Microbiology (2008), 154, 286–295
DOI 10.1099/mic.0.2007/012765-0
Attachment organelle ultrastructure correlates with
phylogeny, not gliding motility properties, in
Mycoplasma pneumoniae relatives
Jennifer M. Hatchel and Mitchell F. Balish
Correspondence
Department of Microbiology, Miami University, 80 Pearson Hall, Oxford, OH 45056, USA
Mitchell F. Balish
[email protected]
Received 28 August 2007
Revised
5 October 2007
Accepted 11 October 2007
The Mycoplasma pneumoniae cluster is a clade of eight described species which all exhibit
cellular polarity. Their polar attachment organelle is a hub of cellular activities including
cytadherence and gliding motility, and its duplication in the species M. pneumoniae is coordinated
with cell division and DNA replication. The attachment organelle houses a detergent-insoluble,
electron-dense core whose presence is required for structural integrity. Although mutant analysis
has led to the identification of attachment organelle proteins, the mechanistic basis for the
activities of the attachment organelle remains poorly understood, with gliding motility attributed
alternatively to the core or to the adhesins. In this study we investigated attachment
organelle-associated phenotypes, including gliding motility characteristics and ultrastructural
details, in seven species of the M. pneumoniae cluster under identical conditions, allowing direct
comparison. We identified gliding ability in three species in which it has not previously been
reported, Mycoplasma imitans, Mycoplasma pirum and Mycoplasma testudinis. Across species,
ultrastructural features of attachment organelles and their cores do not correlate with gliding
speed, and morphological features of cores are inconsistent with predictions about how these
structures are involved in the gliding process, disfavouring a prominent, direct role for the
electron-dense core in gliding. In addition, we found M. pneumoniae to be an outlier in terms of
cell structure with respect to its close relatives, suggesting that it has acquired a special set of
adaptations during its evolution.
INTRODUCTION
The bacterial genus Mycoplasma includes over 100 species.
These organisms lack cell walls and in nature are always
associated with vertebrate hosts. Several mycoplasma
species exhibit a polarized cell morphology, exemplified
by Mycoplasma pneumoniae, a significant causative agent of
assorted respiratory and non-respiratory human disease
conditions (Waites & Talkington, 2004). This surfaceadherent organism exhibits the pleomorphy typical of its
genus, but is characterized by a prosthecal terminal
organelle or attachment organelle at one pole (Balish,
2006). This structure is the site at which adhesins are
clustered, enabling the cell to interact productively with
host cells (cytadhere). The attachment organelle is the
leading end of the cell during gliding motility (Balish &
Krause, 2006) and the location of the gliding motor
(Hasselbring & Krause, 2007). Gliding appears to be
important for spreading from the initial infection site
(Jordan et al., 2007). Duplication of the M. pneumoniae
Abbreviations: SEM, scanning electron microscopy; TX, Triton X-100.
A supplementary figure showing speed distributions for motile cells of
each species is available with the online version of this paper.
286
attachment organelle at a site adjacent to the existing one
accompanies initiation of DNA replication (Seto et al.,
2001).
The cytoplasm within the M. pneumoniae attachment
organelle lacks DNA, is clear of large particles, and contains
a Triton X-100 (TX)-insoluble structure, the electron-dense
core (Biberfeld & Biberfeld, 1970), which appears to be
connected to an ill-defined network of cytoskeletal filaments
present throughout the cell body (Meng & Pfister, 1980;
Göbel et al., 1981). Proper assembly of the core is essential
for formation of a functional attachment organelle (Krause
& Balish, 2004), making the core essential for virulence. The
core consists of several subdomains, including a distal
terminal button which is probably in direct contact with the
attachment organelle tip, a perpendicularly striated double
rod, and a proximal complex that includes a bowl-shaped
component at the base (Henderson & Jensen, 2006; Seybert
et al., 2006). Although studies of M. pneumoniae attachment
organelle mutants have led to the identification of many
attachment organelle proteins and to the outline of an
assembly pathway (Krause & Balish, 2004), how each protein
contributes to the structure of the core remains poorly
understood.
