Vol. 1:32, No. 3 . .4.
,Jot ORNAL. (F BA(CTERL0i().(;Y, Dec. 1977, p. 1024-1026 (opyTight © 1977 American Societv for Microbiology
Printec (i lin
Microtubular Structures in a Stable Staphylococcal L-Form TOHRU FDA, YAYOI KANDA, CHIZTJRIJ MORI, ANI) SAI)AO KIMUJRA* Department of Bacteriology, Teikyo U.ni'ersitv School of Medicine, Itabashi-hk, Kaga 2-11-1, Tokyo,
Japan
Received for Publicationl 30 August 1977
Microtubular structures, which were demonstrated as straight, dense-walled cylinders attached to cell membranes, were found in a stable staphylococcal Lform grown in the absence of antibiotic. There have been a number of reports concerning microtubules found in plant and animal cells and protozoans, but only a few in bacteria. In ordinary bacteria, microtubules have been found in Proteus mirabilis (5) and Azotobacter Linelandii (6). In the L-forms, microtubular structures or microtubules have been observed in group A and D streptococcal L-forms by Cole (1) and Corfield and Smith (2) and in Escherichia coli L-forms by Eda et al. (3). In general, the membrane structures seem to be difficult to find in the L-forms; in fact, there has been no report of membrane structures in staphylococcal L-forms. In this paper, we demonstrate the ultrastructure of microtubular structures, which were
found in staphylococcal L-forms by electron microscopy.
The L-form used was Staphylococcus aureus 209PL, which was induced in our laboratory from S. aureus FDA 209P by the disk method (4), using aminobenzyl penicillin. This strain is
stable in the absence of antibiotics and has been maintained for several years in an L-form broth composed of 3.7'S brain heart infusion broth (Difco), 0.5'% yeast extract (Difco), 4%> NaCl, and 10%- horse serum (Bio 'I'est). This organism reached maximum growth at 48 h in the L-form broth. The L-form, which was cultured at 37°C for 48 h in L-form broth, was prefixed for 2 h at 4°C with 4%7c glutaraldehyde in 0.1 M cacodylate
Fi(o. 1. Thin section of staphylococcal L-form 209PL at the stationary phase of grotwlth. A longitudinal section of microtubular structures is seen. Bar represents I /pm. 1024
VOL. 132, 1977
NOTES
1025
FIG. 2. Thin section of same strain shown in Fig. 1. Cross-section of cluster of microtubular structures is seen. Bar represents 1 wm.
4~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
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.,
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FIG 3.Hge
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1026
NOTES
Fit;. 4. Higher magnification of cross-section of microtubular structures. Microtubular structures are composed of 12 to 13 subunits. Bar represents 100 nnm.
buffer containing 0.1% MgSO4 (pH 7.2) and was then harvested by centrifugation. The pellets were rinsed with several changes of the same buffer and postfixed in 1% osmium tetroxide in the same buffer (pH 7.4) for 2 h at 4°C. After dehydration in a graded acetone series, specimens were embedded in Epon 812. Ultrathin sections were obtained with a Reichert OMU2 ultramicrotome and stained with uranyl acetate and lead citrate. Preparations were examined and photographed with a Hitachi HU-12AS electron microscope at an accelerating voltage of 75 kV. I'he cells of staphylococcal L-form strain 209PL varied in size at the stationary phase of growth. Cells smaller than 1 ,im were almost spherical, and some larger cells were more pleomorphic in shape. Protrusions from part of the cell membrane were seen in some cells (Fig. 1). Some of the cells had cytoplasmic vacuoles containing a few particles (Fig. 2). Each of the cells
