RESEARCH LETTER

Growing Bacillus subtilis tendrils sense and avoid each other

Barry L. James1, Jennifer Kret1, Joyce E. Patrick2, Daniel B. Kearns2 & Ray Fall1
1

Department of Chemistry and Biochemistry, University of Colorado, Boulder, CO, USA; and 2Department of Biology, Indiana University, Bloomington, IN, USA

Correspondence: Ray Fall, Department of Chemistry and Biochemistry, University of Colorado, Boulder, CO 80309-0215, USA. Tel.: 11 303 492 7914; fax: 11 303 492 1149; e-mail: fall@colorado.edu Received 11 December 2008; accepted 6 May 2009. DOI:10.1111/j.1574-6968.2009.01665.x Editor: Steve Diggle Keywords Bacillus subtilis; motility; tendrils; surfactin; gspA; intercellular signaling.

Abstract Growing tendrils of aagellate hag mutants of Bacillus subtilis were found to show an avoidance response when colonizing a semi-solid medium, suggesting a tip-totip communication mechanism between colonies. There may be a second sensing mechanism involved in shaping the morphology of tendrils. Tendril growth in B. subtilis was dependent on and possibly shaped by the release of surfactin, a biosurfactant. Transposon mutagenesis yielded two mutants with touching tendrils, and each had a disrupted gspA gene that encodes a putative glycosyltransferase. Tendrils of gspA mutants, unlike the parental strain, were unresponsive to tendril tip growth by surfactin, suggesting disruption of intercellular signaling. Tendril sensing and avoidance could be physiologically relevant in habitats, such as plant roots, where some limiting nutrient might induce this type of multicellular behavior, promoting avoidance of previously explored areas by sibling colonies.

Introduction

MICROBIOLOGY LETTERS

Aagellate strains of Bacillus subtilis colonize surfaces by sliding motility (Kinsinger et al., 2005; Fall et al., 2006). Sliding motility is dened as surface translocation produced by expansive forces in the growing colony, combined with special surface properties to lower the friction between the monolayer of cells and substrate (Henrichsen, 1972; Harshey, 2003). Colonies of sliding B. subtilis rapidly spread as profuse pellicle-like lms on nutrient-rich soft agar, but spread as radiating tendrils on nutrient-limited media (Fall et al., 2006). Here we explore the basis of the intercellular interaction of these spreading tendrils. It is of interest that sliding motility in various bacteria is promoted by the release of exocellular surfactants, and B. subtilis releases one or more of the surfactin family of cyclic lipopeptides (Peypoux et al., 1999; Julkowska et al., 2005). Mutants defective in the surfactin biosynthetic pathway of B. subtilis are blocked in tendril spreading, and spreading is rescued by surfactin addition (Kinsinger et al., 2003, 2005). Other bacteria use various glycolipid or lipopeptide surfactants to promote colony spreading (Daniels et al., 2004; Caiazza et al., 2005). For example, Recht et al. (2000) and Recht & Kolter (2001) reported that in Mycobacterium smegmatis, formation of acetylated glycopeptidolipids in
FEMS Microbiol Lett ]] (2009) 18

the outermost layer of the cell envelop is necessary for sliding motility. These glycopeptidolipids are the presumed biosurfactants used by M. smegmatis to lower the surface tension of the solidied growth medium. During the course of our work with tendril growth of an aagellate hag mutant, we observed an unusual phenomenon: when plates were inoculated two or three times at different locations, the emerging tendrils rarely touched. Here we present evidence that B. subtilis exhibits complex group behavior in colonizing surfaces with multicellular tendrils, and that there are mechanisms to control tendril avoidance and the shape of elongated tendrils, similar to that observed in Pseudomonas aeruginosa (Caiazza et al., 2005; Tremblay et al., 2007).

Materials and methods

Strains and growth media
Table 1 summarizes the strains used in this work. All strains are derivatives of the undomesticated B. subtilis strain 3610. All strains were maintained on LuriaBertani (LB) medium (Research Products International) containing (L1) 10 g tryptone, 5 g yeast extract, and 10 g NaCl, solidied with 1.5% w/v agar (Difco), and supplemented as needed with
 c

2009 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

B.L. James et al.

