1 Institut de Génétique et Microbiologie, Bât. 409, UMR CNRS 8621, Université Paris-Sud, 91405 Orsay cedex, France
2 Department of Molecular Biology, University of Gdansk, 80-822 Gdansk, Poland
Correspondence
I. Barry Holland
holland{at}igmors.u-psud.fr
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ABSTRACT |
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INTRODUCTION |
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In order to analyse the effect of swarming on the interplay between nutrient concentration and percentage of agar, most previous studies have used complex medium with laboratory strains of B. subtilis, in particular strain 168, grown on peptone (Ben-Jacob et al., 1994; Matsushita, 1997
), beef extract/tryptose (Mendelson & Salhi, 1996
) and LB (Dixit et al., 2002
). These studies revealed different swarming patterns, from amorphous, largely confluent growth zones, through relatively simple radial branching, to highly complex, densely branched structures. Patterns with pronounced chirality, or repeating concentric zones, were also observed. Whilst the concentration of nutrients influenced patterns, the agar concentration appeared most important in determining bacterial swarming per se (Mendelson & Salhi, 1996
), leading to the conclusion that swarming requires the extraction of water from the agar or reduction of surface friction, processes in which surfactants presumably play a role (Matsuyama et al., 1989
; Matsuyama & Matsushita, 2001
; Bees et al., 2000
).
Although there has been speculation on the possible role of different physical factors, nutrient gradients, quorum sensing or chemotactic signalling (Ben-Jacob et al., 1994; Matsushita, 1997
; Mendelson & Salhi, 1996
; Harshey, 1994
; Belas, 1997
; Rauprich et al., 1996
; Bees et al., 2000
) in the control of bacterial swarming, the precise mechanism(s) by which the bacteria sense a suitable surface to be colonized, and in a co-ordinated way then occupy that surface by seemingly different strategies, remains a mystery.
In this study, we sought to establish conditions that would facilitate the systematic analysis of the nature of the activity of B. subtilis that constitutes the swarming process through branching patterns. This involved the use of a non-laboratory strain, 3610 (a natural wild-type), grown under controlled conditions. As we report below, strain 3610 forms a wide range of swarming patterns under different conditions, including so-called fractal branching (Matsuyama et al., 1989). We have established optimal conditions for reproducible swarming patterns with radial branching, and for the first time on a completely synthetic medium. At least two waves of swarming were analysed microscopically and by time-lapse photography. Importantly, we used in situ phase-contrast microscopy, to document a number of surprising and novel features in the development of the swarm colony. These included long filaments and rafts of cells (near normal length) detected in different locations in the developing swarm colony, and following an abrupt outward migration of many cells, new swarm 2 dendrites assembled de novo from nascent tips, beyond the edge of swarm 1, which then elongated backwards towards the colony centre. In an analysis limited to growth on LB-agar, an independent study by Kearns & Losick (2003)
, carried out whilst this work was in progress, has recently described a form of swarming of strain 3610, in which hyperflagellated cells associated in rafts were detected in samples extracted from the swarming edge. Under these conditions no distinguishable architectural features, such as branching, were observable, and no in situ analysis was carried out.
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METHODS |
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Swarming experiments.
Ten millilitres of the medium to be used in a final swarm plate was inoculated with a single colony and shaken overnight at 37 °C. The culture was diluted to OD570 0·1 and grown until OD570
0·2. This procedure was repeated twice and finally the culture was grown to time t4 (4 h after exit from exponential phase), the OD570 was measured and the culture diluted to OD570
0·01. Two microlitres (
104 c.f.u.) of this diluted culture were placed at the centre of an agar plate with the appropriate medium and incubated at 30 °C (relative humidity 40 % saturation), or other temperatures as indicated in the text, for the requisite time. The inoculum formed a spot, diameter 4 mm. Plates (10 cm diameter) containing 25 ml agar medium were prepared 1 h before final inoculation and dried open for 15 min in a laminar-flow chamber. In some experiments, 15 cm diameter plates with 75 ml agar medium, prepared similarly, were also used. For measuring swarming rates, the diameter of the swarm on duplicate plates was measured each hour after swarming commenced. Measurements from replicate plates were essentially identical.
Microscope observations and time-lapse video.
The photographs of the developing swarm were taken in situ with an Axioplan (Zeiss) microscope equipped with a camera (Princeton Instruments). Bacteria were observed on the plates using Neofluar Zeiss objectives x1·25, x5, x20 and x40. Images were captured (by computer) using the IP Lab Spectrum (Princeton Instruments) software version 4.2, exported as TIFF files and adjusted with Adobe Photoshop software. Plates were removed from the incubator at intervals and examined for a minimal time to avoid any delay in further swarming; alternatively several replicate plates inoculated at the same time were employed, discarding the plate after microscope examination. Replicate swarm plates were highly reproducible in both the timing and pattern of the swarming colony.
