BASAL B O D I E S OF B A C T E R I A L F L A G E L L A
IN PROTEUS
MIRABILIS
I. Electron Microscopy of Sectioned Material
WOUTERA
VAN ITERSON, JUDITH
EVA NIJMAN
F. M. H O E N I G E R , and
VAN ZANTEN
From the Laboratory of Electron Microscopy, University of Amsterdam, Amsterdam, The
Netherlands. Dr. Hoeniger's present address is the Department of Microbiology, School of Hygiene,
University of Toronto, Toronto, Canada
ABSTRACT
Years ago (16, 18, 19), in a study of shadowed preparations of Proteus vulgaris that had been
autolyzed in the cold, the observation was madc that the flagella arose from basal bodies.
However, recently (3, 7, 24, 33) doubt has been cast on the conclusion that the flagella of
bacteria emerge from sizable basal bodies. This problem has, therefore, been reinvestigated
with actively developing cultures of Proteus mirabilis, the cell walls of which had been expandcd slightly by exposure to penicillin. Two techniques were applied: ultramicrotomy,
and negative staining of whole mount preparations. This paper deals with the thin sections
of bacteria after the usual fixation techniquc had been altered slightly: the cells were embedded in agar prior to their fixation and further processing. The flagella then remained
attached to the cells and were seen to extend between the cell wall and the plasma membrane. Occasionally, the flagella appeared to bc anchored in the cell by means of a hookshaped ending. In sections of cells rich in cytoplasm, the basal bodies are particularly difficult to visualize due to their small size (25 to 45 m#) and the lack of properties that would
cnablc one to distinguish them from the ribonucleoprotein structures; in addition, their
boundary appcars to bc delicate. However, when the cytoplasm is sparse in thc cells, either
naturally or as a result of osmotic shocking in distilled water, the flagella can bc observed
to emerge from rounded structures approximately 25 to 45 m# wide. Contrary to a previous
suggestion (21), the flagella do not terminate in the peripheral sites of reduced tellurite,
i.c. the chondrioids. The observations in this part of the study agrcc with those described
in the following paper (15) dealing with negatively stained preparations.
INTRODUCTION
Cilia and flagella of all cells examined so far,
with the exception of the bacteria, show a remarkable uniformity in their structure and in the
organization of their basal apparatus, This basal
apparatus, often called for the flagellate protozoa
"blepharoplast" and for the ciliates "kinetosorne,"
can be referred to by the general term "basal
body" (cf. reference 9). Since the last century, it
has been suggested that basal bodies and centrioles
are homologous structures (11, 26). Lwoff (31)
in particular focused attention on these structures
by emphasizing that they may be endowed with
genetic continuity and would provide a beautiful
model of a self-reproducing particle. With the aid
585
of the electron microscope, it has indeed been flagellum terminated in a basal disc, 30 to 35
found that basal bodies are strikingly similar to m/~ wide, just inside the plasma membrane.
centrioles in their fine structure (6, 10, 44). The Abram, Vatter, and Koflter (2) reported that in
expectation, that basal bodies and eentrioles negatively stained preparations of Proteus vulgaris
carry genetic information which may function and in various strains of Bacillus the flagella can
both in instigating their reproduction and in be seen to "arise via hooks from spherical strucforming the protein fibers that constitute the tures, 250 to 350 A in diameter." But Newton
cilium or flagellum and the mitotic spindle, has and Kerridge (33) suggested again in 1965 that
been confirmed, at least for the basal bodies, by the larger "granules" seen in the electron microthe demonstration in them of DNA (38, 40).
scope could be artifacts.
The existence of such bodies at the base of
Because of this uncertainty regarding the basal
flagella and cilia in protozoa and other cells apparatus of bacterial flagella, it remains an open
prompted some early investigators to look for question how far these locomotor organelles
similar structures in bacteria with the light micro- may, in their fundamental features, be compared
scope, and, indeed, so called basal granules to the cilia and flagella of eucaryotic organisms.
(blepharoplasts) were recorded by several authors In their fine structure, bacterial flagella, of course,
(for relevant literature, see reference 16). The differ strikingly from the beautifully symmetrical
first demonstration with the electron microscope "9 + 2" pattern established for cilia and flagella
was made in Proteus vulgaris by one of the authors of diverse origin. Since the over-all width of
in cooperation with Professor C. F. Robinow in bacterial flagella falls between 100 and 190 A,
1948 (16, 18, 19). Astbury (5) confirmed this these filaments can then be compared with the
finding, and subsequently basal granules were
100- to 300-A-wide fibrils that constitute the higher
recorded with the electron microscope for Chromo- cilia and flagella (6, 17, 33, 39). At the molecular
bacterium violaceum and Vibrio metchnikovii (20), level, bacterial flagella were seen to consist of
Rhodospirillum rubrum (8), and Vibrio comma (8, 45), helically or longitudinally arranged rows of
for Vibrio spp. (35) and Spirillum spp. (35, 48). globular subunits of the protein flagellin, there
However, all these observations were made on being between 3 and 10 such rows, depending on
shadowed preparations of cells that either were the organism (1, 25, 27, 28). The fibrils of higher
old or had been allowed to autolyze in order to cilia and flagella may possess comparable features
make them more transparent to the electron (4, 34).