Table 1. Bacillus subtilis strains used in this work Strain 3610 DS64 DS1677 DS2457 MS1 GJMar1 GJMar2 RFGSP4 RFMar3 RF3S11 Genotype/phenotype Wild type hag<MLS Dhag Dhag pMarA kan MLS srfAA<cam Dhag gspA<kan Dhag gspA<kan Dhag gspA<kan Dhag kan; derived from DS2457 as described in Materials and methods srfAAOTn3S11 Spec References/sources Kinsinger et al. (2005) Kearns & Losick (2003) Blair et al. (2008) This work Kinsinger et al. (2003) This work This work This work; SPP1 transductant of GJMar1 in DS1677 This work Kinsinger et al. (2005)

Fig. 1. Tendrils of Bacillus subtilis hag mutant DS64 growing on Mk agarose do not touch. The hag strain was grown overnight at 37 1C on LB MLS plates and then transferred to Mk plates using a round sterile toothpick. The Mk plates were inoculated one, two, and three times as indicated by the colony centers and grown overnight at 37 1C. The plates shown here were prepared in duplicate and represent typical results.

erythromycin (1 mg mL1) and lincomycin (25 mg mL1; referred to as MLS), chloramphenicol (5 mg mL1), or spectinomycin (100 mg mL1). For surface sliding motility, the pellicle medium MSggN described by Branda et al. (2001) and modied by Fall et al. (2006) was used. It was supplemented with 0.1 or 5 mM KCl and solidied with 0.35% w/v agarose (Fisher Scientic; low EEO high gel strength grade) to make Mk (tendril) or MK (lm) medium, respectively. Additionally, in some experiments, tendrils were observed on MK agarose prepared with other limiting nutrients: 1/10th the normal level of sodium glutamate (3 mM) or 1/25th the normal sodium phosphate (138 mM). Plates were routinely inoculated from overnight cultures of B. subtilis strains on LB agar containing suitable antibiotics. Following growth at 37 1C for 1624 h, the plates with tendrils were digitally photographed and the images were adjusted with ADOBE PHOTOSHOP 7.0 (Adobe Systems Inc.).

Aldrich Co.; HPLC analysis [method of Hsieh et al. (2004)] revealed that it contained at least six surfactin isoforms (T. Sivy & R. Fall, unpublished data). The surfactin was prepared as a stock of 10 mg mL1 in 0.01 M NaOH, lter sterilized, and stored in a freezer ( 20 1C). Surfactin (or water as a control) was pipetted onto sterile paper discs and placed on 60 mm Mk agarose plates. The surfactin was allowed to diffuse over the surface of the media simultaneously as the colonies grew, typically with the discs 12 cm from the point of inoculation. The plates were then inoculated with the appropriate hag strain using the toothpick method above. In all the experiments presented here, plates were prepared in duplicate or triplicate.

Transposon mutagenesis
The temperature-sensitive vector carrying TnYLB, pMarA, was delivered into the hag strain by SPP1-mediated generalized transduction to generate DS2457, which was used for transposon mutagenesis as described in the Supporting Information.

Tendril growth and avoidance

In some experiments, Mk plates were inoculated singly or multiple times (as seen in Fig. 1), and then grown overnight at 37 1C; this allowed visualization of the tendril avoidance behavior. To further investigate tendril avoidance, we used sterile paper discs (6-mm diameter; Becton Dickinson Co.) containing B. subtilis surfactin, purchased from Sigma c

Other methods
In order to verify that the hag DS64 strain produced normal amounts of surfactin, a modied drop collapse assay was used (Bodour & Miller-Maier, 1998; Kinsinger et al., 2005). Using hag DS64 or its parent 3610 strain grown in MK broth
FEMS Microbiol Lett ]] (2009) 18