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Mycoplasma attachment organelles
Mycoplasma gliding is poorly understood compared to
other prokaryotic forms of motility. The M. pneumoniae
motility mechanism has been addressed through characterization of mutants with differences in colony-spreading phenotypes, resulting in the identification of a
moderate number of genes of both known and unknown
function (Hasselbring et al., 2006b). In Mycoplasma mobile,
a distant relative whose adherence, motility and terminal
organelle components are unrelated to those of M.
pneumoniae (Miyata, 2005), gliding is proposed to involve
the cyclical binding and release by the adhesin Gli349
(Uenoyama et al., 2004) of carbohydrate moieties present
on a wide variety of host-cell proteins, including those
deposited on the surface of the microscope slide from
serum in vitro (Nagai & Miyata, 2006). A similar bindingand-release process might occur in M. pneumoniae, albeit
through the use of an unrelated set of adhesins. In support
of this hypothesis, the adhesins P30 (Hasselbring et al.,
2005) and P1 (Seto et al., 2005a) have both been implicated
in gliding motility of M. pneumoniae. Alternative hypotheses have focused on the electron-dense core as the major
component of the M. pneumoniae motor, citing differences
in fine features of individual M. pneumoniae cores
(Henderson & Jensen, 2006) or postulating that the core
is capable of twisting within the attachment organelle
(Hegermann et al., 2002). Core-centred models require
that the morphology of the core would change during
motility, such as by shortening or twisting, and one might
further predict that organisms with different motile
properties would exhibit corresponding differences in the
morphology of the core. The ability to distinguish among
these models is predicated on an understanding of the
relationship between motile properties and attachment
organelle features. One approach to the basis for
mycoplasma motility is the characterization of relatives of
M. pneumoniae that have a similar motility mechanism but
differing motility characteristics, thereby constituting a
series of ‘naturally occurring mutants’.
Eight species are currently assigned to the M. pneumoniae
cluster based on 16S rRNA sequence similarity (Johansson &
Pettersson, 2002) and the presence of an attachment
organelle that contains a core (Balish & Krause, 2005).
Gliding has been described for some of these species,
including M. pneumoniae, Mycoplasma genitalium,
Mycoplasma gallisepticum and Mycoplasma amphoriforme,
while Mycoplasma alvi has been reported to be non-motile
(Bredt, 1979; Hatchel et al., 2006). Mean speeds reported in
this cluster range from 50 nm s21 for M. amphoriforme to
.300 nm s21 for M. pneumoniae (Hatchel et al., 2006).
Homologues of the M. pneumoniae attachment organelle
proteins required for cytadherence and motility are
restricted to the M. pneumoniae cluster (Tham et al., 1994;
Dhandayuthapani et al., 1999; Papazisi et al., 2002), making
it likely that a common fundamental mechanism involving
this set of proteins underlies attachment organelle-mediated
functions in each of these organisms. For most of
these species, detailed analysis of the dimensions of the
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electron-dense cores has not been performed. Correlating
molecular and structural aspects of attachment organelle
components with cytadherence and motility-related properties in each species will reveal which components are likely
participants in the motility process, directing future research
on the mechanistic aspects of mycoplasma gliding motility.
We have therefore examined each species of the M.
pneumoniae cluster with respect to gliding motility and,
for motile species, the morphological features of attachment
organelles and cores. Time-lapse microcinematographic
analysis revealed gliding in all species of the M. pneumoniae
cluster except M. alvi. However, scanning electron microscopy (SEM) revealed substantial differences in cell and
attachment organelle dimensions among all species which,
while largely associated with phylogenetic relatedness,
correlated poorly with gliding motility characteristics, not
supporting a direct role for the core in motility. In addition,
SEM revealed evidence of DNA association with the
proximal end of the core in most species.
METHODS
Growth and culture conditions. The strains used in this study were:
M. pneumoniae strain M129; M. genitalium strain G37; M.
gallisepticum strain Rlow; Mycoplasma imitans strain 4229; M.
amphoriforme strain A39; Mycoplasma testudinis strain 01008;
Mycoplasma pirum strain 70-159; and M. alvi strain Ilsley. Cells were
grown in plastic tissue-culture flasks from frozen stocks for 2–10 days
at 37 uC (30 uC for M. testudinis) in SP-4 broth (Tully et al., 1979) to
mid-exponential phase (phenol red indicator was orange). M. alvi was
also grown in a modified SP-4 formulation containing a 1 : 1 mixture
of fetal bovine and porcine sera (HyClone) and grown until slightly
turbid. Motility stocks were prepared as previously described (Hatchel
et al., 2006). For M. testudinis and M. pirum, cells were passaged three
to five times in SP-4 broth to enrich for plastic-attached subpopulations prior to further use and analysis.