J. BAc,rERXIOL.
was bounded by a unit membrane without a cell wall and had dispersed ribosomes and fibrillar nucleoid materials. In some of the larger cells, microtubular structures were found in the cytoplasm (Fig. 1 and 2). These structures, which had dense walls and hollow cores, were located at the center and periphery of the cytoplasm, and some of them attached to the cell membrane (Fig. 1 and 3). They were straight and cylindrical in shape, and arranged in one or more parallel bundles (Fig. 3). Individual structures measured approximately 25 nm in external diameter, 15 nm in internal diameter, and 200 nm to 1 ,im in length (Fig. 3 and 4). Figures 2 and 4 show a crosssection of this structure. As shown in Fig. 4, it appears that the microtubular structure is composed of 12 to 13 subunits. These results indicate that microtubular structures observed in stable staphylococcal L-forms correspond to "microtubules, because they are straight, dense-walled cylinders structurally similar to those found in bacteria and L-forms (1, 2, 5, 6). Our results suggest that microtubules may be found in many species of L-forms if there are conditions adequate for their formation. LITERATURE CITED 1. Cole, R. M. 1971. Some implications of the comparative ultrastructure of bacterial L-forms, p. 49-83. In S. Madioff (ed.), Mycoplasma and L-forms of bacteria. Gordon & Breach Science Publishers, New York. 2. Corfield, P. S., and D. G. Smith. 1968. Microtubular structures in group I) streptococcal L-forms. Arch. Mikrobiol. 63:356-361. 3. Eda, T., Y. Kanda, and S. Kimura. 1976. Membrane structures in stable 4-forms of Escherichia coli. J. Bacteriol. 127:1564-1567. 4. Eda. T., S. Matsuoka, and I. Tadokoro. 1972. Studies on staphylococcal L-forms. I. Induction and morphological characteristics of staphylococcal L-forms. Jpn. J. Bacteriol. 27:657-664. 5. van Iterson, W., J. F. M. Hoeniger, and E. N. van Zanten. 1967. Microtubule in a bacterium. J. Cell Biol. 32:1-10. 6. Pope, L. M., and P. Jurtshuk. 1967. Microtubule in Azotobacter Luinelandii strain 0. J. Bacteriol. 94:2062-2064.
,Jot ORNAL. (F BA(CTERL0i().(;Y, Dec. 1977, p. 1024-1026 (opyTight © 1977 American Societv for Microbiology
Printec (i lin
Microtubular Structures in a Stable Staphylococcal L-Form TOHRU FDA, YAYOI KANDA, CHIZTJRIJ MORI, ANI) SAI)AO KIMUJRA* Department of Bacteriology, Teikyo U.ni'ersitv School of Medicine, Itabashi-hk, Kaga 2-11-1, Tokyo,
Japan
Received for Publicationl 30 August 1977
Microtubular structures, which were demonstrated as straight, dense-walled cylinders attached to cell membranes, were found in a stable staphylococcal Lform grown in the absence of antibiotic. There have been a number of reports concerning microtubules found in plant and animal cells and protozoans, but only a few in bacteria. In ordinary bacteria, microtubules have been found in Proteus mirabilis (5) and Azotobacter Linelandii (6). In the L-forms, microtubular structures or microtubules have been observed in group A and D streptococcal L-forms by Cole (1) and Corfield and Smith (2) and in Escherichia coli L-forms by Eda et al. (3). In general, the membrane structures seem to be difficult to find in the L-forms; in fact, there has been no report of membrane structures in staphylococcal L-forms. In this paper, we demonstrate the ultrastructure of microtubular structures, which were
found in staphylococcal L-forms by electron microscopy.
The L-form used was Staphylococcus aureus 209PL, which was induced in our laboratory from S. aureus FDA 209P by the disk method (4), using aminobenzyl penicillin. This strain is
stable in the absence of antibiotics and has been maintained for several years in an L-form broth composed of 3.7'S brain heart infusion broth (Difco), 0.5'% yeast extract (Difco), 4%> NaCl, and 10%- horse serum (Bio 'I'est). This organism reached maximum growth at 48 h in the L-form broth. The L-form, which was cultured at 37°C for 48 h in L-form broth, was prefixed for 2 h at 4°C with 4%7c glutaraldehyde in 0.1 M cacodylate
Fi(o. 1. Thin section of staphylococcal L-form 209PL at the stationary phase of grotwlth. A longitudinal section of microtubular structures is seen. Bar represents I /pm. 1024
VOL. 132, 1977
NOTES
1025
FIG. 2. Thin section of same strain shown in Fig. 1. Cross-section of cluster of microtubular structures is seen. Bar represents 1 wm.
