Gesellschaft für Biotechnologische Forschung mbH, Bereich Mikrobiologie, Mascheroder Weg 1, D-38124 Braunschweig, Germany1
Zentrum für Molekulare Biologie der Universität Heidelberg, Im Neuenheimer Feld 282, D-69120 Heidelberg, Germany2
Author for correspondence: H. Lünsdorf. Tel: +49 531 6181495. Fax: +49 531 6181411. e-mail: Lunsdorf{at}gbf.de
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ABSTRACT |
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Keywords: gliding motility, myxobacteria, frozen motion
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INTRODUCTION |
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The present study describes surface features of actively gliding versus non-gliding cells from the fruiting gliding bacteria M. xanthus and Stigmatella aurantiaca and the non-fruiting gliding bacteria Flexibacter filiformis and Flavobacterium johnsoniae (formerly Cytophaga johnsonae), supplemented by studies on gliding motility mutants of M. xanthus. For the first time, the in toto structure of the motility apparatus and its plasticity, revealed from the gliding-motility-associated cell topography, could be observed at high resolution. This process of cellular translocation is based on a rather dynamic helical superstructure, built as a circularly closed continuum.
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METHODS |
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Cryo-preparation and electron microscopy.
Gliding motility as a dynamic state was fixed physically by shock freezing. In order to reduce the sample volume to a size optimal for freezing, cover-glasses were briefly spun off with the aid of a mini sawmill (Proxxon, Minimot 40). The cover-glass was fixed to double-adhesive tape, mounted on an aluminium stub used in scanning electron microscopy, and was plugged to the mini sawmill. After a 1 s run, the cover-glass was immediately dipped into melting nitrogen slush (-210 °C), taking care that air-drying was reduced to a minimum. After temperature equilibration the cover-glass with the shock-frozen cells was transferred into a storage vessel filled with liquid nitrogen. Samples were mounted to the stage of a freeze-drying facility (Baltec MED 020 unit) at a temperature of -120 °C. Freeze-drying was done in two steps at a sublimation pressure of 3 x10-6 mbar: (a) 30 min at -90 °C and (b) 30 min at -80 °C. The freeze-dried samples were subjected to shadow-casting at 90° with platinumcarbon, and warmed to ambient temperature before they were withdrawn from the freeze-drying unit. The freeze-dried samples were stabilized by additional sputter coating with a 4 nm layer of gold (sample to target distance 50 mm, sputter current 30 mA, coating period 30 s) in a sputter coater device SCD 040 from Balzers Union. Freeze-dried bacteria were examined in a DSM 982 Gemini scanning electron microscope equipped with a field emission gun (Zeiss). Micrographs were taken at a primary magnification from x3000 to x20000 at an acceleration voltage range from 0·5 to 10 kV and a working distance from 4 to 5 mm.
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RESULTS |
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In general, by following the traits of the helical folds, the in toto topography of this dynamic state can be described as a circularly closed band-shaped continuum which is twisted along the longitudinal axis of the cell. The proximal end of a cell may present the active helical principle and the distal part of the cell body appears smooth, as can be recognized from Fig. 3(b) (asterisk). This interruption of continuous helicity may be indicative of an only partially active gliding machinery or even different states of gliding activity.
Morphological evidence of the progression of gliding force vectors and surface dynamics
The question arises of how the translational force vectors that drive the cell ahead are established. Do they just follow the traces of an either left- or right-handed rotating spiral driller as a rigid construction with a constant wavelength, i.e. equidistant nodes, or are they based on an apparatus which in its overall complexity produces varying internodal distances at variable wavelengths? The latter mode would imply that gliding motility is based on oscillation of the twisted closed continuum.
Here, the filamentous form of F. filiformis was a suitable multicellular system to test and elaborate these details. In Fig. 1(e) and Fig. 3(e, f, g)
(twin arrowheads), several nodes at varying distances can be observed along the filaments. Nodes do not appear to be restricted to individual cells within the trichome but uniformly cross the intercellular septum (Fig. 3f
, circle). If two trichomes contact each other and are oriented lengthwise, their helical nodes appear aligned in phase, thus giving evidence for synchronization (Fig. 3e
, open arrows). A schematic presentation of internodal distances relative to the cells apex is outlined in Fig. 3(h)
, encompassing 14 individual filaments. The mean value of 101 internodal distances of F. filiformis trichomes was found to be 2·27 µm±1·09 µm. The overall wavelength
is 5·54 µm as a mean and minimally 2·36 µm versus maximally 6·72 µm. The standard deviation of 1·09 µm underlines the great variance of the internodal distances. This characterizes the underlying apparatus as flexible rather than rigid in nature. Further, the distribution of nodes along the filaments indicates that they are mainly found at the apex (see filaments 2, 3, 6, 9, 10, 11 and 13 in Fig. 3h
), though nodes appear further downwards, separated by a region free of intersections (Fig. 3h
, filament 1). However, the apex is not the only region presenting nodes. This disproves the assumption that gliding activities along the filament were restricted to its leading edge. Nodes were found scattered generally along the whole filament (see Fig. 3h
, filaments 8, 12 and 14).