Phylogenetic tree. The 16S rRNA sequence for each species was
entered into BioEdit Sequence Alignment Editor v. 7.0.5.2 (Hall,
1999) for trimming and neighbour-joining alignment by CLUSTAL_X v.
1.8 (Thompson et al., 1997). The phylogenetic tree was generated
from these data by NJPlot (Perrière & Gouy, 1996).
SEM. SEM was performed as previously described (Hatchel et al.,
2006) with minor modifications. Briefly, stocks were grown in 24-well
plates containing glass coverslips for 3 h to 1 day before processing. For
analysis of TX-insoluble structures, coverslips were incubated for
30 min at 37 uC in a solution containing 2 % TX in TN buffer (20 mM
Tris/HCl, pH 7.5, 150 mM NaCl). For DNase treatment, cells were
grown for 1–4 days on coverslips before processing. TX extraction was
followed by a 30 min incubation with 5 U bovine pancreas DNase I
(Sigma-Aldrich) in 500 ml 16 DNase reaction buffer (10 mM Tris/
HCl, pH 7.6, 2.5 mM MgCl2, 0.5 mM CaCl2) at room temperature.
For RNase A treatment, TX-extracted coverslips were incubated 30 min
in 500 ml TE buffer (10 mM Tris/HCl, pH 8.0, 1 mM EDTA)
containing 100 mg RNase A (Qiagen). For whole cells, coverslips were
fixed 30 min in fixative A (1.5 % glutaraldehyde/1 % formaldehyde/
0.1 M sodium cacodylate, pH 7.2; M. pneumoniae, M. genitalium, M.
gallisepticum, M. imitans and M. pirum) or fixative B (1 %
glutaraldehyde/2 % formaldehyde/0.1 M sodium cacodylate, pH 7.2;
M. amphoriforme and M. testudinis). For visualization of TX-insoluble
material, fixative A was used for all species. Dehydration, critical-point
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J. M. Hatchel and M. F. Balish
only M. alvi did not attach to glass and exhibit gliding
motility, as previously reported (Bredt, 1979). M. alvi did
not attach under any conditions we tested, and consequently we were unable to investigate it further. For M.
pirum and M. testudinis, only a small minority of the
population derived from the initial stocks attached to glass.
In these cases, all further studies were performed on
plastic-attached subclones. Efforts to enrich for a similar
population of M. alvi cells were unsuccessful.
Fig. 1. Phylogenetic tree based on 16S rRNA sequences of
mycoplasmas within the M. pneumoniae cluster. Scale bar, 0.1
substitutions per site.
drying and gold coating were performed as previously described
(Hatchel et al., 2006). Images were viewed and captured on a Zeiss
(Carl Zeiss) Supra 35 FEG-VP scanning electron microscope operating
at 5 kV. Dimensions of whole cells, TX-insoluble structures, and
structures following DNase treatment were measured individually
using SPOT software (Diagnostic Instruments).
Time-lapse microcinematographic analysis. Motility sequences
were captured as previously described (Hatchel et al., 2006). Briefly,
cells were inoculated into chamber slides containing SP-4+3 %
gelatin. After 3 h incubation at 37 uC, cells attached to the slide were
visualized using a Leica DM IRB inverted microscope (Leica
Microsystems). The sample was held at 37 uC in a heated chamber.
Twenty-seven consecutive phase-contrast images were taken at
appropriate time intervals and later merged using Adobe Photoshop
CS version 8.0. The speed of each cell was computed by measuring the
distance travelled and dividing it by the duration of movement. Cells
moving in fewer than ten frames were scored motile, but were not
included in the calculation of mean speed because below this
threshold we regarded the speed measurements as unreliable.
RESULTS
Gliding motility
Of the eight species found in the M. pneumoniae cluster
based on 16S rRNA sequences (Fig. 1; Pitcher et al., 2005),
Gliding characteristics, which were previously determined
by our method for M. pneumoniae, M. amphoriforme and
M. gallisepticum, were assayed for the remaining species
(Hatchel et al., 2006). Mean speeds ranged from 28 nm s21
for M. pirum to 2970 nm s21 for M. testudinis (Table 1; see
also Supplementary Fig. S1, available with the online
version of this paper). M. testudinis was the fastest, though
with the lowest percentage of motile cells. Although cells
glided in the direction of the attachment organelle, largescale cellular movement appeared generally random for
most species. However, ~80 % of M. genitalium and M.
testudinis cells moved in nearly circular patterns (not
shown). There was no relationship between phylogeny and
gliding speed.
