JOURNAL OF BACTERIOLOGY, Sept. 2003, p. 5627–5631 Vol. 185, No. 180021-9193/03/$08.00�0 DOI: 10.1128/JB.185.18.5627–5631.2003Copyright © 2003, American Society for Microbiology. All Rights Reserved.

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Rapid Surface Motility in Bacillus subtilis Is Dependent onExtracellular Surfactin and Potassium Ion

Rebecca F. Kinsinger, Megan C. Shirk, and Ray Fall*Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309-0215

Received 7 February 2003/Accepted 30 June 2003

Motility on surfaces is an important mechanism for bacterial colonization of new environments. In thisreport, we describe detection of rapid surface motility in the wild-type Bacillus subtilis Marburg strain, but notin several B. subtilis 168 derivatives. Motility involved formation of rapidly spreading dendritic structures,followed by profuse surface colonies if sufficient potassium ion was present. Potassium ion stimulated surfactinsecretion, and the role of surfactin in surface motility was confirmed by deletion of a surfactin synthase gene.Significantly, this motility was independent of flagella. These results demonstrate that wild-type B. subtilisstrains can use both swimming and sliding-type mechanisms to move across surfaces.

There is increasing interest in how bacteria establish symbi-otic or pathogenic biofilms on plant or animal cell surfaces.Several recent reviews (5, 15, 16, 24, 27) describe an importantrole for bacterial motility in such interactions, including mo-tility as a component of the initial development of microbialbiofilms. Many types of bacterial motility are known, as re-viewed by Henrichsen (8), allowing increased access to nutri-ents, movement to favorable colonization sites, or avoidance ofantimicrobial substances produced by the host.

We are interested in the possible presence and role of Ba-cillus subtilis biofilms on plant roots (R. Fall, R. F. Kinsinger,and K. A. Wheeler, submitted for publication). B. subtilis isnormally considered as a soil bacterium that can be transferredto associated plants and plant materials as well as animals andaquatic habitats (20), but there are also few reports of its directassociation with roots (17, 26). While testing the presence of B.subtilis and related strains on plant roots, we observed twotypes of rapid surface motility on semisolid media, including (i)dendritic growth, reminiscent of the branched colony morphol-ogy exhibited by some Bacillus, Paenibacillus, Serratia, andSalmonella isolates on agar surfaces, as reviewed by Kozlovskyet al. (9); and (ii) profuse surface colony formation if themedium was supplemented with a source of K� ion. Further-more, the rapid surface motility of our Bacillus isolates mightbe explained by a lubricating model, in which secretion ofbiosurfactants is a component of this type of surface growth(9). These considerations led us to examine the role of surfac-tin, an extracellular lipopeptide with exceptional surfactantproperties (19), in the surface motility of wild-type B. subtilis.

Media for rapid surface motility. In this work, the wild-typeB. subtilis Marburg strain was used (strain 6051, Table 1), anda distinctive feature of its growth on semisolid media is den-dritic surface motility. To routinely demonstrate this, a casein

digest-based agarose medium was used, as typically preparedwith 0.3% (wt/vol) agarose (low electroendosmosis grade;Fisher Scientific) in 60-mm-diameter petri dishes; this mediumis described in detail elsewhere (Fall et al., submitted). Invarious experiments described here, different carbon sourceswere used with very similar results, including 0.3% (wt/vol)glucose or maltose or 1% (wt/vol) mannitol: termed CG,CMal, and CM media, respectively. The same media weresupplemented to contain a source of K� ion, such as KCl orK2HPO4, and then used to demonstrate extensive surface col-ony formation. Figure 1 (upper left plates) shows results onCMal versus CMal-KCl agarose media. In the absence ofadded potassium ion, and with plates inoculated in the centerwith a sterile toothpick, surface growth appeared as brancheddendrites, and the surface was not completely covered (16 h,40°C). Gram staining of cells in these dendrites showed tightlyclustered cells. In contrast, addition of a potassium source,typically 2 to 14 mM KCl or 7 mM K2HPO4, to the caseindigest medium resulted in coverage of the agarose surfacewithin 8 h at 40°C (or overnight at 37°C) with a pellicle-likecolony (Fig. 1, upper center) similar to pellicles seen on thesurfaces of liquid cultures (3). These surface colonies often hada wrinkled appearance, and if grown on 100-mm-diameterpetri dishes usually collapsed onto the lid of the dish withvisible bubbles indicative of the presence of a surface-activeagent. Very similar results were obtained with all three of thecarbon sources mentioned above.