2009 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

Bacillus subtilis: tendril sensing and avoidance

overnight at 37 1C, the supernatants were found to contain very similar levels of surfactant, ranging from 780 to 910 mg mL1 surfactin equivalents in different experiments. The Sigma-Aldrich surfactin was used to prepare the standard curve. For analysis of the general stress response of two gspA mutants and the 3610 parent strain, the growth of each in MK broth with and without 4% v/v ethanol was analyzed as described elsewhere (Antelmann et al., 1995).

ions, but limiting for either phosphate or glutamate (Fig. 2). Thus, tendril avoidance was not dependent on the hag allele or on the limiting nutrient in the media. In contrast, a plate with the complete dened medium (MK agarose) is shown in Fig. 2 (top left) to illustrate the profuse pellicle-like, wrinkled lm that forms, instead of tendrils, when nutrients are not limiting as described previously (Fall et al., 2006). The formation of such wrinkled colonies in wild-type B. subtilis has been reviewed in Lemon et al. (2008).

Results
Approaching B. subtilis tendrils rarely touched during growth on a defined medium with limiting nutrients
We observed that plates inoculated at multiple sites with the DS64 hag mutant resulted in tendrils that avoided each other. Figure 1 shows that when the center of the plate was inoculated and allowed to grow overnight at 37 1C, the tendrils spread to the edges of the plate in all directions. However, if the plates were inoculated two or three times, the tendrils spread, but rarely touched one another even upon prolonged incubation. Figure 1 also shows a second phenomenon, namely that on this growth medium, there is limited lateral spreading of the individual tendrils (see Discussion). This tendril avoidance behavior was observed with various hag mutants, and on media with sufcient K1

Tendril growth is redirected by a surfactin source

We tested whether puried surfactin would produce the tendril avoidance response. Paper discs containing various concentrations of surfactin (or sterile water as a control) were placed on either side of the point of inoculation with the hag DS64 strain. On the Mk agarose medium used at 37 1C, surfactin diffused at the rate of about 1 cm h1 from paper discs. Supporting Information, Fig. S1 shows the typical results after overnight growth at 37 1C: discs containing surfactin diverted the growth of approaching tendrils. The minimum amount of surfactin required for tendril diversion was determined using the paper disc method; the amount of surfactin required for tendril diversion was found to be in the range 1020 mg of surfactin per disc. At these lower surfactin concentrations, hag DS64 tendrils grew

Fig. 2. Bacillus subtilis hag mutant DS64 tendrils do not touch even on different tendril induction media. Top left: MK; control for complete MK medium. Top right: Mk medium (low KCl). Bottom left: MK with 1/10 of the normal sodium glutamate (3 mM). Bottom right: MK with about 1/25 of the normal sodium phosphate (138 mM). All plates above were prepared with 0.35% w/v agarose in duplicate and inoculated with DS64. After 1824 h growth at 37 1C, the plates were photographed; these plates represent typical results from replicated experiments.

FEMS Microbiol Lett ]] (2009) 18

 c

2009 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

B.L. James et al.

Mar3

3S11

hag

3S11

Mar3

MS1

hag

MS1

Fig. 3. Growing tendrils of two different hag mutants do not avoid colonies of surfactin mutants. Mk plates were prepared with 0.4% w/v agarose. They were inoculated (sterile toothpicks) with hag DS64 (hag on the gure) or hag RFMar3 (Table 1; Mar3 on the gure), both aagellate hag strains, and then again at a different site with 3S11 or MS1, srfAA mutant strains, at about 1.5 cm apart. After growth for 1824 h at 37 1C, plates were photographed. Similar results were obtained with duplicate plates and in different experiments.

towards and beyond the discs but did not touch them. When higher concentrations of surfactin (Z60 mg on a disc) were tested, as can be seen in Fig. S1, fewer tendrils formed and they avoided growing toward the disc. Tendril diversion by surfactin was also seen when tendrils were induced by low concentrations of phosphate or glutamate, instead of low concentration of potassium ions.