Attachment organelle and cell morphology
To test whether gliding characteristics correlated with
ultrastructural features of the attachment organelle, we
analysed both whole cells and TX-insoluble electron-dense
cores in each of the seven adherent, motile species by SEM.
Attachment organelles were divided into substructures for
common reference: the distal moiety was termed the knob,
and the proximal portion, the shaft (see Fig. 2h).
Dimensions of individual attachment organelles and cells
(Table 2) were measured.
M. gallisepticum (Fig. 2a), M. imitans (Fig. 2b), M.
amphoriforme (Fig. 2c), and M. testudinis (Fig. 2d)
exhibited short, wide attachment organelles, with mean
lengths from 120 to 150 nm, and widths from 130 to
150 nm (Table 2). The knob was distinct from the shaft in
M. gallisepticum and M. amphoriforme, sometimes in M.
Table 1. Gliding motility parameters at 37 6C
Mycoplasma species
M.
M.
M.
M.
M.
M.
M.
pneumoniae str. M129*
genitalium str. G37
gallisepticum str. Rlow*
imitans str. 4229
testudinis str. 01008
amphoriforme str. A39*
pirum str. 70-159
Mean speed
(nm s”1)±SD
Range (nm s”1)
336±59
111±22
131±38
107±31
2970±570
49±19
28±8
197–538
62–172
50–286
41–194
2180–4740
15–133
12–58
Percentage of cells moving
per frame (total cells
analysed)
51
72
64
65
37
53
45
(149)
(126)
(421)
(173)
(100)
(1034)
(289)
*Data from Hatchel et al. (2006).
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Mycoplasma attachment organelles
Fig. 2. Scanning electron micrographs of mycoplasma cells grown on glass coverslips. Three representative cells are shown
for each species. Images are aligned so that the attachment organelle is at the top. (a) M. gallisepticum; (b) M. imitans; (c) M.
amphoriforme; (d) M. testudinis; (e) M. pirum; (f) M. genitalium; (g) M. pneumoniae; (h) schematic of mycoplasma cell. Open
and black arrows indicate a ridge-like feature at the base of the attachment organelle shaft in M. gallisepticum and M.
amphoriforme respectively; white arrows indicate striations on the M. pirum attachment organelle; black arrowheads indicate
the curvature of M. testudinis and M. genitalium attachment organelles. Scale bar, 250 nm.
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J. M. Hatchel and M. F. Balish
Table 2. Whole-cell dimensions (nm)±SD, mean of 30 cells
Whole-cell length
M.
M.
M.
M.
M.
M.
M.
ND,
pneumoniae
genitalium
gallisepticum
imitans
amphoriforme
testudinis
pirum
1500±420
590±50
830±120
620±50
770±70
680±100
720±110
Cell body
Attachment organelle
Length
Width
Length
Width
520±90
420±50
710±110
500±50
620±70
560±100
480±90
220±30
280±50
330±70
350±30
360±60
280±20
230±20
290±40
170±20
120±20
120±10
150±20
120±10
250±30
80±10
80±10
150±20
140±20
150±20
130±20
100±10
Knob
Length
Width
ND
ND
80±10
100±20
80±10
150±20
ND
90±10
ND
140±20
ND
ND
100±20
100±10
Not determined because knob and shaft were not uniformly distinguishable.
testudinis, but not in M. imitans. The M. pirum attachment
organelle, which also featured a prominent knob, was long,
at 250 nm, and moderately wide, at 100 nm (Table 2), and
uniquely exhibited three or four parallel striations
approximately perpendicular to the long axis (Fig. 2e).
M. genitalium (Fig. 2f) and M. pneumoniae (Fig. 2g) had
narrow shafts, only 80 nm in width. The M. genitalium
structure, at 170 nm, was substantially shorter than that of
M. pneumoniae, which at 290 nm was the longest of the
cluster, consistent with previously noted differences (Lind
et al., 1984). Knobs were indistinct in M. pneumoniae
attachment organelles (Fig. 2g). The two species that
exhibited circular gliding paths had curved attachment
organelles. For M. testudinis, ~25 % of attachment
organelles were bent (Fig. 2d), whereas 100 % of M.
genitalium attachment organelles were curved an average of
20u±10u from the long axis of the cell (Fig. 2f), and the
knob, prominent in this species, was off-centre in the
direction of this curvature.