Time-lapse video films of swarming colonies were produced using a Proscope digital camera with a Computar zoom TV 8·5 mm lens. The camera was positioned above the Petri dish and the light source below it in a constant-temperature environment. Images were recorded with USB Shot software and adjusted with the Adobe Photoshop program.
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RESULTS |
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The effect of the size of the inoculum was investigated over the range of 50 to 5x104 c.f.u. Swarm colonies were obtained in all cases, with the onset of swarming from the central inoculation point clearly later when fewer cells were used. For example, with an inoculum of 50 c.f.u., the delay was close to 24 h (data not shown). Under the standard inoculum conditions finally adopted (104 c.f.u.), swarming was first visible (at 30 °C) after 1112 h, that is, approximately 7 generations, based on a doubling time of 100 min in cultures grown in liquid B-medium.
Finally, the effect of growth temperature (on B-medium) on the rate of swarming was examined over the range 2242 °C. Periodic rounds of swarming appeared to occur under all conditions, although this was less obvious at high temperatures. This may be due to the constraints of the 9 cm plate when swarming is accelerated, and the masking or blurring of the transitions from one swarm to another by the more rapid growth of the bacteria at the higher temperatures. Temperature also had some effect on the pattern of branching, with particularly dense and intricate branching at 37 and 42 °C, and a central zone of thicker branches increasingly obvious at lower temperatures (see photographs in Fig. 2, and also the higher magnification, Fig. 1E
). The expansion of the colony appeared to increase linearly over most of the time-course at all temperatures.
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The swarming pattern is not dependent upon the presence of glucose or amino acids
The standard B-medium contains glucose as the carbon source and is also supplemented by three amino acids: lysine, glutamate and tryptophan. First we compared swarming (on 0·7 % agar) with glucose or glycerol as carbon source. As expected with the cells growing more slowly on glycerol, swarming was slower but the final results were essentially the same (data not shown). This is in contrast to swarming in E. coli (Harshey & Matsuyama, 1994) or Pseudomonas aeruginosa (Kohler et al., 2000
), which is abolished by growth on glycerol. In the presence or absence of tryptophan or lysine, the swarming rate and patterns obtained were indistinguishable. In the absence of glutamate, which resulted in an increase of the doubling time of cultures in liquid medium (120 min compared with 100 min at 30 °C), the rate of swarming appeared to be reduced. Moreover, supplementation of the basic B-medium simply by glutamate was sufficient to restore the growth rate and the rate of swarming to that obtained with all three amino acids present (all data not shown). Therefore, under these conditions, a supply of amino acids in the medium does not appear to be specifically required for swarming. We also noted that B. subtilis grows in the full B-medium without glucose, presumably using the amino acids as carbon source (generation time 285 min at 30 °C in liquid medium). In the absence of any added sugar (data not shown), the same swarming pattern was still observed, although developing much more slowly.
The swarming zone on B-medium is preceded by a bacteria-free area bounded by a narrow ring
Swarming colonies were examined with the Proscope digital camera or by phase-contrast microscopy. As shown in Fig. 3, when colonies are photographed in reflected light, it is possible to see that the swarm zone is always preceded by a transparent zone devoid of bacteria and delimited several millimetres from the swarm by a narrow ring, approximately 1 mm wide (see also Wakita et al., 1998
). We suggest that this zone, and the ring surrounding the swarm, may contain the wetting agent surfactin (a lipopeptide: Nakano et al., 1992
), secreted by the bacteria to aid in some way their translocation across the surface. Our view that this zone contains surfactin is based on our analysis of several other B. subtilis strains and their ability to swarm on LB-agar, where surfactin is not essential for swarming. This analysis demonstrated a strict correlation between the presence of surfactin and the ability to produce the ring preceding the swarm. Moreover a drop of surfactin (Sigma) placed at the centre of an agar plate produces an identical spreading ring in the absence of bacteria (unpublished data).
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Successive swarming waves were not limited to the dendritic patterns observed on B-medium, but were also evident when swarming on LB was monitored, especially at intermediate times, 515 h (data not shown), but the trace of them was not always visible in the mature swarm after 24 h.
Filamentous cells and many multicellular rafts' are present at different stages of the swarming process
Swarm 1.