beam. Pijper (36) and Kerridge (24) therefore
The aim of the present study was to reinvestisuggested that the basal bodies might be artifacts gate by means of current techniques of electron
caused by cytoplasmic coagulation.
microscopy whether the bacterial flagella arise
Since Weibull's observation (47) that, in Bafrom structures comparable to the basal bodies
cillus megaterium, flagella remain attached to the
(kinetosomes or blepharoplasts) in other cells.
naked protoplasts after the cell wall had been reThe thin-sectioning technique has been applied,
moved with lysozyme, it is generally agreed that
flagella terminate within the plasma membrane. together with negative staining of whole mounts
But the question whether basal bodies really exist with potassium phosphotungstate (46). In order
still remained unanswered. Recently several groups to preserve in the thin sections the flagella atof workers have looked for the exact site of im- tached to the ceils, the usual procedure had to be
plantation of bacterial flagella in sectioned ma- altered by embedding the organisms in agar
terial and in cells negatively stained with po- prior to fixation and further processing.
After this work had been completed, an article
tassium phosphotungstate. Murray and BirchAndersen (32) could see in thin sections of Spirillum appeared by Abram, Komer, and Vatter (3)
serpens that the polar flagella penetrate the cell containing beautiful illustrations of the basal
wall and terminate within the plasma membrane, structures of the flagella of Proteus vulgarls. They
but the detailed mode of anchorage was not ap- examined negatively stained and shadow-cast
parent. Glauert, Kerridge, and Horne (7), preparations of ghost cells and "long forms" obexamining the sheathed flagellum of Vibrio tained by treating cultures in the cold for 2 days
metchnikovii, found that although the sheath was to 6 wk, and concluded that the flagellum arises
continuous with the cell wall, the core of the from a small body, i.e. a spherical structure 11
586
T ~ JOURNALOF C]~LL BIOLOOV • VOLVME81, 1966
to 14 m~ in diameter, associated with the plasma
membrane.
This first communication deals with the sectioned cells. T h e organisms studied were actively
motile swarmers of Proteus rnirabilis, since they
possess multitudes of flagella (13). T h e cells were
treated with penicillin, with a view to loosening
the cell wall and the texture of the cytoplasm so
as to facilitate tracing the flagella to their cellular
origin. I n order to ascertain whether a relation
exists between the chondrioids and the sites of
flagellar implantation, some cells were incubated
with potassium tellurite, a treatment which enhances the contrast of the peripherally located
chondrioids by the incorporation of reduced product (21).1
In this study of flagellation in Proteus, and in the
next (15), the term "chondrioid" is used to denote
The second article (15) will deal with negatively
stained material from Proteus mirabilis cells.
the rounded areas contiguous with the plasma membrane in which the reduced product of potassium tellurite first appears following incubation under suitable conditions. Obviously, one difficulty in staining
the chondrioids by means of tellurite reduction is
how to determine the moment at which to stop the
experiment. Therefore, the length of the incubation
period must be arbitrary; so also is the delineation of
the true sites of reductive activity. During this unnatural treatment, the optical density of the culture
gradually stops rising (21); the cells autolyze and
gross morphological changes take place, including the
formation of large electron-scattering crystals. As described previously (21), the structure of the chondrioids in Proteus differs fundamentally from that in
Bacillus subtilis.
X~mtrRE 1 An oblique section through a cell in which the plasma membrane has partly receded from
the cell wall, leaving an empty space within which four rounded structures are to be seen. A fifth body
lies outside the cell. The arrows point to flagella emerging from the bodies; Te indicates deposit of reduced tellurite. Note the tubules in the lower part of the cell; such organelles are the subject of a separate paper (~8). X 180,000.
