Department of Molecular and Cellular Biology1 and Department of Physics2, University of Arizona, PO Box 210106, Tucson, AZ 85721-0106, USA
Gonville and Caius College, Cambridge CB2 1TA, UK2
Author for correspondence: Neil H. Mendelson. Tel: +1 520 621 3617. Fax: +1 520 621 3709. e-mail: nhm{at}u.arizona.edu
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ABSTRACT |
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Keywords: bacterial fibres, motions on surfaces, rolling, walking, pivoting
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INTRODUCTION |
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At all stages of macrofibre formation the structures have the same helix hand and twist, suggesting that individual cells grow with constant twist in a given environment and that the supercoiling process responsible for fibre morphogenesis is always negative supercoiling (Mendelson et al., 1984 ). Macrofibre helix hand and degree of twist are governed by both genetic and environmental factors. Many strains are capable of forming structures ranging from tight right-handed to tight left-handed fibres depending upon environmental conditions (such as temperature, nutrient composition and the concentration of certain ions) and can be made to change from one form to another by changing the environment (Mendelson & Thwaites, 1988
). Although macrofibre helix hand and twist state are thought to reflect conformation states of the cell wall polymers, presumably established during peptidoglycan incorporation into the fabric of the wall, a direct relationship between polymer and cellular structure remains to be shown.
The observation that macrofibres sediment when transferred into fresh fluid medium of the same composition as that in which they are produced suggests that fibre morphogenesis occurs on the floor of the growth chamber rather than in the bulk phase of the fluid growth medium. If so, the contact fibres make with the floor is likely to be a significant factor in fibre assembly because impediment of the twisting produced when cells grow triggers supercoiling, the key process in fibre morphogenesis. Macrofibres on a surface may be influenced in two ways: by friction and by the constraining effect of the surface. Whether friction or boundary effects do indeed influence macrofibres growing on a surface was sought in the experiments described here.
To examine fibresurface interactions, a new microscopy system was constructed that provides both a lateral view of fibres and the floor of a glass growth chamber, as well as a conventional view of the structures from above. Analysis of video films obtained using the new system show that fibres make contact with the floor during their growth and undergo motions such as rotation about a centre located near the centre of a fibres length, rolling over the surface and stepping. In addition, fibres assume configurations not previously observed when they were viewed either from above or below: they perch on points such as the loops at their ends or side branches that protrude from the fibre shaft.
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METHODS |
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Media and growth conditions.
The complex medium TB, consisting of 10 g Bacto Tryptose (Difco), 3 g Bacto Beef Extract (Difco) and 5 g NaCl (l deionized water)-1 (Mendelson & Favre, 1987 ) was used in static cultures housed either in standard 100 mm diameter plastic Petri dishes or in glass growth chambers constructed from 75x25 mm glass microscope slides. The chamber dimensions were 56x25x13 mm (length, width, height). Right-handed FJ7 fibres were produced by overnight growth in 10 ml TB containing 50 mM MgSO4 at 20 °C. Left-handed RHX fibres were produced in a similar manner using TB containing 50 mM (NH4)2SO4. A single intact fibre was transferred into medium of the same composition in the glass growth chamber on which the top was covered with two glass slides. For Petri dish cultures, a single fibre was disrupted by toothpick transfer into medium of the same composition. Both glass chamber and Petri dish cultures used for studies of growth dynamics were grown on a microscope stage at 24 °C.
Video film production and analysis.
Three sorts of time-lapse films were produced: low magnification films showing the view of Petri dish cultures from above; side-view films showing contact of the fibres with the floor of the glass growth chamber; and dual-view films that simultaneously show the view from above and from the side of structures in the glass growth chamber. For side-view films the growth chamber was illuminated at an oblique angle using a series 180 high intensity Fibre-Lite (Dolan-Jenner, VWR Scientific Products). Dual-view films were illuminated simultaneously from below and from the side with the same light source using two light pipes.