Specificity of K�-dependent surface motility and its relationto surfactin. To determine the specificity of dendritic surfacegrowth for monovalent cations, the chloride salts of LiCl,NaCl, KCl, CsCl, or NH4Cl were tested; CG agarose mediumwas used for these experiments. Very similar growth, althoughwith subtle differences in the dendritic patterns, was seen if themedium was supplemented with common monovalent cations,2 to 20 mM LiCl, NaCl, or NH4Cl, and no growth was seen inthe presence of 20 mM CsCl (data not shown). In contrast,addition of as little as 0.25 mM KCl converted dendritic growth

* Corresponding author. Mailing address: Department of Chemistryand Biochemistry, University of Colorado, Boulder, CO 80309-0215.Phone: (303) 492-7914. Fax: (303) 492-1149. E-mail: fall@colorado.edu.

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to nearly full coverage of the plate with a surface colony, withfull coverage at KCl concentrations of 0.5 to 1.0 mM. Theseresults were each scored visually, but the extent of surfacebiomass was not quantified. The finding of film growth stimu-lation by such low levels of K� ion may simply represent athreshold for the putative high-affinity K� transporter in B.subtilis (22) or could signify induction of potassium-dependentgenes that control colony growth.

If plates lacking added K� ions like those shown in Fig. 1(upper left) were observed at intermediate times (6 to 8 h), theappearance of a fluid preceding each dendritic “finger” ofgrowth could be seen. Placement of 10-�l drops of water nearsuch fluid regions, but not on other bare portions of the plates,led to a rapid spreading of the water drop—indicative of thepresence of a surfactant. It is known that wild-type B. subtilis

strains secrete surfactin (13). In addition, as mentioned above,growth on casein digest agarose medium with K� ions addedsuggested the presence of a biosurfactant.

To determine if strain 6051 produces extracellular surfactinand if addition of K� ion affects growth and/or surfactin for-mation, 6051 was grown in liquid CM medium with or withoutaddition of 7 mM K2HPO4. With or without the addition of theK2HPO4, in duplicate experiments, there were similar rates oflogarithmic growth at 37°C and similar final optical densities at600 nm after 6 h (�10%) (data not shown). However, theproduction of extracellular surfactin was stimulated by thepresence of 7 mM K2HPO4, as determined by the drop col-lapse method (2). The cell-free culture media of early-station-ary-phase cultures contained 5.7 to 8.6 �g of surfactin ml�1

(CM broth) and 38.4 to 48.6 �g of surfactin ml�1 (CM plus 7

FIG. 1. Effect of KCl on the surface growth of B. subtilis 6051and OKB105 and visualization of the stages of surface growth with X-Glc. Shownare CMal agarose plates (pH 7.5) with no added KCl (left), with 2 mM KCl added (center), or with 2 mM KCl and 80 �g ml�1 X-Glc added tovisualize �-glucosidase activity (right). The arrows indicate (i) some of the many blue-stained dendrites of cells growing out from the central pointof inoculation and (ii) a slower-growing surface film that fills in between the individual dendrites. It should be noted that in the presence of X-Glcin the CMal-KCl agarose medium, both dendritic growth and surface film growth were slower than in its absence (center plates). All plates wereinoculated in the center and grown overnight at 37°C, and the results were replicated in three independent experiments.