VAN ITERSON, HOENIGER, AND NIJMAN VAN ZANTEN Ba~al Bodies o.f Flagella. 1
587
MATERIALS
AND METHODS
T h e strain of Proteus mirabilis used in this work was
isolated f r o m a stool s p e c i m e n a n d m a i n t a i n e d o n egg
slopes as described previously (12-14). O r g a n i s m s
w h i c h h a d b e e n passed t h r o u g h a motility t u b e of
semisolid a g a r (heart infusion b r o t h + 0 . 3 % agar)
were s u s p e n d e d in physiological saline to a concent r a t i o n of a b o u t 109 b a e t e r i a / m l . O n e m l of this susp e n s i o n was spread uniformly over the surface of h e a r t
infnsion a g a r (Difco) in a 15-cm d i a m e t e r Petri dish;
s u c h plates were i n c u b a t e d for 3}3 to 5 hr at 35°C,
i.e. to the peak of s w a r m e r differentiation w h e n
checked with the l i g h t microscope (13). T h e o r g a n isms were r e m o v e d from the plates in h e a r t infusion
broth, c o n t a i n i n g 2000 I U / m l penicillin G, 5 % horse
serum, 0.25 M sucrose, a n d 0.003 M MgCI~; they were
left in this m e d i u m for 75 m i n at r o o m temperature.
I n m a n y cases, the bacteria were i n c u b a t e d with
0 . 0 5 % p o t a s s i u m tellurite (K~TeO3) u n d e r semianaerobic conditions for 60 to 75 m i n in order to enh a n c e contrast of the chondrioids (21). T h e cultures
were t h e n cemrifugcd, a n d e m b e d d e d before fixing in
1 % agar m a d e u p in modified Michaelis buffer containing 0.25 M sucrose (43). T h e fixation procedure
a n d p o s t t r e a t m e n t with u r a n y l acetate of R y t e r a n d
Kellenberger (43) was e m p l o y e d o n the a g a r - e m b e d d e d bacteria; they were d e h y d r a t e d t h r o u g h
g r a d e d series of alcohol, transferred to propylene
oxide, a n d e m b e d d e d in E p o n 812 (30).
I n later e x p e r i m e n t s parallel to the work of Part II
(15), the cell walls were loosened d u r i n g 60 rain with
2000 I U / m l penicillin, w h e r e u p o n the ceils were
shocked in distilled water with 0.01 M MgCI~, a n d
t h e n e m b e d d e d in agar a n d p r e p a r e d for u l t r a m i c r o t o m y as usual. T h e s e preparations (Figs. 15, 17,
a n d 18) were e m b e d d e d in Vestopal W.
T h i n sections were c u t o n a n L K B U l t r o t o m e with
a glass knife, a n d stained with lead citrate (41). Electron m i e r o g r a p h s were t a k e n with a Philips E M 200
o p e r a t i n g at 80 kv, with a double condenser lens
system a n d a n objective aperture of 50 t~. Most microg r a p h s were m a d e with the specimen-cooling device
in operation.
FIGuaEs ~ to 6 Five serial sections through the tip of an elongated organism rich
arrows in Figs. ~ and 3 indicate faintly distinguishable rounded structures; those
point to flagellar extensions which can be traced in the cytoplasm. I n Fig. 3, a
teIlurite is marked Te. The dense track in Fig. 5 m a y represent an arrangement of
rein structures. )< 180,000.
588
TH~ ~OURNAL OF CELL BIOLOGY - VOLUME 31, 1966
in cytoplasm. T h e
in Figs. 5 and 6
deposit of reduced
the ribonucleopro-
vx~ I T ~ s o ~ , H O ~ G ~ a , AND Nxa~z-~ vx~ ZA~TEN Basal Bodies of Flagella. 1
589
590
T~IE JOURNAL OF CELL BIOLOGY • VOLUME 31, 1966
OBSERVATIONS
Embedding of bacteria prior to their fixation
proves to be a good method for keeping f a g e l l a
attached to the cells so that in thin sections one can
study their mode of insertion. In the sections, they
are found to be distributed either singly (Figs.
1 to 12) or occasionally in small bundles, as in
Fig. 7 (cf. reference 20). In many instances (Figs.
6 to 8, and l0 to 18), the flagella do not stop at
the cell wall, but extend between the loosened
cell wall and the plasma membrane, and sometimes even farther, as will be described below.
The width of the flagella in the sectioned material
was calculated from some 200 measurements:
after penicillin treatment only, it was found to be
88 ± 21 A (mean -4- SD) ; after penicillin treatment
and water shocking, it was found to be 97 ± 17 A.
The considerable variation in the two sets of data
may be due either to individual differences between the flagella or to inaccuracies in measurement. Moreover, there was apparently some
shrinkage of the flagella during fixation and
embedding (cf. diameter of 133 ± 16 A for flagella
in negatively stained preparations--reference I5).
Since our work concerned mainly Proteus
swarmers, observations of the type of short rodshaped cell illustrated in Fig. 1 were rarely made.