Measurements of cell bodies revealed a positive correlation
between cell width and attachment organelle width (Table 2).
M. pneumoniae and M. pirum cells were distinctly narrow in
comparison to the others (Fig. 2e, g; Table 2). In contrast,
cell length appeared unrelated to other features (Table 2). M.
pneumoniae cells were unique in the prominence of the
trailing filament found at the pole opposite the attachment
organelle (Fig. 2g). This structure, whose length varied
greatly, drove the mean length of the M. pneumoniae cell to
1500 nm (Table 2). M. gallisepticum and M. amphoriforme
cell bodies often had a ridge-like feature in the vicinity of the
base of the attachment organelle shaft (Fig. 2a, c).
Cell division
We observed SEM fields of cells from each of the seven
adherent species of this cluster (Fig. 3) to characterize cells
that appeared to be in the process of dividing. Either the
appearance of two attachment organelles at the same pole
on a single cell or two attachment organelles pointing in
different directions was taken to indicate that cell division
was in progress. All species except M. imitans (Fig. 3b) were
290
observed to have cells exhibiting one or both of these
phenotypes. Dividing M. pirum cells were only found to
have attachment organelles at opposite poles (Fig. 3e), and
in M. gallisepticum, adjacently paired attachment organelles
were not observed (Fig. 3a), suggesting that in these species
the new attachment organelle might not form immediately
adjacent to the old one as in M. pneumoniae.
TX-insoluble structures
Substructures of the electron-dense core of the attachment
organelle, namely the terminal button, the rod, the base
and a tuft of fibrous material (Fig. 4h; Hatchel et al., 2006),
have been described. Although we previously had difficulty
resolving structures in M. gallisepticum (Hatchel et al.,
2006), the use of a fresh preparation of TX resulted in
consistent observation of core-like structures in all seven
species (Fig. 4).
Rod length was conserved across species over a narrow
range from 130 to 170 nm; inclusion of the terminal
button raised the values to 180–240 nm (Table 3).
However, rod width varied substantially among species,
from 30 to 80 nm (Table 3), and the variation correlated
well with phylogenetic relatedness. The length of the M.
imitans rod (Table 3) is underestimated on account of
being variably obscured by the unusual base (see below).
The slight difference between the current results and those
previously reported for rod width in M. amphoriforme
might be due to the use of a different fixative preparation
(Hatchel et al., 2006). Terminal button length and width
varied somewhat across species, and although the variation
appeared unrelated to gliding speed, width appeared to
have a phylogenetic component, with M. pneumoniae and
M. genitalium exhibiting the narrowest terminal buttons
(Table 3). The M. pneumoniae terminal button was
frequently difficult to distinguish from the rod (Fig. 4g),
as previously described (Hatchel et al., 2006). The M.
genitalium rod exhibited a pronounced lateral curvature,
and the terminal button was consistently offset in the
direction of this curvature (Fig. 4f). Curvature of the
electron-dense core was also visible in ~25 % of M.
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Mycoplasma attachment organelles
Fig. 3. Representative scanning electron micrographs of mycoplasma cells grown on glass coverslips. Each image is a
representative field of cells. (a) M. gallisepticum; (b) M. imitans; (c) M. amphoriforme; (d) M. testudinis; (e) M. pirum; (f) M.
genitalium; (g) M. pneumoniae. Arrows indicate cells that have two attachment organelles pointing at opposite poles;
arrowheads indicate cells with two attachment organelles not yet at opposite poles on a single cell. Scale bar, 1 mm.
testudinis cells, though in this species the curvature was
more abrupt and located close to the terminal button
(Fig. 4d). Although we could not image the non-adherent
M. alvi, it is known to have an electron-dense core
(Gourlay et al., 1977).