TABLE 1. B. subtilis strains used and some of their properties

B. subtilis strain Relevant phenotype or genotype Source or referencea

Phenotype score

Surfactinproduction(hemolysis)

Dendritic on CMagarose/surface filmon CMK agarose;

no added surfactinb

6051 Wild type (Marburg) ATCC � �/�M1 Surfactin negative, �srfA-A This work � �/�1A1 (168) trpC2 BGSC � �/�JH642 Surfactin negative pheA1 trpC2 P. Zuber (14) � �/�OKB105 Surfactin-positive transformant of JH642, pheA1 sfp P. Zuber (14) � �/�OKB120 Surfactin negative, pheA1 srf::Tn917 sfp P. Zuber (14) � �/�1A139 Flagellumless, flaA4 hag-1 lys trpC2 BGSC � �/�

a ATCC, American Type Culture Collection (http://www.atcc.org/); BGSC, Bacillus Genetic Stock Center (http://bacillus.biosci.ohio-state.edu/).b Phenotypes were scored as shown in Fig. 1 after 16 h, of growth at 37°C and, as described in the text.

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mM K2HPO4 broth) (data from three separate experiments).Smaller stimulation of surfactin formation was obtained byaddition of 14 mM KCl with or without control of the pH of themedium (pH 6.6 or 7.3 to mimic the absence or presence of 7mM K2HPO4, respectively). Thus, the stimulation of surfacemotility and colony formation by potassium salts like that seenin Fig. 1 and 2 could to be due in large part to different levelsof extracellular surfactin, controlled by K� levels in the me-dium.

Isolation and characterization of a �srfA-A mutant. To ver-ify the role of surfactin in the two phases of surface motility, weused insertional disruption of an essential gene, sfrA-A, of thesurfactin operon (25). We used the pJM103 integration vector(18) to disrupt the surfactin synthase srfA-A gene in the B.subtilis Marburg genetic background. The protocols used byShirk et al. (23) were followed. Primer sequences were ob-tained by using the published srfA-A sequences of Schneider etal. (21). The forward primer 3�-srfA-BamHI (5�-CGCGGATCCGTGAGCTTGATGTTGAAAGG-3�) and the reverseprimer 3�-srfA-EcoRI (5�-GCGGAATTCGAGTACGAATGTAAGCCC-3�) were located within the 10,764-bp wild-typegene, generating a 1,040-bp DNA fragment. All primers weredesigned with BamHI or EcoRI tails for directional cloning.Primers were purchased from Invitrogen (Frederick, Md.).The integration vector, pJM103, with a chloramphenicol resis-tance element, was obtained from M. Perego (18). Chemicallycompetent DH5� cells, purchased from Invitrogen, were trans-

formed according to the manufacturer’s protocol. B. subtilischromosomal DNA was isolated from the Marburg strain asdescribed by Cutting and Vander Horn (4). B. subtilis 6051cells were prepared and transformed as described by Anagnos-topoulus and Spizizen (1), using chloramphenicol (5 �g/ml;Sigma) added to tryptose blood agar base (TBAB) plates toselect and maintain the mutant strain. The srfA-A mutant wasdesignated strain M1, and was maintained on TBAB agar con-taining 5 �g of chloramphenicol ml�1.

Mutant M1 no longer produced surfactin, as assessed byhemolysis of erythrocytes, as described by Nakano et al. (14),and by high-performance liquid chromatography analysis oflipopeptides in growth medium (H. Pal Bais, J. Vivanco, andR. Fall, unpublished observations). As shown in Fig. 2 (centerplates), the resulting �srfA-A mutant (M1) exhibited no signif-icant dendritic structures on low-potassium medium or com-plete surface colony coverage on high-potassium medium com-pared to its wild-type parent (Fig. 2, left plates). To confirmthat the growth phenotype effects of the �srfA-A mutationwere specific to loss of surfactin production, the M1 mutantwas grown on plates supplemented with authentic B. subtilissurfactin (Sigma-Aldrich). As shown in Fig. 2 (right plates),addition of surfactin to the surface of the plates restored boththe low-potassium dendritic growth and the high-potassiumsurface colony coverage.