In this oblique section the plasma membrane has
partly receded from the cell wall; in the empty
space between the two integuments four rounded
structures can be seen which strongly resemble
those observed years ago in a shadowed, autolyzed swarmer (16, 18). The arrows in Fig. 1
point to sections of flagella emerging from the
rounded structures. A similar rounded element is
also seen outside the cell in Fig. 1; presumably it
had been released from a broken part of the cell.
The bacteria examined had been exposed to
penicillin. It was to be expected that this treatment would decrease the rigidity of the cell wall,
thus enabling the contents of the cell to swell.
Yet the cytoplasm, in m a n y cases, remained so
compact that few structures can be discerned
between the ribonucleoprotein. Figs. 2 to 6 are
examples of serial sections through the end of such
a compact cell. There are indications of rounded
structures near the cell periphery (see arrows in
Figs. 2 and 3) but these are faint, and indeed one
could have reason to question their existence on
the basis of such evidence alone. In Figs. 5 and 6
the direction of the sectioning is such that several
flagellar extensions can be traced in the cytoplasm
at the tip of the cell. In Fig. 5, the electron-opaque
track (see arrows) through the cytoplasm and
probably extending from a flagellum on the
lower surface may prove of particular interest
(cf. Discussion) at a more advanced stage in our
research. In this series of five sections, deposits of
reduced tellurite (labeled Te) are so few that one
cannot decide whether any correspondence exists
between such sites and the implantation of the
flagella, as originally suggested by van Iterson and
Leene (21).
Some swarmers were found to be unusually
poor in cytoplasmic material, and to have a m u c h
swollen nucleoplasm. Figs. 7 and 8 together depict
a single longitudinal section from such an organism. In the periphery of this very transparent
swarmer there are rounded structures, labeled B.
The borders of these rounded bodies are distinct
from the plasma membrane proper. In most electron micrographs, the bodies lack a distinctly
visible limiting m e m b r a n e (Figs. 12 to 18), and
this makes them difficult to recognize. The width
of the rounded structures in the sections is about
25 to 45 m/z. The reduced tellurite can be recognized by its contrast (arrows, Te), and at the
arrow in Fig. 8 b a flagellum seems to come out of
the cell in the neighborhood of such an area.
However, these and similar pictures do not seem
to support the contention (21) that the deposits
of reduced tellurite coincide with the sites of the
rounded bodies.
Figs. 9 to 11, representing three serial sections
through a swarmer, further illustrate the difficulty
of analyzing the structures at the bases of the
flagella in cells with a more or less normal content
of cytoplasm. (It will be noted that the sister cell
on the left of the swarmer became swollen during
the penicillin treatment.) Flagella that can be seen
emerging from the long bacterium in one of these
three sections are numbered 1 to 8. Several features should be noted. O f interest is the cytoplasmic
structure at the site of penetration of flagellum 1
in Fig. 10 (see arrows). In the previous section
(Fig. 9), this location may correspond with a
deposit of reduced tellurite. T o the right of this
flagellum in Fig. 9 is a delicately striated area
surrounded by reduced tellurite; it is unlikely that
this area would correspond to the basal granule
of the flagellum. Flagellum 8, in Fig. 11, shows,
between the cell wall and the plasma membrane,
a bend above which the outlines of a basal granule
may perhaps be recognizable in the cytoplasm
VAN ITERSON, HOENIGER, AND NIJMAN VXN ZA~TEN Basal Bodies of Flagella I
591
FIGURES 7 and 8 Longitudinal sections from a Proteus swarmer which is unusually poor in cytoplasm.
It should be noted that both extremities of this cell show on their left side signs of compression during
sectioning due to defective polymerization of the Epon. In the periphery of this organism deposits of
reduced tellurite (Te) can be seen, as well as rounded structures (B) which are interpreted as possibly
representing the basal bodies of the flagella. However, here, contrary to their condition in cells from
592
which the cytoplasm had been released artificially (see Figs. 15 to 18), the basal bodies appear to be
more or less empty. I n Fig. 8 a, the flagellum at h is seen to have a hook-shaped bend. At the arrow
in Fig. 8 b, a flagellum appears to extend in the direction of a deposit of reduced tellurite, b u t this was
not usually found to be the case. Figs. 7 and 8, X 75,000; Figs. 8 a and b. X 130,000.
593
(see arrows). O n the whole, the details to be discerned in the serial sections are disappointing,
and this is not surprising, since the basal structures
are likely to be smaller (25 to 45 m/z) than the
section thickness, which has not been estimated
here. Moreover, contrast in the sections and the
properties of the boundary of the bodies seem to
militate against their clear demonstration in
thin sections.