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Except in M. pneumoniae, the base of the electron-dense
core was obscured by irregular material. Treatment of
adherent TX-insoluble fractions with DNase I resulted in
highly consistent loss of this material, exposing the base of
the core (Fig. 4). Incubation in buffer alone had no effect,
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J. M. Hatchel and M. F. Balish
Fig. 4. Scanning electron micrographs of mycoplasma electron-dense cores. Under ‘TX only’, cells were grown on glass
coverslips and extracted with 2 % TX for 30 min at 37 6C (see Methods). Under ‘TX+DNase I’, cells were grown on glass
coverslips, extracted with TX as above, and then treated with DNase I for 30 min at room temperature (see Methods). Two
representative cores are shown for each species with and without DNase treatment, with terminal buttons aligned at the tops of
images. (a) M. gallisepticum; (b) M. imitans; (c) M. amphoriforme; (d) M. testudinis; (e) M. pirum; (f) M. genitalium; (g) M.
pneumoniae; (h) schematic of the mycoplasma electron-dense core substructures; (i) M. gallisepticum treated with TX and then
RNase A. Arrows point to notches in M. gallisepticum and M. imitans bases. Scale bar, 250 nm.
nor did treatment with RNase A (Fig. 4i). Pronase caused
loss of all visible structures (data not shown). In M.
genitalium (Fig. 4f) and M. amphoriforme (Fig. 4c) the
DNase-sensitive material was predominantly filamentous,
with some clumpier regions; in M. gallisepticum (Fig. 4a),
M. imitans (Fig. 4b), and M. testudinis (Fig. 4d) this mass
was mostly nodular, with occasional filaments; and in M.
pirum (Fig. 4e) the material had a smooth appearance with
some short filaments extending from it. These filaments are
consistent with the appearance of loops of DNA that have
been locally denuded of protein (Cunha et al., 2001). Taken
together, these data are consistent with the identification of
the core-associated material as DNA, rendered condensed
by treatment with detergent and salt (Cunha et al., 2001),
although whether the interaction between the DNA and the
core is physiologically relevant is unclear. Treatment with
DNase had no significant effects on measurements of other
292
core substructures, except for occasional lengthening of the
rod due to its being less obscured.
The dimensions of the exposed bases were highly variable
across species (Table 3; Fig. 4). The base of M. pneumoniae
was by far the smallest in both length and width. At the
other extreme was M. imitans, which had a very large and
highly irregularly shaped base (Fig. 4b). The DNase-treated
bases of M. gallisepticum and M. imitans consistently had a
notch-like feature of irregular orientation (Fig. 4a, b),
suggesting that the removal of DNA was equally complete
in each species. No relationship between base dimensions
and gliding speed or phylogeny was apparent across
species. In fact, although attachment organelle morphology
generally correlated well with phylogeny, no relationship
was detected between gliding speed and any element of
attachment organelle morphology.
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Mycoplasma attachment organelles
Table 3. Electron-dense core dimensions (nm)±SD, mean of 30 cells
Terminal button
M.
M.
M.
M.
M.
M.
M.
ND,
pneumoniae
genitalium
gallisepticum
imitans
amphoriforme
testudinis
pirum
Rod
Terminal button+rod
length
Length
Width
Length
Width
ND
60±10
60±20
90±20
60±20
90±20
80±10
90±10
ND
160±20
140±30
130±60
160±20
170±20
130±10
50±10
50±10
30±0
40±10
60±10
80±10
80±10
50±10
90±10
60±20
70±10
80±10
90±10
240±20
220±20
230±30
180±70
220±20
240±20
210±10
Base
Length
Width
70±10
110±20
120±40
240±60
90±20
150±40
100±20
70±10
120±20
140±40
160±50
140±30
120±20
110±10
Not determined because rod and terminal button were difficult to distinguish.
DISCUSSION
Gliding motility and ultrastructure
In the present study, all species of the M. pneumoniae
cluster except M. alvi exhibited gliding motility, raising the
total number of motile mycoplasma species to nine with
the addition of M. imitans, M. testudinis and M. pirum.
These seven organisms exhibited a wide range of speeds
and proportions of cells moving at a given time. M.
testudinis populations had a low frequency of motile cells,
but interestingly, the motile cells glided as rapidly as M.
mobile, whose gliding speed was previously unchallenged
among mycoplasmas (Miyata, 2005). Its ultrastructure and
phylogenetic position suggest that M. testudinis has a
mechanistically similar motor to that of its close relatives,
but that it and M. mobile have evolved, perhaps under
similar unidentified pressures, to glide rapidly.