The leading region of a swarm colony on B-medium at 30 °C was examined at different times in situ, without disturbing the structure of the colony, by low-magnification microscopy and the Proscope digital camera (see Methods). First, we observed that within 34 generations (46 h) division is blocked in a proportion of the cells, with the consequent appearance of long filamentous cells already at 1112 h, in particular detectable in the rim of the central colony (Fig. 5C). The first indication of the onset of swarming itself (swarm 1) was detected at 1011 h with the appearance of the ring of presumed surfactin around the colony, followed 12 h later by the initiation of dendrite formation (see below).
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At 2122 h, which coincides with the completion of the first swarm and the beginning of the second swarm (Fig. 4, upper left panel), the mature dendrites of swarm 1 were also examined in situ; representative results are shown in Fig. 6(A)
. Each dendrite is bordered along its length by a wide monolayer formed by a palisade of rafts, each containing 45 cells side by side, with in many cases the poles at one end rather precisely aligned (Figs 6B, 6C and 8B
). The multilayered centre of these dendrites is covered by many filamentous cells, some of which also congregate in rafts, where the internal region of the dendrite merges into the palisade (Fig. 6C
).
Swarm 2.
In many experiments with strain 3610, the initiation of swarm 2 displayed the surprising characteristics shown most clearly in Fig. 3(B). Thus, between 18 and 21 h into the swarm, and only possible to observe by reflected light, the edge of the second swarm rather abruptly appears more than 1 cm beyond the edge of swarm 1, with the intervening region seemingly lacking any cellular structures visible by eye without magnification. However, phase-contrast microscopy shows that this region in fact contains a thinly dispersed population of cells, mostly of normal size distribution. In contrast, the visible edge of the swarm, in particular after 2122 h, appears to be constituted by cells assembling or coalescing de novo into new dendrites, commencing at the tips and progressively assembling backwards towards the colony centre. This is shown in Fig. 7
.
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DISCUSSION |
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In B. subtilis, in which the swarming process has been little studied so far, the swarms appear in different forms, from highly intricate branching to carpet-like coverage of the surface, depending on the conditions. However, the mechanisms that trigger swarming behaviour or the formation of different patterns, including dendritic branching, remain completely unknown. Secretion of the protease Epr was reported to be required for swarming of the laboratory strain 168 on LB (Dixit et al., 2002). The presence of both flagella and a detergent-like lipopeptide (surfactin), secreted by the bacteria and subject to regulation by quorum sensing in planktonic culture (Solomon et al., 1995
), has also been implicated in swarming in this organism (Ohgiwari et al., 1992
; Wakita et al., 1998
). Some reports, however, have indicated that swarming can occur independently of either flagella or surfactin (Dixit et al., 2002
; Kinsinger et al., 2003
; Mendelson & Salhi, 1996
). In our studies with the laboratory strain 168 (Trp), the bacteria, although SrfA, still swarmed efficiently on LB agar, albeit less rapidly than strain 3610. Swarming, however, was apparently dependent on flagella since a hag mutant failed to swarm at all on LB. Notably, however, on the synthetic B-medium, strain 168 only swarmed in the presence of surfactin (all unpublished data), indicating that this is absolutely essential for the swarming of this strain under these conditions.
Rafts of hyperflagellated cells of close to normal size distribution, in contrast to the filamentous P. mirabilis swarmer cells, were detected at the swarm edge of strain 3610 on LB (Kearns & Losick, 2003) and a similar grouping of cells into such rafts was observed on B-medium in our in situ analysis. However, the localization of the rafts in this study suggested a role in maintaining the structure of the dendrites, in addition to any possible role they may have in swarming movement per se (see below).
In order to establish a more amenable system for the analysis of the regulation of this complex process of co-ordinated behavioural activity, we considered it important to employ a defined medium under optimized conditions, together with a natural wild-type strain, 3610. This strain has not so far been subjected to extensive culture in the laboratory, which can lead, in particular, to the inactivation of dispensable, so-called social functions (Velicer et al., 1998; Branda et al., 2001
). In addition, the development of the swarm colony was examined in situ, without disrupting the organization of the swarming colony, in our attempts to define some of the important stages in the process under these conditions, including any morphological changes to the cells.
The results obtained in this study indicated a surprisingly complex series of events when 3610 was examined on the synthetic medium. The events include, at the very initial stage of swarm 1, the appearance of bud-like structures at the edge of the inoculation point, composed of a loose association of normal-sized cells. More strikingly, the initiation of swarm 2 is accompanied by rapid migration of individual, non-filamentous cells 12 cm beyond the end of swarm 1. These cells then begin to form de novo the tips of new dendrites which subsequently appear to assemble backwards towards the colony centre. Other notable features include the formation of rafts of cells at the edges of dendrites and filamentous cells in the centre of dendrites. Our analysis also detected an expanding, bacteria-free zone of material preceding the swarm front throughout the swarming process. We suggest this material to be the biosurfactant surfactin, but further studies are required to confirm its nature and its possible role.