The optimal concentration of surfactin needed to supportsurface motility was determined according to dendritic growth

FIG. 2. Insertional inactivation of the srfA-A gene in B. subtilis M1 blocks dendritic growth on a low-potassium medium and K�-dependentsurface film formation on a high-potassium medium, and addition of authentic B. subtilis surfactin restores rapid surface growth. (Top row) Platesof CMal agarose (low potassium) were inoculated in duplicate with 6051 (B. subtilis Marburg), the �srfA-A mutant, or the �srfA-A mutant plussurfactin. For the latter two inoculations, the center of each plate was spotted with 10 �l of water or a surfactin solution (10 mg ml�1 in 20 mMNaOH) and dried 45 min before inoculation. The bottom row is the same as the top row, but plates containing 7 mM K2HPO4 (CMalK agarose)were used. The center of the plates was inoculated with sterile toothpicks from cultures grown on CMal agarose plates, and then the plates wereincubated for 8 h at 40°C. All of the results shown are from multiple, independent experiments; similar results were seen if surfactin was dissolvedin 20 mM Tris base.

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on semisolid CM agarose medium and also on CM mediumsolidified with agar (0.6% [wt/vol]; Difco). On the latter me-dium, surface spreading was more compact, and it was easier tomeasure the diameter of the surface colony. On either agaroseor agar surfaces, the effects of surfactin could be seen with aslittle as 0.2 �g of surfactin applied per plate: the approximateeffective concentration to give a 50% response (EC50) in dif-ferent experiments ranged from 0.3 to 0.6 �g of surfactin perplate (data not shown). If the applied surfactin had diffusedthroughout the medium in the plate (10 ml), this would give anEC50 of 0.03 to 0.06 �g ml�1. This is much below the reportedcritical micellar concentration of 7.8 �g ml�1 for surfactin (10).However, when CM agarose plates were prepared with surfac-tin added at the time the plates were poured, the EC50 wasapproximately five times higher (2.5 to 4 �g per plate). It isnoteworthy that typical values for surfactin secretion in liquidbroth experiments with various B. subtilis strains range wide-ly—from zero to 3,000 �g ml�1 depending on the growthconditions (see reference 25)—and that during the surfactinproduction phase, these concentrations are usually muchhigher than the levels necessary for effective surface translo-cation seen here.

Surface motility defects in B. subtilis 168 strains. The com-monly used genetic strain JH642, derived from widely used B.subtilis strain 168, does not produce surfactin, as a result of adefect in the sfp gene (13), and when casein digest agarosemedium was used with various carbon sources, we could notdetect the type of surface motility seen here with the 6051strain (data not shown). We found that addition of surfactinwas sufficient to restore dendritic motility and K�-dependentsurface colony formation to both strains JH642 and 168 (datanot shown). In addition, the surface motilities of OKB105(sfp�) and its surfactin-negative derivative, OKB120, which hasa Tn917 insertion in the srfA-B gene (13), were also tested.OKB105, as shown in Fig. 1 (lower left and lower center),showed both dendritic motility and K�-stimulated surface col-ony formation without addition of surfactin. Surfactin-negativeOKB120 only showed these surface growth phenotypes if sur-factin was added to the plates (data not shown), confirming theresults we obtained with JH642, 168, and the surfactin-negative�srfA-A mutant (Fig. 2). However, the formation of K�-de-pendent surface colonies was less extensive in the 168-derivedstrains (in the presence of surfactin) than with the Marburg

strain or the M1 �srfA-A mutant, which may reflect additionalmutations in the 168 parent, which was derived by X-ray mu-tagenesis of the Marburg strain or a close relative (1).

Stages of surface motility. It was possible to visualize theintermediate stages of surface motility and colony formation byhydrolysis of a colorimetric substrate as described by Mendel-son and Salhi (11). CMal agarose was prepared and supple-mented with X-Glc (5-bromo-4-chloro-3-indolyl-�-glucoside;80 �g ml�1; Sigma-Aldrich). X-Glc forms a blue product uponhydrolysis of the glycosidic bond, which helps visualize thedendritic stages of surface motility. As shown in Fig. 1 (upperright), for B. subtilis 6051, addition of X-Glc to these platesslowed the surface spreading and led to blue staining of thecentral rib of each dendrite and a central colony that had muchless of the blue stain. Even after plates were covered with thesurface colony that grew between the dendritic structures, theblue-stained features of the underlying dendrites remained(when plates were observed from the bottom; data not shown).These findings correspond to visual inspection of plates with-out X-Glc during the time course of surface motility. Earlyrapid dendritic growth, which could reach 5 mm h�1, was seenon several carbon sources, such as glucose, mannitol, sorbitol,maltose, and cellobiose, and was always followed by slower filmformation that eventually covered the plate, as long as a sourceof K� ion (�1 mM) was added.