Alternatively, despite its close relatedness to M. pneumoniae and its similar appearance to its relative, M. testudinis
might have acquired a different mechanism for motility,
perhaps through horizontal gene transfer. However, the
absence of a relationship between the motor mechanism
and gliding speed is observed in Mycoplasma pulmonis,
which appears to have the same gliding machinery as M.
mobile (Seto et al., 2005b), despite its mean speed being
much slower (Bredt & Radestock, 1977).
Aside from the curvature of the rod in relation to largescale near-circular movement in M. genitalium and M.
testudinis, no measured dimension of the attachment
organelle appeared to correlate with gliding characteristics.
Curved paths were previously indicated for M. genitalium
(Pich et al., 2006). The curvature of these structures is quite
different in the two species, with the rod of the M.
genitalium core exhibiting a shallow curvature throughout
(Fig. 4f), and that of the M. testudinis core, when curved,
being sharply bent at a single location (Fig. 4d).
Importantly, this curvature is distinct from the bend
observed in M. pneumoniae cores in cells that are not
attached to the substrate (Henderson & Jensen, 2006). We
do not observe this bend under our conditions, as
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confirmed by high-angle tilt SEM (not shown), perhaps
because when attached to the substrate the attachment
organelle is under tension.
In no case did we observe cores that were twisted, as
predicted by one model (Hegermann et al., 2002).
Although the M. pneumoniae core is clearly suggested to
have a broad and a narrow side (Regula et al., 2001;
Hegermann et al., 2002; Henderson & Jensen, 2006), there
was little variation in rod width within any species. The
inchworm model, an alternative model for core function
during gliding (Henderson & Jensen, 2006), invokes
repeated contraction and expansion of the core along its
length, which might have been borne out by substantial
differences in rod length of cells within a single species.
However, except for M. imitans, whose rod was difficult to
measure because of obscuration by the large, irregular base,
distributions of the length of the terminal button plus the
rod were not dramatic. It is possible that the degree of
contraction is too small to be measured by our techniques,
but also possible that contraction does not occur. Also,
observations of gliding cells did not suggest any quantized
movement, though the step size could be smaller, or the
steps faster, than our ability to detect. The consistency in
the dimensions of cores within each species leads us to
propose that the electron-dense core, though an important
determinant in attachment organelle biogenesis, is not
especially dynamic, and not directly involved in motility.
Cell division
Whether or not the electron-dense core has direct
involvement in gliding motility, it might well play a direct
role in some other process. Its roles in normal attachment
organelle formation and adhesin localization in M.
pneumoniae are well documented (Krause & Balish,
2004), though other core-lacking mycoplasmas such as
M. mobile (Shimizu & Miyata, 2002) form head-like
structures that appear to function analogously to attachment organelles. The special relationship between attachment organelle formation and DNA replication in M.
pneumoniae might highlight a role for the electron-dense
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J. M. Hatchel and M. F. Balish
core in coordination between the two events. An increase
in the amount of cellular DNA is accompanied by
appearance of a second attachment organelle adjacent to
the first one in M. pneumoniae, and the distance between
the two organelles increases with continually increasing
DNA levels (Seto et al., 2001). Furthermore, time-lapse
microcinematography of dividing M. pneumoniae cells
reveals that a new attachment organelle generally appears
adjacent to the old one, and the new one anchors the cell in
place while the old one glides away from it (Hasselbring
et al., 2006a), resulting in the two structures being present
at opposite poles. Finally, in M. gallisepticum, newly
synthesized DNA is specifically enriched in a biochemical
fraction itself enriched for attachment organelles (Maniloff
& Quinlan, 1974). Although it could be artefactual, the
physical interaction observed in this study between the
cellular DNA and the electron-dense core is intriguing and
will be a subject of future studies.
dimensions; as such, it might constitute a more general
model for attachment organelle function in species of the
M. pneumoniae cluster.
ACKNOWLEDGEMENTS
This work was supported by funds from Miami University. SEM was
performed at the Miami University Electron Microscopy Facility. We
thank the following for strains: L. Duffy and K. Waites, University of
Alabama-Birmingham (M. genitalium); S. Kleven, University of
Georgia (M. imitans); M. Brown, University of Florida (M. testudinis);
M. Davidson, Mollicutes Culture Collection, Purdue University (M.
alvi, M. pirum). Thanks also to R. Balish and E. Bridge for suggestions
about the manuscript.
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