4~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
fWNZ
V
.,
4,1.I'dr
FIG 3.Hge
anfcto
ftecl
hw
ude des-aldclnes ragdi. y~~~~~~~~~~~~~~~~~~~~~':
n
nFg
irtblrsrcue
tahdt h cel
m
r
bevda
brn. Brrpeet
tagt m
1026
NOTES
Fit;. 4. Higher magnification of cross-section of microtubular structures. Microtubular structures are composed of 12 to 13 subunits. Bar represents 100 nnm.
buffer containing 0.1% MgSO4 (pH 7.2) and was then harvested by centrifugation. The pellets were rinsed with several changes of the same buffer and postfixed in 1% osmium tetroxide in the same buffer (pH 7.4) for 2 h at 4°C. After dehydration in a graded acetone series, specimens were embedded in Epon 812. Ultrathin sections were obtained with a Reichert OMU2 ultramicrotome and stained with uranyl acetate and lead citrate. Preparations were examined and photographed with a Hitachi HU-12AS electron microscope at an accelerating voltage of 75 kV. I'he cells of staphylococcal L-form strain 209PL varied in size at the stationary phase of growth. Cells smaller than 1 ,im were almost spherical, and some larger cells were more pleomorphic in shape. Protrusions from part of the cell membrane were seen in some cells (Fig. 1). Some of the cells had cytoplasmic vacuoles containing a few particles (Fig. 2). Each of the cells
J. BAc,rERXIOL.
was bounded by a unit membrane without a cell wall and had dispersed ribosomes and fibrillar nucleoid materials. In some of the larger cells, microtubular structures were found in the cytoplasm (Fig. 1 and 2). These structures, which had dense walls and hollow cores, were located at the center and periphery of the cytoplasm, and some of them attached to the cell membrane (Fig. 1 and 3). They were straight and cylindrical in shape, and arranged in one or more parallel bundles (Fig. 3). Individual structures measured approximately 25 nm in external diameter, 15 nm in internal diameter, and 200 nm to 1 ,im in length (Fig. 3 and 4). Figures 2 and 4 show a crosssection of this structure. As shown in Fig. 4, it appears that the microtubular structure is composed of 12 to 13 subunits. These results indicate that microtubular structures observed in stable staphylococcal L-forms correspond to "microtubules, because they are straight, dense-walled cylinders structurally similar to those found in bacteria and L-forms (1, 2, 5, 6). Our results suggest that microtubules may be found in many species of L-forms if there are conditions adequate for their formation. LITERATURE CITED 1. Cole, R. M. 1971. Some implications of the comparative ultrastructure of bacterial L-forms, p. 49-83. In S. Madioff (ed.), Mycoplasma and L-forms of bacteria. Gordon & Breach Science Publishers, New York. 2. Corfield, P. S., and D. G. Smith. 1968. Microtubular structures in group I) streptococcal L-forms. Arch. Mikrobiol. 63:356-361. 3. Eda, T., Y. Kanda, and S. Kimura. 1976. Membrane structures in stable 4-forms of Escherichia coli. J. Bacteriol. 127:1564-1567. 4. Eda. T., S. Matsuoka, and I. Tadokoro. 1972. Studies on staphylococcal L-forms. I. Induction and morphological characteristics of staphylococcal L-forms. Jpn. J. Bacteriol. 27:657-664. 5. van Iterson, W., J. F. M. Hoeniger, and E. N. van Zanten. 1967. Microtubule in a bacterium. J. Cell Biol. 32:1-10. 6. Pope, L. M., and P. Jurtshuk. 1967. Microtubule in Azotobacter Luinelandii strain 0. J. Bacteriol. 94:2062-2064.
