not present in the vegetative rod (unpublished observations and 7). A closer examination of the vesicular area revealed the following features. The vesicles are free from ribosomes and appear to lie external to the cell wall but beneath the 250 A thick outer layer (Fig. 2). In whole cells these structures are just visible with the phase contrast microscope. In such preparations they appear as one or more highly refractile regions. The individual vesicles are bounded by a triple-layered membrane structure about 50-75 A across. We describe these structures as vesicles because tubules have rarely been seen in sections through the area. A few consecutive sections about 500 A thick through several groups of cells consistently revealed vesicles and not tubules. In addition, observations of grazing sections of the cells also suggest that the structure is vesicular (Fig. 1). Although the kinetics of appearance of these vesicles has not yet been systematically determined, a few have been observed as early as 60 minutes after induction. It also appears that the microcysts m a y have an internal membrane system not visible in vegetative cells (unpublished observations). The location and appearance of the structure appears to be unique among bacteria. In the case of cyst formation in Azotobacter vinelandii peripheral bodies have been described, but they are definitely internal to the cell wall (8). Published micrographs
F~. 2. Microcysts preserved 5 hours after induction. The nuclear region (NR) and microcyst capsule (C) are indicated. Note the location of the vesicular structure (VS) between the cell envelope (E) and capsule.
UNIQUE STRUCTURE IN MYXOCOCCUS XANTHUS
381
382
KAREN BACON AND F. A. EISERLING
of microcysts of M. xanthus produced on solid medium (7) have not shown the vesicular structures we describe here, and we believe them to be a new structure. This investigation originated as part of a more general program of research on differentiation in Myxococcus xanthus in the laboratory of Dr. E. Rosenberg. We gratefully acknowledge his valuable assistance and encouragement in this project.
REFERENCES 1. BACON, K. and ROSENBERG,E., aT. Bacteriol. in press. 2. DWORKIN, M., J. Bacteriol. 84, 250 (1962). 3. DWORKIN, M. and GIBSON, S., Science 146, 243 (1964). 4. ROSENBERG, E., KATARSKI, M. and GOTTLI~, P., Y. Bacteriol. 93, 1402 (1967). 5. RYTER, A. and KELLENBERGER,E., Z. Naturforsch. 131, 597 (1958). 6. SCHREIL, W. H., J. Cell. Biol. 22, 1 (1964). 7. VOELZ, H. and DWORKIN, M., J. Bacteriol. 84, 943 (1962). 8. WYss, O., NEUMANN,M. and Socor-oFsrcv, M., J. Biophys. Biochem. Cytol. 10, 555 (1961).