T h e penetration of a flagellum through the cell
wall and into the cytoplasm is shown particularly
well in Fig. 12. Again, the basal structure is only
faintly distinguishable because of its delicacy and
its lack of conspicuous properties and because the
section is comparatively thick. O f special interest
is the electron-opaque, very thin track in the
cytoplasm (indicated by arrows) which m a y
originate from the basal body.
Finally, in Figs. 13 to 18, a selection of cell
fragments is shown in order to obtain further
proof for the true existence of specific cytoplasmic
structures at the bases of the flagella. With the
exception of Figs. 13, 14, and 16, these electron
micrographs were made parallel with the work
described in Part II (15), i.e. from cells that had
released most of their cytoplasm after being
osmotically shocked in distilled water. In all of
these illustrations, flagella can be seen entering
the cell and terminating in basal bodies. In Fig.
13, there appear to be two such bodies about 30
m~ wide. The presumably double body in Fig. 14
again is difficult to distinguish from the surrounding cytoplasm. In cells from which the
cytoplasm had been released by osmotic shock
the bodies sometimes--but not always (Fig. 18)-appear incomplete, like those indicated by arrows
in Fig. 15. The one indicated by two arrows
(Fig. 15) may represent a more complete basal
body. To its left can be seen a circular, clearly
membranous structure. Fig. 17 is of interest
because it is a section through a cell comparable
to the one in Fig. 4 of Part II (15) in which an
elongated structure inside a negatively stained
cell is interpreted as a fragment of the plasma
membrane "caught up by the flagellar endings
and folded around them." T h a t this interpretation holds true, and that "this enfolding of the
fragmented membrane probably hides the basal
bodies" can be deduced from Fig. 17, in which the
arrow indicates a basal structure attached to a
flagellum which passes both the cell wall and the
fragmented plasma membrane.
In Fig. 18, several bodies are shown at high
magnification. It is interesting to note that in
this young cell, quickly released of its contents
by osmotic shocking, there remains around the
basal bodies no clearly visible limiting m e m b r a n e
of a structure comparable to that of the plasma
membrane. T h i n filaments appear to interconnect remnants of the cellular content, including
the basal bodies (cf. reference 22).
DISCUSSION
Fixation through agar is an easy way to obtain
satisfactory embeddings for the sectioning of material in which the flagella are well preserved.
The usual failure to demonstrate flagella in thin
sections is probably due to their being broken off
during centrifuging after fixation. In thin sections
of swarmers of Proteus mirabilis prepared in this
manner, numerous flagella are seen to penetrate
the cells and to extend between the cell wall and
the plasma membrane. When serial sections were
superimposed, the flagella could be traced over
considerable distances.
The present state of the electron microscope
technique does not yield preparations in which the
basal granules on Proteus flagella are easily demonstrated. But the evidence is sufficiently convincing
when considered together with the results of negative staining (15). One of the difficulties in demon-
FIGURES 9 to 11 Three serial sections through a swarmer. The sister cell just visible on
the left in this series was observed to be quite enlarged, presumably as a result of penicillin treatment. Flagella emerging from the cell in at least one of these three sections have
been numbered 1 to 8; their position is indicated in the other two sections so that the corresponding cytoplasmic areas can be examined. For example, note in Fig. 10 the cytoplasmic fine structure (arrows) at the site of penetration of flagellum 1; in Fig. 9 this location may correspond to a deposit of reduced tellurite, while in Fig. 11 it looks like a
basal granule. The thickness of the section, when compared with the diameter of the basal
granules (~5 to 45 m/z), is too great to permit really profitable study. > 78,000.
594
THE JOURNAL Or" CELL BIOLOGY • VOLUME 31, 1966
VAN ITERSON, HOENIGER, AND NITMAN VAN ZANTEN Basal Bodies
of Flagella. I
595
F m v l ~ 1~ Note the penetration of a flagellum through the cell wall and into the cytoplasm at the
left side of this organism. The basal structure can be distinguished only faintly (at tile lower arrow)
owing to the relative thickness of the section, and to the lack of properties which would make it stand
out from the surrounding cytoplasm. The upper arrows indicate a very thin track in the cytoplasm;
deposits of reduced tellurite are marked Te. >( 81,000.
596
strating such bodies in sections of young cells is
that in the electron micrographs limiting membranes of a structure comparable to that of the
plasma m e m b r a n e cannot readily be recognized
around them. There are possibly two reasons for
this: first, the bodies may not be bounded by a
membrane similar in thickness and construction
to the plasma membrane; second, such a membrane, when present, m a y not show up well in the
sections, due to curvature of the small bodies.
These "membranes" are best shown in Figs. 7, 8,
and 18. They should perhaps be studied in cells
that have passed beyond the logarithmic phase.
