Comparative Analysis of the Development of Swarming Communities ofBacillus subtilis168 and a Natural Wild Type: Critical Effects of Surfactin and the Composition of the Medium

Abstract

The natural wild-type Bacillus subtilis strain 3610 swarms rapidly on the synthetic B medium in symmetrical concentric waves of branched dendritic patterns. In a comparison of the behavior of the laboratory strain 168 (trp) on different media with that of 3610, strain 168 (trp), which does not produce surfactin, displayed less swarming activity, both qualitatively (pattern formation) and in speed of colonization. On E and B media, 168 failed to swarm; however, with the latter, swarming was arrested at an early stage of development, with filamentous cells and rafts of cells (characteristic of dendrites of 3610) associated with bud-like structures surrounding the central inoculum. In contrast, strain 168 apparently swarmed efficiently on Luria-Bertani (LB) agar, colonizing the entire plate in 24 h. However, analysis of the intermediate stages of development of swarms on LB medium demonstrated that, in comparison with strain 3610, initiation of swarming of 168 (trp) was delayed and the greatly reduced rate of expansion of the swarm was uncoordinated, with some regions advancing faster than others. Moreover, while early stages of swarming in 3610 are accompanied by the formation of large numbers of dendrites whose rapid advance involves packs of cells at the tips, strain 168 advanced more slowly as a continuous front. When sfp ؉ was inserted into the chromosome of 168 (trp) to reestablish surfactin production, many features observed with 3610 on LB medium were now visible with 168. However, swarming of 168 (sfp ؉ ) still showed some reduced speed and a distinctive pattern compared to swarming of 3610. The results are discussed in terms of the possible role of surfactin in the swarming process and the different modes of swarming on LB medium.We have recently described a wide range of swarming patterns, including successive waves of dendritic branching, followed by consolidation, of the wild-type strain 3610 on the surface of a synthetic agar medium (9). A simpler, apparently nondendritic swarming pattern of Bacillus subtilis on LuriaBertani (LB) agar has also been described (9, 10), in particular, with the wild-type strain 3610, although the details of the intermediate stages of these swarm communities during development have not so far been reported. In contrast, we showed that the formation of the complex branching communities on the synthetic B medium, as detected by an in situ microscope analysis, is unexpectedly complex, with a precise developmental program accompanied at different stages of the swarming process, for example, by the appearance of long filamentous cells and rafts of tightly aligned cells. There was no evidence, however, that these types of cells were directly involved in swarming per se (9). Moreover, in our previous study we also detected the apparent migration of large numbers of individual cells prior to their aggregation into the tips of nascent dendrites. This suggested that, in at least some stages of the swarming process, migration does indeed proceed independently of the morphologically d...

Single-cell analysis in situ in a Bacillus subtilis swarming community identifies distinct spatially separated subpopulations differentially expressing hag (flagellin), including specialized swarmers

“…The long-chain forms could already be detected at 10 h, and accumulated in large numbers at the edge of the MC at around 12-16 h post-inoculation, as we have previously shown for the non-domesticated strain 3610 (Julkowska et al, 2004). These cells expressed high levels of eps (extracellular polysaccharide), but had no detectable hag expression.…”

“…We have observed in both the domesticated and nondomesticated strain (168 sfp + swrA and 3610, respectively) that this early growth of the MC is accompanied by the appearance of long cell forms on both LB and B medium plates (Julkowska et al, 2004;Hamze et al, 2009). We now show that staining of such cells in situ in the MC with a membrane stain (see Supplementary Methods) shows that the long cell forms are septated ( Supplementary Fig.…”

“…Thus, a drop of water placed on these swarm plates did not spread (Banaha et al, 2009). Note that about 11 h of growth of the central MC occurred before eight to 12 monolayered primary dendrites radiated outwards from the edge of the MC at a constant speed of approximately 3.5 mm h 21 (1 mm s 21 ) at 30 uC (Julkowska et al, 2004).…”

“…On Luria broth (LB) agar at low humidity and 37 u C (primarily analysed with nondomesticated strains such as 3610), migration proceeds as an expanding confluent field, headed by small groups of bacteria, apparently with little change in individual size (Kearns & Losick, 2003;Julkowska et al, 2004). In contrast, we have described the formation of hyper-branched dendritic patterns by strain 3610 on the minimal B medium at both high and low humidity, which develop through a series of well-defined stages (Julkowska et al, 2004). We have also observed very similar behaviour for the sfp + derivative of the laboratory strain 168 (able to produce surfactin) at high humidity, although the cells fail to swarm reproducibly at low humidity.…”

“…We also show that both 168 sfp + swrA and an swrA + derivative produce very similar dendritic migration at high humidity. In addition, we present several lines of evidence to show that dendritic migration of the laboratory strain 168 sfp + swrA, like the migration of swrA + strains, does not involve swimming of individuals in a large volume of surface water, but instead is like classical swarming in being restricted to a thin film.Dendritic migration of strain 168 sfp + swrA involves distinct morphological and genetically defined stages at high humidity (Julkowska et al, 2004(Julkowska et al, , 2005 Hamze et al, 2009). Thus, following the first visible indication of initiation of migration, a spreading zone of surfactin emanating from the mother colony (MC; the site of inoculation), several small bud-like structures burst outwards from the edge of the MC.…”