Growing tendrils did not avoid colonies of surfactin biosynthetic mutants

The observations that tendrils avoided each other and were diverted by a surfactin source supported the idea that surfactin plays a key role in tendril growth behaviors. To further test this idea, we showed that when plates were inoculated both with different hag mutants and strains unable to produce surfactin, the latter did not divert tendrils of surfactin-producing hag mutants (Fig. 3). In each case, the hag mutant tendrils radiated over or around the circular colonies of the surfactin-decient B. subtilis strains.

Tendril phenotypes of mutants obtained by transposon mutagenesis

To identify genes required for the tendril avoidance phenotype, a transposon mutant library was generated and colonies were screened for altered tendril growth behavior
 c

using gridded Mk agarose plates (Supporting Information). After screening c. 1000 mutated colonies, we were able to identify several recurring, distinct colonial phenotypes (Fig. 4, left) that were grouped into (1) tendril-makers and (2) lm formers. Most interesting was the observation that upon subculture on Mk agarose plates, tendrils of some mutants did not show the avoidance response. Subculture of two such mutants, GJMar1 and GJMar2, illustrates this effect (Fig. 4, top right and bottom right respectively). Analysis of the GJMar1 and GJMar2 mutants by Touchdown PCR (Levano-Garcia et al., 2005; Blair et al., 2008) and comparison with the B. subtilis genome (http://genolist. pasteur.fr/SubtiList/) identied the gene insertion sites. In each mutant, the gspA gene, a general stress-induced gene and potential glycosyltransferase (Antelmann et al., 1995; Petersohn et al., 1999), was disrupted. This was conrmed by the sensitivity of both mutants to growth in 4% v/v ethanol in MK broth, and the ability to backcross the disrupted gspA gene with SPP1 phage into the hag mutant DS1677. The resulting transductants produced tendrils that grew into each other, like those seen in Fig. 4 (lower). Figure S2 (lower) illustrates this phenotype for one of the mutants (GJMar1) and its transductant [labeled (4B)]. In addition, Fig. S2 indicates that coinoculation of Mk agarose plates with a gspA mutant with a parental hag strain (DS1677) results in suppression of the hag strains tendrils;
FEMS Microbiol Lett ]] (2009) 18

2009 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

Bacillus subtilis: tendril sensing and avoidance

interestingly, the gspA mutants tendrils did not touch tendrils of the wild type.

Discussion
The work presented here identies another case of cellular communication in bacteria (von Bodman et al., 2008), in particular, the colonization of semi-soft media by B. subtilis strains with multicellular tendrils that do not touch. A review of the literature suggests that the rst case of tendriltendril communication was reported by OToole and coworkers (Caiazza et al., 2005) working with P. aeruginosa. They proposed a model for this phenomenon based on the idea that exocellular rhamnolipids and 3-(3-hydroxylalkanoyloxy) alkanoic acids (HAAs) released from the growing ends of tendrils coordinate group behavior and tendril avoidance. This model was further rened by Tremblay et al. (2007), who determined that rhamnolipids (with two rhamnosyl groups) attract swarming cells, while HAAs act as repellants. Here we show analogous tendril avoidance behavior of B. subtilis hag strains aagellate mutants that use sliding motility to move across a dened, semi-solid medium. This avoidance response occurs whether tendril initiation is triggered by nutritional depletion of the macronutrients glutamate or phosphate ion, or the micronutrient K1 ion. In contrast, if these strains are grown on the complete dened medium, and inoculated at two or more sites, a robust cellular lm overgrows the plate with no evidence of an avoidance response between the pellicle-like lms (Fig. 2). These results lead to the simple notion that nutritional deprivation of B. subtilis changes the group growth behavior