The " m e m b r a n e " may be derived from the plasma
membrane, but is separated from it (cf. insets,
Fig. 8). The impression obtained from cells with
a well developed cytoplasm suggests that the
boundary is more delicate than the plasma membrane, and that this makes it nearly impossible
technically to observe it in such comparatively
thick sections. The results obtained by negative
staining, as shown in particular by Fig. 8 in
Part II (15), appear to confirm the present suggestion that the basal bodies have very delicate
boundaries.
The study of serial sections is of interest in
view of the relative positions of the sites of flagellar implantation; but it is of no help in shedding
light on the structure of the bodies themselves,
their size being presumably in the same range
as the section thickness. Contrary to previous expectation (21), we did not find the sites of flagellar
origin in Proteus to correspond with the sites of
tellurite reduction believed to be chondrioids
(Figs. 9 to 11).
Particularly in ceils in which the cytoplasm is
sparse, the flagella are seen to emerge from
rounded structures approximately 25 to 45 m/z
wide. This value is somewhat lower than that for
basal bodies in negatively stained preparations,
i.e. 50 m # (of. reference 15); but so also is the
diameter of the flagella. Presumably, both flagella
and basal bodies shrink during fixation and embedding. O u r present estimates for the diameter of
the basal bodies are lower than that mentioned
earlier (16), i.e. 100 m#. T h a t value was, however,
a very rough one made on a flattened and shadowed preparation.
Further, the value of 25 to 45 m # given here
agrees fairly well with both the original measurement made by Abram et al. of 25 to 35 m/z (2)
and with their more recent determination of 20
to 70 m/z (3) ; but it is significantly higher than the
11 to 14 m/z (3) which these authors interpret as
representing the genuine basal body. We believe
that the value, 11 to 14 m/z, applies only to a part,
i.e. a small "knob," of the whole basal structure
(of. reference 15).
Thus, the agreement between the present resuits and those made on autolyzed material of
Proteus (5, 16, 18, 19) is quite remarkable. The
results obtained with other autolyzed organisms
(8, 35, 45, 48) now requires reinvestigation with
more modern techniques (cf. reference 7).
The flagella are sometimes seen to be anchored
to the cell by means of a hook (Figs. 1, 8 a, 11,
17), which agrees with earlier observations (3,
13, 16, 20, 29, 42).
I n some electron micrographs (Figs. 8 a and b,
12, 16, and 18), thin filamentous structures, which
may contain nucleic acids (22), appear to be in
touch with the basal bodies. But it is considered
premature to speculate on the possible contact of
these bodies with nucleic acids, particularly DNA,
although this would make their comparison with
self-reproducing particles of great interest, as
mentioned in the Introduction.
We wish to thank Mr. P. J. Barends, Mrs. J. RaphaalSnijer, Miss N. M. Slikker, and Miss E. Bon for their
capable assistance. One of us (J.F.M.H.) is indebted
to the Medical Research Council of Canada for a
travel grant.
Received for publication 31 March 1966.
VAN ITERSON, HOENmER, AND NIX~L~NVAN ZANTEN Basal Bodies of Flagella. [
597
FIGURE 18 At the arrows two basal bodies may be seen, each about 30 m~t in diameter.
X 160,000.
FIGURE 14 The arrows indicate what is presumed to be a double basal body. X 156,000.
FI(~URE 15 Fragment of a cell from which most of the cytoplasm has been released by
osmotic shocking in distilled water. The large, single arrows point to the origins of flagella,
and these are interpreted as being fragments of basal bodies. The smaller arrows indicate
a complete basal body, to the left of which a rounded fragment of membrane can be seen.
X 156,000.
FIouRE 16 A flagellum appears to be emerging from a basal body which has dimensions
~7 X 40 m#. Note the fine, central fibril which lies within or over the basal body. X
147,000.
FIGURE 17 This micrograph is interesting because it represents in section the same sort
of situation as is seen in the negatively stained preparation of Fig. 4 in Part II (15). A
fragment of the plasma membrane has been caught up by the flagcllar endings and appears
to be folded around them. The arrow points to a basal structure of a flagellum which penetrates both the cell wall and the plasma membrane; note the hooklike bend in this flagellum. The fine dots which speckle the micrograph are due to dirt picked up during preparation when the section was floated. X 110,000.
FIGURE 18 As in Figs. 16 and 17, most of the cytoplasm has been released by osmotic
shocking: this figure can thus be compared with those of negatively stained preparations
in Part II (15). Several basal granules appear to be present which resemble in size and situation those illustrated in Part II (15). No distinct limiting membrane can be distinguished, even at this high magnification. The bodies appear to be in contact with fibrillar
material remaining from the cytoplasm. X 190,000.