In general, the distribution of nodes along the filaments thus strongly implies gliding activity to be based on oscillating translational force vectors, as is outlined in a model (Fig. 3i).
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DISCUSSION |
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To minimize the shortcomings mentioned above, our rationale led us to halt and fix gliding motility of four different bacterial species, i.e. M. xanthus, S. aurantiaca, F. filiformis and F. johnsoniae, by shock freezing under optimal vitrifying conditions in melting nitrogen at -210 °C without the addition of cryoprotectants. This is a prerequisite to maintaining the dynamic states of surface features of actively gliding cells, since these are highly sensitive and respond immediately to alterations of the surrounding milieu.
To differentiate gliding-associated surface patterns from those of non-gliding cells it was necessary to arrest gliding motility as a control. This was achieved effectively by using cyanide and azide as potent inhibitors of electron transport (Ridgway, 1977 ): these are weak proton ionophores and cause a partial breakdown of the membrane potential (Harold, 1972
, 1977
; Duxburry et al., 1980
; Dzink-Fox et al., 1997
). Our data show for the first time the obvious impact of electron flow inhibition on surface features of actively gliding cells. These cells appear as rods with a smooth surface and, furthermore, the close contact of adjacent cells within filaments is no longer observed (Fig. 1b, d)
. This may represent the morphological counterpart of impaired cell cohesion, which was found to be energy-dependent in myxobacteria (Gilmore & White, 1985
; Shimkets, 1986
).
Actively gliding cells show a distinct surface pattern that is obviously created by macromolecular structures which are organized in toto as a supertwisted circularly closed band. A clearly helically arranged surface pattern at the end of a cell from M. fulvus assumed to be actively gliding has been described in negatively stained specimens. However, this pattern faded away further down the cell and no data on the helix pitch were obtained (Lünsdorf & Reichenbach, 1989 ). So-called strands, composed of rings and elongated elements, were described as fragments from mechanically broken cells and they were shown to end up in a so-called belt, about 300 nm in width, which was wrapped around the cell. Similar strands were found in M. xanthus (Freese et al., 1997
). It is assumed that by conformational changes of rings relative towards the elongated elements along the strands of the belt, gliding motility of M. fulvus is performed. The present study substantiates the existence of a belt that represents the morphological equivalent of the circularly closed bands seen in relief (Fig. 3i)
. It is observed in all gliding cells examined in the present study and shows a width of 170380 nm. We suppose these circularly closed bands or belts to be directly associated with the peripheral part of the gliding machinery within the periplasmic space. So-called strands, as integral parts of the belt, were found to be situated within the periplasmic space and in direct contact with the outer membrane, as was shown by in situ freeze-fracture studies of M. fulvus (Freese et al., 1997
). This is consistent with the helicity, inferred from light microscopical studies of movement patterns of latex beads and ink particles of cytophagas and flexibacters (Lapidus & Berg, 1982; Ridgway & Lewin, 1988
; Beatson & Marshall, 1994
) or the appearance of slime threads that are helically wrapped around gliding filaments (Reichenbach, 1980
; Halfen & Castenholz, 1970
). These observations fit the presented ultrastructural data of dynamic states of actively gliding cells.
The ultrastructural appearance of unicellular and filamentous gliding bacteria gives an impression of the flexibility of the supertwisted gliding apparatus as a circularly closed continuum, and some general considerations can be made.