to that of tendril growth. However, the tendril growth phenotype is likely to have elements of surface conditioning with surfactin, tendril tip response to surfactin gradients, and lateral inhibition with mechanisms of communication between adjacent or approaching tendrils. As mentioned above, P. aeruginosa releases exocellular metabolites to control tendril growth behavior, and we reasoned that in B. subtilis the exolipopeptide, surfactin, may play a similar role. The hag strains used produce surfactin(s), and based on analysis of surfactin mutants in the same genetic background (Fig. 3), it is likely that this lipopeptide or that lipopeptide isoforms (Peypoux et al., 1999) are required for sliding motility on semi-soft media. However, surfactins may also act as morphogen-like signals or cues, analogous to the peptides seen in Myxococcus that control aspects of A- and C-signaling (Kroos, 2007). At present, it appears that growing B. subtilis tendrils release a sufcient concentration of surfactin to decrease surface tension, allowing greater sliding motility, while release of a greater concentration or a different pattern of isoforms may exert an inhibitory effect on tendriltendril touching and the lateral expansion of tendril arms. Supporting this view are our results showing that: (1) strains incapable of synthesizing surfactin (signal-negative) are unable to produce tendrils and colonize the surface of the semi-solid media; (2) addition of exogenous surfactin to these strains restores their tendril phenotype, allowing them to colonize the plate; (3) strains with normal surfactin production avoid each others tendrils, but overgrow mutants that cannot produce surfactin (srfAA mutants, Fig. 3); (4) creating a surfactin gradient near the point of inoculation prevents tendrils from growing in the

Fig. 4. Typical phenotypes seen in the screening of mutants obtained by Mariner transposon mutagenesis, and for two isolated tendril-touching mutants. The Mk grid plate (left) shows two clear phenotypes, lms or tendrils, for colonies picked from mutagenized cells. When further subcultured, these colonies were all predisposed to the colonial phenotypes found on this plate. The Mk agarose plates (right) show mutants GJMar1 (above) and GJMar2 (below) obtained from this screen; points of inoculation are shown by white Xs, and each mutant exhibited a lack of tendril avoidance in numerous replicated experiments.

FEMS Microbiol Lett ]] (2009) 18

 c

2009 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

B.L. James et al.

direction of the surfactin source (Fig. S1); and (5) gspA mutants that produce surfactin, but do not respond to it (signal-blind), lose the tendril avoidance response. Possibly, cells in the tips of the tendrils sense surfactin gradients, analogous to sensing of exosurfactants by P. aeruginosa (Tremblay et al., 2007), such that the increased concentration of surfactin between approaching tendrils is higher and results in an avoidance response. Because surfactins or other extracellular factors may suppress the lateral spreading of the individual tendrils from a single colony, there may be a second sensing mechanism involved in shaping the morphology of tendrils. This could be considered as a type of intratendril communication. Clearly, more work is needed to understand this mechanism. Notably, Debois et al. (2008) have recently shown that imaging MS can be used to quantify surfactin isoforms in situ on the surface of an agar medium that induces swarming and dendrite formation in B. subtilis. In addition, these investigators demonstrated surfactin gradients along dendrites and that longer chain surfactins are present between dendrites. Application of imaging MS to the tendril-sensing phenotypes studied here could be very informative. We demonstrated that transposon mutagenesis can be used to disrupt the tendril avoidance response, and identied a required gene, gspA, which is a general stress-induced gene regulated by the s(B)-dependent stress regulon (Antelmann et al., 1995; Petersohn et al., 1999). It is unclear as to how gspA is related to the B. subtilis tendril avoidance response. The annotation of the gspA gene product as a potential glycosyltransferase related to the lipopolysaccharide glycosyltransferases of Gram-negative bacteria (Petersohn et al., 1999) suggests that glycosylation of cell wall components (i.e. lipoteichoic acids; Weidenmaier & Peschel, 2008) may be necessary for communication between cells in multicellular structures such as growing tendrils. The gspA mutant phenotype may be a result of disrupting (1) a tendril sensing or signaling mechanism, (2) a membrane pump that could sample the level of exocellular surfactin(s), (3) the hydrophobic nature of the cell surface, or (4) some other cellular function, such as regulation of exopolysaccharide production. We note that recently Lopez et al. (2009) have proposed a new functional linkage between cellular potassium ion concentration, surfactin, a membrane protein kinase (KinC), and biolm formation. They propose that KinC governs the expression of genes required for biolm formation, and it is tempting to speculate that there might be a convergence between our work and theirs. The tendril phenotype we have investigated here requires a low potassium ion concentration in the medium (Kinsinger et al., 2005; Fall et al., 2006), the presence of surfactin, and involves the formation of multicellular structures that could be considered a type of surface biolm (see Lemon et al., 2008).
 c