Dual-view films were obtained using the system shown in Fig. 1. Two Cohu charge-coupled device cameras simultaneously captured images from above and from the side. One camera was fitted to an Olympus SZ-Tr zoom stereo microscope positioned above the growth chamber, the other to a Bausch & Lomb monocular compound microscope tube aimed horizontally at the growth chamber. Both images were transferred to a Phase Eight screen splitter (Vicon Industries). The synchronized output was then sent to a GYYR time-lapse VHS tape deck (Odetics). Both images were recorded simultaneously on the same film, and a time and date stamp was printed on each frame by the GYYR tape deck. The two images on each frame were arranged so that the image from the camera positioned above is displayed on the top half of the frame and that showing the side view appears on the bottom half of the frame.
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RESULTS |
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Macrofibre pivoting has been studied at higher magnification on glass surfaces using the growth chambers described earlier. Dual-view films of right-handed FJ7 show that pivoting involves both rolling and stepping over the surface. Rolling is caused by the twisting of a fibres shaft about its axis as it grows in contact with the surface (a link is available at http://mic.sgmjournals.org for an example of the rolling of a terminal loop described by Mendelson et al., 2000 ). The fibre shaft itself may also rotate about its axis by transferring its weight from one side branch to another as the shaft twists, or by alternating from an elevated position when perched on a branch to lying flat on the surface until twisting brings the branch into contact with the surface again. Such motions take place on a short time scale: that of a single revolution of some section of the macrofibre, i.e. much less than the period of a pivoting revolution. Corresponding variations can be seen in the rate of pivoting (Fig. 2
). There are particularly rapid variations at 32·5 min and at 53 min. The mean pivoting rate from 20 min onwards is 17·6° min-1, so that these rapid variations are separated by almost exactly one revolution of pivoting. There are other smaller short term variations in pivoting rate. Most of these appear however, to be ramifications of the raising and lowering of the fibre shaft as it either perched on short side branches or rested directly on the glass when the branch upon which it had perched was no longer positioned beneath it.
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Fig. 5 shows the variation with time in the elevation of the 1 end of the macrofibre of Fig. 4
. Comparison of Figs 5
and 2
reveals that the major variations in pivoting rate occurred at times when the 1 end took large steps from the floor.
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Moving large structures
Bacterial macrofibres that matured into ball-form structures (Mendelson et al., 1997 ) also perched on short macrofibre legs that grew from the ball surface (Fig. 8
; links to supplementary data for this figure are available at http://mic.sgmjournals.org) The legs supporting these structures twisted and slid over the surface but they were unable in general to cause the entire structure to move. When, however, long fibres grew out of the surface of a ball structure and made contact with the floor, they were seen to pivot about the point of attachment. Motions produced by protruding fibres were observed to cause ball structures to pivot. Pivoting of the larger structure appears to result either from dragging by the pivoting fibre or in smaller structures because the torque on the pivoting fibre due to the drag of the fluid medium is reacted on the ball causing it to rotate in the same direction. (Fig. 9
; links to supplementary data for this figure are available at http://mic.sgmjournals.org) Peripheral fibres eventually supercoiled and collapsed onto the ball surface, thereby contributing to its mass.
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DISCUSSION |
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A simple twisting cylinder is not an ideal model however for a mature macrofibre. Macrofibres are not at all uniform, contact is made at a few points only (a mean of less than 4), and there is a constantly changing pattern to their distribution (Fig. 4). Quite long branches extend from the fibre shaft, the results of past supercoiling involved in morphogenesis, and the tips of branches are often contact points with the surface below (Fig. 3
). As a result, it is not surprising that there are substantial variations in pivoting rate (Fig. 2
, Table 1
). What is surprising is that the mean pivoting rate remains constant during the changing contact pattern and even during supercoiling itself. Pivoting is a remarkably robust phenomenon.
Local translation of macrofibres is achieved when the position of the centre of mass is changed. This occurs by the sweeping of an arm (Fig. 6) and, more significantly, by supercoiling. Suppose that a uniform fibre supercoils by folding and loop formation at its centre; the centre of mass, in this also the centre of pivoting, moves to a point that was formerly one quarter of the length from one end. Similar situations with perhaps smaller displacements arise in the supercoiling of normal macrofibres, i.e. those that are already non-uniform because of asymmetric supercoiling. Macrofibre motion is linked to cell growth, which in itself is quite slow: a 2 mm long fibre growing under the conditions described elongates at about 0·3 µm s-1. This elongation rate is similar to the rate at which eukaryotic cells move by polymerization of filamentous proteins. A nematode sperm cell crawls at about 1 µm s-1 by assembling the major sperm protein into filaments (Italiano et al., 2001
; Mahadevan & Matsudaira, 2000
). Macrofibres move by insertion of new cell wall polymers into the fabric of the wall (Thwaites & Mendelson, 1991
) in cells throughout the entire fibre.