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THE JOUI~NALOF CELL BIOLOGY • VOLUME 31, 1966
VAN •TERSON, HOENIGER, AND N I J ~ N VAN ZANTEN Basal Bodies of Flagella. I
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REFERENCES
1. ABRAM, D., MATTER, A. E., and KOFFLBR, H.,
Fine structure of bacterial flagella, Abstracts
Biophysic. Soc., 1964, WC 5.
2. ABRAM, D., MATTER, A. E., and KOFFLER. H.,
Some specialized structures of bacterial flagella,
Bact. Proc., 1964, G 68.
3. ABRAM, n., KOFFLER, H., and MATTER, A. E.,
Basal structure and attachment of flagella in
cells of Proteus vulgaris, J. Bact., 1965, 90, 1337.
4. ANDRe, J., and THI~RY, J.-P., Mise en 6vidence
d'une sous-structure fibrillaire dans les filaments axon6matiques des flagelles, J. Micr.,
1963, 2, 71.
5. ASTBURY, W. T., Some recent adventures among
proteins, Pubbl. Staz. Zool. Napoli, 1951, 23,
suppl. 18.
6. FAWCETT, D., Cilia and flagella, in The Cell, (J.
Bracbet and A. E. Mirsky, editors), New York,
Academic Press Inc., 1961, 2,217.
7. GLAOERT, A. M., KERRIDOE, D., and HORNE, R.
W., The fine structure and mode of attachment
of the sheathed flagellum of Vibrio metchnikovii,
J. Cell Biol., 1963, 18, 327.
8. GRACE, J. B., Some observations on the flagella
and blepharoplasts of Spirillum and Vibrio spp.,
J. Gen. Microbiol., 1954, 10, 325.
9. GRIMSTONE, A. V., Fine structure and morphogenesis ill protozoa, Biol. Rev., 1961, 36, 97.
18.
19.
20.
21.
22.
23.
24.
I0. HARVEN, E. DE, and BERNHARD, W., Etude au
11.
12.
13.
14.
15.
16.
17.
600
microscope 61ectronique de l'ultrastructure du
centriole chez les vert6br6s, Z. Zellforsch., 45,
1956, 378.
HENNEOUY, L. F., Sur les rapports de ells vibratiles avec les centrosomes, Arch. Anat. Micr.,
1898, 1, 482.
HOENICER, J. F. M., Cellular changes accompanying the swarming of Proteus mirabilis. I.
Observations of living cultures, Canad. J.
Mierobiol., 1964, 10, 1.
I-~[OENIGER,J. F. M., Development of flagella by
Proteus mirabilis, J. Gen. Microbiol., 1965, 40, 29.
HOENmER, J. F. M., Cellular changes accompanying the swarming of Proteus mirabilis. II.
Observations of stained organisms, Canad. J.
Microbiol., 1966, 12, 113.
HOENICER, J. F. M., ITERSON, W. VAN, and
NIJMAN VAN ZANTEN, E., Basal bodies of
bacterial flagella in Proteus mirabilis. II. Electron microscopy of negatively stained material,
J. Cell Biol., 1966, 31,603.
HOUWlNK, A. L., and ITERSON,W. VAN, Electron
microscopical observations on bacterial cytology. II. A study on flagellation, Biochim. et
Biophysica Acta, 1950, 5, 10.
INOU~, S., Motility of cilia and the mechanism of
25.
26.
27.
28.
29.
30.
31.
mitosis, in Biophysical Science. A Study
Program, New York, J o h n Wiley and Sons,
1961, 402.
ITERSON, W. VAN, Some observations on bacterial
flagellation, Proceedings of the Conference on
Electron Microscopy, Hoogland Co., Delft,
Netherlands, 1950, 117.
ITERSON, W. VAN, Discussion on bacterial surface,
flagella and motility, in The Nature of the
Bacterial Surface, Symp. Soc. Gen. Microbiol.,
1949, 172.
ITERSON, W. VAN, Some remarks on the present
state of our knowledge of bacterial flagellation,
in Bacterial Cytology, Symposium 6th International Congress of Microbiology, Bacterial
Cytology, Rome, Fondazione E. Paterno, 1953,
24.
ITERSON,W. VAN, and LEENE, W., A cytochemical
localization of reductive sites in a Gramnegative bacterium, Tellurite reduction in
Proteus vulgaris, J. Cell Biol., 1964, 20,377.
ITERSON, W. VAN, The fine structure of the ribonucleoprotein in bacterial cytoplasm, J. Cell
Biol., 28, 1966, 563.
ITERSON, W. VAN, HOENIGER, J. F. M., and N UMAN VAN ZANTEN, E., A "microtubule" in a
bacterium, J. Cell Biol., 1967, 32, 1.