The use of transposon mutagenesis to identify other required genes for intercellular communication in B. subtilis is in progress. In addition, future work is needed to explain why gspA mutant tendrils are much more inhibitory to tendrils of its parental strain and do not touch the latter (Fig. S2), and why many of the surviving colonies in the transposon screening produced pellicle-like colonies rather than tendrils (Fig. 4, upper). Identication of genes that alter the tendril phenotype to that of pellicle formation might also reveal controls on these types of colony growth. Various gene disruption methods been used successfully to identify large numbers of genes essential for the formation of B. subtilis multicellular communities (Branda et al., 2004; Kobayashi, 2007); these genes could be related to the tendril/ pellicle phenotypes seen in our work. It is of interest that Gibbs et al. (2008) have shown that in the bacterium Proteus mirabilis, boundaries form between swarming colonies of different strains, but those of a single strain exhibit touching and overlap. This suggests another type of self-identity mechanism in approaching bacterial colonies. It is not yet known whether our nding of the opposite phenomenon in B. subtilis (i.e. tendrils of the same strain do not touch) is related to a self-identity mechanism, but this phenomenon could be relevant to the behavior of B. subtilis colonization of plant roots (Bais et al., 2004). For example, the growth of tendrils on laboratory media might be indicative of the exploratory colonization of soil/root habitats by B. subtilis, where there is likely to be some deciency of essential macro- or micronutrients, an idea that we have expressed elsewhere (Kinsinger et al., 2005). A tendril avoidance behavior may be a strategy for more efcient exploration of a root surface before commitment to the metabolically expensive process of biolm production. In addition, with the ndings that various B. subtilis strains produce different families of exocellular lipopeptides, including surfactins, iturins, and fengycins (Lecl ere et al., 2006), future work may reveal whether specic lipopeptides (e.g. particular surfactins) are responsible for the signaling that controls tendril growth and avoidance behavior.

Acknowledgements
This work was supported in part by a grant from The University of Colorado, Innovative Grant Program, and by NSF grant MCB-0721187 to D.B.K. We thank Tami Sivy for help with HPLC analysis of surfactins.

References
Antelmann H, Bernhardt J, Schmid R & Hecker M (1995) A gene at 333-degrees on the Bacillus subtilis chromosome encodes

2009 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

FEMS Microbiol Lett ]] (2009) 18

Bacillus subtilis: tendril sensing and avoidance

the newly identied sigma(B)-dependent general stress protein GspA. J Bacteriol 177: 35403545. Bais HP, Fall R & Vivanco JM (2004) Biocontrol of Bacillus subtilis against infection of Arabidopsis roots by Pseudomonas syringae is facilitated by biolm formation and surfactin production. Plant Physiol 134: 307319. Blair KM, Turner L, Winkelman JT, Berg HC & Kearns DB (2008) A molecular clutch disables agella in the Bacillus subtilis biolm. Science 320: 16361638. Bodour AA & Miller-Maier RM (1998) Application of a modied drop-collapse technique for surfactant quantitation and screening of biosurfactant-producing microorganisms. J Microbiol Meth 32: 273280. Branda SS, Gonz alez-Pastor JE, Ben-Yehuda S, Losick R & Kolter R (2001) Fruiting body formation by Bacillus subtilis. P Natl Acad Sci USA 98: 1162111626. Branda SS, Gonzalez-Pastor JE, Dervyn E, Ehrlich SD, Losick R & Kolter R (2004) Genes involved in formation of structured multicellular communities by Bacillus subtilis. J Bacteriol 186: 39703979. Caiazza NC, Shanks RMQ & OToole GA (2005) Rhamnolipids modulate swarming motility patterns of Pseudomonas aeruginosa. J Bacteriol 187: 73517361. Daniels R, Vanderleyden J & Michiels J (2004) Quorum sensing and swarming migration in bacteria. FEMS Microbiol Rev 28: 261289. Debois D, Hamze K, Guerineau V et al. (2008) In situ localisation and quantication of surfactins in a Bacillus subtilis swarming community by imaging mass spectrometry. Proteomics 8: 36823691. Fall R, Kearns DB & Nguyen T (2006) A dened medium to investigate sliding motility in a Bacillus subtilis agella-less mutant. BMC Microbiol 6: 3142. Gibbs KA, Urbanowski ML & Greenberg EP (2008) Genetic determinants of self identity and social recognition in bacteria. Science 321: 256259. Harshey RM (2003) Bacterial motility on a surface: many ways to a common goal. Annu Rev Microbiol 57: 249273. Henrichsen J (1972) Bacterial surface translocation: a survey and a classication. Bacteriol Rev 36: 478503. Hsieh FC, Li MC, Lin TC & Kao SS (2004) Rapid detection and characterization of surfactin-producing Bacillus subtilis and closely related species based on PCR. Curr Microbiol 49: 186191. Julkowska D, Obuchowski M, Holland IB & Seror SJ (2005) Comparative analysis of the development of swarming communities of Bacillus subtilis 168 and a natural wild type: critical effects of surfactin and the composition of the medium. J Bacteriol 187: 6576. Kearns DB & Losick R (2003) Swarming motility in undomesticated Bacillus subtilis. Mol Microbiol 49: 581590.