Although macrofibre movement by growth extension alone is slow in comparison to the rates at which bacterial cells can move using other motility mechanisms, macrofibres can move at speeds approaching those of single cell swimming or gliding (Amsler & Matsumura, 1995 ; Berg & Brown, 1972
; Henrichsen, 1972
; Macnab, 1996
). Rapid speeds in macrofibres are achieved because the collective growth behaviour of all the cells within a single macrofibre contribute to its motion and the magnitude of forces resulting from their helical growth is much in excess of that needed to overcome resistances such as viscous drag of the fluid and friction with a solid surface. A 2 mm long macrofibre pivoting at 17·6° min-1, for example, has a tip speed of only 5 µm s-1, but the tip speed of the moving arm during the step (Figs 6
and 7
) is 46 µm s-1. The distances traversed by an individual macrofibre are much shorter however than those travelled by cells using conventional motility.
Touching a solid surface during growth strongly influences macrofibre motion and may also play a major role in macrofibre morphogenesis. It is difficult to estimate the magnitude of the friction forces involved. The density of macrofibres is only about 1·015 g ml-1 so that, even if the whole weight of a fibre were borne by one surface contact, the normal load would be small. For a fibre of 30 µm diameter and length 2 mm it would be a mere 0·2 nN, in which case friction could hardly exceed 0·1 nN. However, the normal force at a contact also depends on the geometry of the macrofibre when bent, as bent beams react substantial forces at their supports. The friction forces may be as large as 1 nN. If such a friction force were applied to the tip of a branch such as that making contact in Fig. 3(a and d), which is about 400 µm long, the torque transmitted to the macrofibre shaft would be 0·4 pNm. If this were applied to a macrofibre of 30 µm diameter, the torsional rigidity of which is estimated to be 0·056 pNm2, it would twist it by about 0·4° mm-1. In contrast, the fibre grows with a twist of 36 turns mm-1, approximately 30000 times that resulting from friction. Torques reacted on a fibre shaft due to fluid friction (viscosity) are also of the order of pNm (for example those developed by a sweeping arm as in Fig. 6
and Mendelson et al., 2000
). Clearly these estimated torques are only a tiny fraction of those required to fully block the twisting with growth that is presumed to be the prime cause of macrofibre morphogenesis. The mechanism by which contact with a solid surface influences macrofibre morphogenesis remains therefore to be elucidated. The forces associated with these estimated torques (of the order of nN) can however be measured using specially designed cantilever transducers. This is the subject of current research.
The inferred magnitude of forces and torques associated with growth of macrofibres on a solid surface suggest that only a tiny fraction of the potential power of growth is used in macrofibre motion and morphogenesis. The full power available can be estimated as follows. If the twisting with growth of a (cylindrical) macrofibre of torsional rigidity C and twist N is resisted by a torque Q, the relative (angular) velocity of the ends is L· (N-Q/C) and the power transferred to the resisting load is Q times this. The maximum power transfer is (when Q=1/2CN) 1/4CL· N2. For the macrofibre considered above (diameter 30 µm, length 2 mm and twist 36 turns mm-1) this is approximately 200 pW. For comparison, the power produced by a flagellar motor when fully energized is about 2x10-3 pW (Berg, 1995 ), and that of a pilus retracting (and consequently causing cells to move over a glass surface) is about 8x10-5 pW (Merz et al., 2000
). The difference between macrofibre power and that of these other two bacterial motion engines, factors of 105 and 108 respectively, is very great, but note that a macrofibre contains approximately 5x105 cells. This cellular concentration brings the macrofibres growth power within the realm of possible uses. Twist and writhe can transduce the power of macrofibre growth creating a miniature machine that does work by moving objects. Some examples can be seen in Mendelson (1999)
and in sequence 1 at http://research.biology.arizona.edu/mendelson/enviromicro. Whether such motion serves an adaptive purpose for macrofibres remains to be determined.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 31 October 2000;
revised 5 January 2001;
accepted 8 January 2001.
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