KERRIDGE, D., The effect of environment on the
formation of bacterial flagella, in Microbial
Reaction to Environment, Symp. 5'oc. Gen.
Microbiol., 1961, 11, 41.
K~RRIDOE, D., HORNE, R. W., and GLAUERT, A.
M., Structural components of flagella from
Salmonella typhimurium, J. Mol. Biol., 1962, 4,
227.
LENHOSSEI% M. VON, Ueber Flimmerzellen,
Verhandl. Anat. Gesdl., Kiel, 1898, 12, 106.
LowY, J., and HANSON, J., Structure of bacterial
flagella, Nature, 1964, 202, 538.
LowY, J., and HANSON, J., Electron microscope
studies of bacterial flagella, J. Mol. Biol., 1965,
11,293.
LowY, J., Structure of the proximal ends of
bacterial flagella, J. Mol. Biol., 1965, 14,297.
LUYT, J. H., Improvements in epoxy resin embedding methods, J. Biophysic. and Biochem.
Cytol., 1961, 9, 409
LWOFF, A., Problems of Morphogenesis in Ciliares, New York, J o h n Wiley and Sons, 1950.
32. MURRAY, R. G. E., and BIRCH-ANDERSEN, A.,
Specialized structure in the region of the
flagella tuft in Spirillum serpens, Canad. J.
Microbiol., 1963, 9, 393.
33. NEWTON, B. A., and KERRIDGE, D., Flagellar and
ciliary movement in micro-organisms, in
TEE ffOI:ENAL OF CELL BIOLOGY - VOLUME 31, 1966
Function and Structure in Micro-organisms,
Syrup. Soc. Gen. Microbiol., 1965, 15,220.
34. PEASE, D. C., The nltrastrueture of flagellar fibrils, J. Cell Biol., 1963, 18, 313.
35. PEASE, P., Some observations upon the development and mode of attachment of the flagella in
Vibrio and Spirillum species, Exp. Cell Research,
1956, 10, 234.
36. PIjPER, A., Bacterial flagella and motility,
42.
43.
Ergebn. Mikrobiol. Immunitiitsforsch. Exp. Therap.,
1957, 30, 37.
37. PITELKA, D. R., Electron-Microscopic Structure
of Protozoa, New York, The Macmillan Company, 1963.
38. RANDALL,J. T., and JACKSON, S. F., Fine structure and function ill Stentor polymorphus, J.
Biophysic. and Biochem. Cytol., 1958, 4, 807.
39. RANDALL,SIR J., HOPKINS,J. M., EADIE,J. M.,
and BUTCHER, R. W., Studies of cilia, basal
bodies and some related organelles. Part I:
Observations on fine structure, Proc. Linn. Soc.
London, 1961-1962, 174, 31.
40. RANDALL,SIR J., and DISBREY, C., Evidence for
the presence of DNA at basal body sites in
Tetrahymena pyriformis, Proc. Roy. Soc. London,
Series B, 1965, 162,473.
41. REYNOLDS, E. S., The use of lead citrate at high
44.
45.
46.
47.
48.
pH as an electron-opaque stain in electron
microscopy, J. Cell Biol., 1963, 17,208.
ROOERS, G. E., and FILSHm, B. K., Some aspects
of the ultrastructure of a-keratin, bacterial
flagella, and feather keratin, in Ultrastructure
of Protein Fibres, (R. Borasky, editor), New
York, Academic Press Inc., 1963, 123.
RYTER, A., and KELLENBEROER, E., Etude au
microscope 61ectronique de plasmas eontenant
de l'acide d6soxyribonucleique. I. Les nucl6oides des baet6ries en eroissanee active, Z.
Naturforsch., 1958, 13b, 597.
SLEIGH, M. A., The Biology of Cilia and Flagella,
New York, The Macmillan Company, 1962.
TAWARA, J., Electron-microscopic study on the
flagella of Vibrio comma, J. Bact., 1957, 73, 89.
THORNLEY, M. J., and HORNE, R. W., Electron
microscope observations on the structure of
fimbriae, with particular reference to Klebsiella
strains, by use of the negative staining technique, J. Gen. Microbiol., 1962, 28, 51.
WEIBULL, C., The isolation of protoplasts from
Bacillus megaterium by controlled treatment
with lysozyme, J. Bact., 1953, 66, 688.
WILLIAMS,M. A., and CHAPMAN,G. B., Electron
microscopy of flagellation in species of Spirillum,
J. Bact., 1961, 81, 195.
VAN ITERSON, HOENIGER,ANn NIJMAN VAN ZANTEN Basal Bodies of Flagella. 1
601