(1) The helical pitches from different cells of the same species vary to a certain degree, inferring that gliding is an individual cell process and not strictly synchronized, on the assumption that single cells are not moving in rafts (Fig. 1a, b). One may speculate whether, relative to the degree of gliding intensity of a single cell, the profile of the cells surface is accordingly modulated. Since the observed cells were all prepared from liquid cultures and were brought into contact with the substratum for only a few minutes, all cells were in the Such-situation, i.e. were coordinating and arranging one another before building the gliding swarm. Furthermore, it has to be considered that cells are continuously pressed against the substratum by surface tension during drying of the surrounding liquid. Such flattening has been observed by phase-contrast light microscopy of F. filiformis trichomes as a change in cell width from thin in liquid-saturated surroundings to broad in liquid-deficient surroundings (data not shown). As a consequence of this situation, the topographic profile of the cell surface changed from pronounced to smooth. Therefore, care was taken to analyse only those areas with actively gliding cells that were completely covered with a thin liquid layer immediately before shock freezing. Thus alterations of gliding-associated surface features by surface tension were prevented.
(2) It is obvious from gliding trichomes of F. filiformis that the gliding motility process is often restricted to different parts of the filament, as is outlined by the distribution of helical nodes (Fig. 3h). This demonstrates a gliding correlated alteration of the wavelengths and consequently of the helical pitches along an individual trichome. It seems obvious that a group of equidistant nodes may move along the trichome during gliding similar to travelling waves. In accordance with this impression, nodes appear to cross the septa without being split during transition (Fig. 3f)
. It seems plausible that the nodes of the travelling waves may correlate with the dark adhesion zones that have been described by Godwin et al. (1989) from interference reflection microscopic studies and time-lapse video recording. Furthermore, if two adhesion zones or focal contacts of a trichome or single cell have different translational speeds relative to one another, the interspatial non-adhesive part of the trichome will equilibrate the growing imbalance of tension by bending out and forming a curved trichome, which often could be observed with F. filiformis. If both adhesion zones move with the same speed, the curvature of the trichome will be maintained. In this state, the trichome will glide forward as long as the speed synchronization is maintained and force vectors of both adhesive zones run strictly in parallel. In this way, the diverse curvilinear features of gliding cells, as have been described for M. xanthus (Spormann & Kaiser, 1995
) and cyanobacterial trichomes of Phormidium uncinatum (Häder & Vogel, 1991
) by time-lapse video, could be explained.
(3) The nodes of the supertwisted continuous band may act as physical points in cell alignment, useful for synchronization of the gliding process of several cells laterally in contact (Fig. 3e, open arrows).
(4) The irregular distribution of nodes along the trichomes implies that different motility speeds, possibly correlated with appropriate frequencies, may influence and alter the pitch height.
(5) The presence of right- and left-handed helicity of the continuous band (Fig. 3a, b) may be linked to forwards and/or backwards movements of the cells.
(6) The observed nodes of the actively gliding wild-type cells of M. xanthus are obviously under the regime of the adventurous multigene system (Hodgkin & Kaiser, 1979a , b
), since they are only present in A+S- mutants (Fig. 2a)
and not in A-S+ (Fig. 2b)
or A-S- (Fig. 2c)
mutants and a mgl mutant.
One main feature of the supertwisted circularly closed continuum is its anti-parallelism, as indicated in the model (Fig. 3i, arrowheads). If one assumes the gliding apparatus to move helically along the cell body within the periplasmic space, the force vectors are oppositely oriented. If the adhesive zones of these force vectors have the same intensity of contact with the substratum, the translocating forces will neutralize each other and no net movement will occur. Ridgway & Lewin (1988)
have already mentioned this problem. A simple solution is to postulate an uncoupling of those adhesive zones from the substratum which are oppositely oriented to the direction of cell locomotion. Weak support for this suggestion is obvious from interference reflection microscopy (Godwin et al., 1989
). Here, the bright-shining parts of gliding cells that are not in contact with the substratum represent those regions of oppositely directed force vectors and thus do not hinder forward translocation. However, these different adhesion intensities of cells with the substratum imply a rather complex regulation and coordination of the gliding machinery, and in consequence demand a force sensing capability, which still has to be verified.
The high-resolution data presented, obtained from four different species of gliding bacteria and an A+S- gliding mutant of M. xanthus, for the first time give strong evidence of the in toto topographic appearance of the gliding machinery in action upon freezing the various states of this complex, dynamic motility process. However, its design at the macromolecular level could not be elucidated and high-resolution electron microscopy data at this level remain fragmentary (Pate & Chang, 1979 ; Lünsdorf & Reichenbach, 1989; Freese et al., 1997
). In particular, the question of how the gliding machinery is organized at the septa of filamentous gliding bacteria is enigmatic. Nevertheless, all these morphological data are of value as pieces in this mosaic, termed bacterial gliding motility.
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ACKNOWLEDGEMENTS |
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Received 2 September 2000;
revised 6 December 2000;
accepted 11 December 2000.
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