For comparison, a B. subtilis strain derived from the 168genetic background was tested on CMal plates with X-Glc. Asshown in Fig. 1 (lower right), the surfactin-producing strainOKB105 described above also showed the blue staining ofsurface dendrites in the presence of X-Glc. No such dendriteformation or staining was seen in surfactin-negative strainsOKB 120, 168, and JH642 (data not shown). These resultsdemonstrate that the dendritic stage of surfactin-dependentsurface motility in B. subtilis strains can be readily visualizedwith X-Glc.

Rapid surface motility can be independent of flagella. Ba-cillus species are well known to use flagella for surface motility(6), and in the case of B. anthracis, it is reported that thesebacilli can use sliding motility (8). To determine if the rapidmotility seen here is due to flagella, we used Ryu stain as usedby Heimbrook et al. (7) to attempt to detect flagella in den-dritic and surface colonies of the Marburg strain and the�srfA-A mutant. Motile cells from the leading edge of these

FIG. 3. Flagellar staining of motile B. subtilis 6051cells from the leading edges of growth on motility agar (Bsa) (a) versus CM-K2HPO4 agarose(c). Gram stains are also shown for motile cells from Bsa (b) and CM-K2HPO4 agarose (d). Polar flagella are readily visible on motile cells fromBsa motility agar, but not on those from CM-K2HPO4 agarose. In numerous images like that shown in panel c, faint background structures weredue to components of the stain and not detached flagella.

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surface structures were stained, and there was no consistentdetection of flagella in these bacteria on CM or CMK agarose(Fig. 3). As a control, these strains were grown in a semisolidswimming medium (Bsa) used to measure swimming motilityin B. subtilis (12). These cells contained readily visualized polarflagella (Fig. 3). For comparison in each case (Fig. 3), Gram-stained cells show the typical gram-positive rods.

As a further test of the independence of surface motilityfrom expression of flagella, we obtained B. subtilis 1A139, withmutations in the flaA4 and hag-1 genes that control flagellarexpression and assembly (12). The flagellum-negative pheno-type (determined by flagellar staining) and tryptophan andlysine requirements of 1A139 (Table 1) were confirmed, andwhen the strain was grown on CG or CG-KCl agarose, theresults were essentially identical to those seen with the�srfA-A, as in Fig. 2. That is, dendritic motility and surfacecolony formation were only seen when the plates were supple-mented with surfactin or surfactin-KCl, respectively. We con-clude that dendritic motility and surface colony formation in B.subtilis are independent of flagella.

Concluding comments. A striking feature of the surface mo-tility of wild-type B. subtilis shown here is that in the presenceof K� ion, the cells rapidly spread to cover the surface of thecasein digest agarose media used. This spreading occurs in twophases, including initial dendritic growth of tightly clusteredcells, followed by complete surface film formation that oftencontinues over the edge of the petri dish onto the lid. Both ofthese stages of surface motility are independent of flagella andthus are not examples of typical swarming motility, which isusually associated with the movement of rafts of multiflagel-lated cells (6). Instead, this surface spreading is likely to bemore akin to sliding motility, which was defined by Henrichsen(8) as a kind of translocation produced by expansive forces incombination with reduction of friction between the cell and thesurface. Our finding that the biosurfactant surfactin is requiredfor the surface motility seen here is consistent with this view.Secretion of surfactin at the edges of growing surface dendriteswould result in lowering the surface tension, allowing rapid cellspreading. It remains to be determined how addition of K� ionstimulates surfactin production, which in liquid-phase growthof B. subtilis is under the control of a complex quorum-sensingsystem (25).

This work was supported by grant DE-FG03-97ER20274 from theU.S. Department of Energy, Office of Basic Energy Sciences.

We thank Daniel Kearns and Peter Zuber for advice and for pro-viding strains.

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