In the wild-type strain, the initial buds of swarm 1 in the edge of the central colony develop into the observed dendrites, although how this occurs remains to be analysed. The elongating dendrites then branch in some way into unexpectedly complex patterns. After 18 h, the more mature dendrites, thickened by multiplication of cells, are seen to be bordered by a wide monolayer zone of randomly arranged, closely packed rafts, enclosing a central multilayered area, containing many filamentous cells. Thus, in some remarkable but ordered way, the division cycle of cells in very close proximity in the swarm community is regulated differentially. Concerning the rafts, we have so far only been able to observe a palisade at the edges of the mature dendrites. However, from analysis of the way the nascent dendrites of swarm 2 develop, we suggest that the entire lower surface of each dendrite, abutting onto the agar surface, may initially be laid down as a complete monolayer of such rafts. The tightly packed nature of the uniformly sized rafts and their persistence as a monolayer at least at the edges of dendrites suggests, intriguingly, that growth may have been arrested in these cells. Future studies will address these points. Finally, in some experiments, the swarm 1 dendrites were observed at the end of a period of swarming to terminate at the tips as even more complex, floret-like formations (see Fig. 3A), whose function and detailed structure remain to be analysed.
On the B-medium, the manifestation of the second swarm provides a further surprisingly complex behaviour. In this case, an organized structure, corresponding to the disconnected tips of new dendrites, was first detected at least 12 cm outward from the edge of the first swarm (see Fig. 3B), with the intervening region containing no organized structures, simply an area filled with a low-density population of individual cells. Subsequently, although the precise order of events needs to be confirmed, the new full-length dendrites appeared to form through progressive coalescence or aggregation of cells, commencing from the tips and continuing to assemble towards the colony centre (shown in Fig. 7A
). This presumably occurs by recruiting cells from the intervening region (i.e. between the boundary of swarm 1 and the edge of the new swarm 2). Further studies are also now required to establish the nature of the switch or signalling mechanism(s) that gives rise to the second swarm and the subsequent ability to assemble into the new structures.
As in the very earliest stage of development of swarm 1, in the initial formation of the tips of the swarm 2 dendrites no rafts were detected. However, a monolayer of rafts soon appears in the nascent swarm 2 tips, which progressively widens and spreads backwards towards the colony centre as assembly proceeds. Whilst the cells internal to this palisade continued to move actively in random directions until the increasing population density seemed to preclude this the rafts were quite immobile relative to each other, even at the earliest stages. In their studies of 3610 swarming on LB-agar, Kearns & Losick (2003) observed similar rafts of cells on LB-agar, which they showed were hyperflagellated. However, since the samples were removed from the swarm edge for microscopy the actual location of these rafts in the swarm colony was not established in that study. Rafts of hyperflagellated cells suggest that such groups collaborate to promote movement over the surface, similar to the role of filamentous swarmers in P. mirabilis. In fact our observations of the positioning of the rafts around the entire borders of dendrites, and apparently on the lower surface of dendrites, could indicate another role for rafts, not mutually exclusive, in maintaining the integrity of these branching structures. On the other hand, on LB medium, as the first swarm of cells of strain 3610 emerges from the colony centre, we have detected (unpublished data) very large groups of rafts that are, quite dramatically, all aligned in the same direction, exactly as described previously for P. mirabilis swarmers (Harshey, 1994
). Nevertheless, in relation to the complex structures that we observe on the synthetic B-medium, a role for the rafts or the large bundles of filamentous cells in driving the rapid movement of the swarm front and the elongation of the dendrites is not yet clear.
Considerable complexity in behaviour and in the concomitant structural organization of the B. subtilis swarming colony has been revealed in this study. This is presumably orchestrated at different stages by a variety of molecular switches responding to signalling mechanisms that should now be amenable to genetic and biochemical analysis. We note, however, that if quorum sensing is important, for example, in controlling processes such as inhibition of cell division or surfactin synthesis (which appears to be associated with the initiation of swarm 1), then this occurs whilst separate micro-colonies containing only hundreds to thousands of bacteria are still detectable at the site of the original inoculum. Therefore, in contrast to conditions in liquid cultures, quorum sensing may be manifest at extremely low cell densities, facilitated perhaps by the close packing of a very small number of cells.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 27 January 2004;
revised 2 March 2004;
accepted 4 March 2004.
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