Kinsinger RF, Shirk MC & Fall R (2003) Rapid surface motility in Bacillus subtilis is dependent on extracellular surfactin and potassium ion. J Bacteriol 185: 56275631. Kinsinger RF, Kearns DB, Hale M & Fall R (2005) Genetic requirements for potassium ion-dependent colony spreading in Bacillus subtilis. J Bacteriol 187: 84628469. Kobayashi K (2007) Bacillus subtilis pellicle formation proceeds through genetically dened morphological changes. J Bacteriol 189: 49204931. Kroos L (2007) The bacillus and myxococcus developmental networks and their transcriptional regulators. Annu Rev Genet 41: 1339. Lecl ere V, Marti R, Bechet M, Fickers P & Jacques P (2006) The lipopeptides mycosubtilin and surfactin enhance spreading of Bacillus subtilis strains by their surface-active properties. Arch Microbiol 186: 475483. Lemon KP, Earl AM, Vlamakis HC, Aguilar C & Kolter R (2008) Biolm development with an emphasis on Bacillus subtilis. Curr Top Microbiol 322: 116. Levano-Garcia J, Verjovski-Almeida S & da Silva ACR (2005) Mapping transposon insertion sites by touchdown PCR and hybrid degenerate primers. Biotechniques 38: 225229. Lopez D, Fischbach MA, Chu F, Losick R & Kolter R (2009) Structurally diverse natural products that cause potassium leakage trigger multicellularity in Bacillus subtilis. P Natl Acad Sci USA 106: 280285. Petersohn A, Bernhardt J, Gerth U, Hoper D, Koburger T, Volker U & Hecker M (1999) Identication of sigma (B)-dependent genes in Bacillus subtilis using a promoter consensus-directed search and oligonucleotide hybridization. J Bacteriol 181: 57185724. Peypoux F, Bonmatin JM & Wallach J (1999) Recent trends in the biochemistry of surfactin. Appl Microbiol Biot 51: 553563. Recht J & Kolter R (2001) Glycopeptidolipid acetylation affects sliding motility and biolm formation in Mycobacterium smegmatis. J Bacteriol 183: 57185724. Recht J, Martinez A, Torello S & Kolter R (2000) Genetic analysis of sliding motility in Mycobacterium smegmatis. J Bacteriol 182: 43484351. Tremblay J, Richardson AP, Lepine F & Deziel E (2007) Selfproduced extracellular stimuli modulate the Pseudomonas aeruginosa swarming motility behaviour. Environ Microbiol 9: 26222630. von Bodman SB, Willey JA & Diggle SP (2008) Cellcell communication in bacteria: united we stand. J Bacteriol 190: 43774391. Weidenmaier C & Peschel A (2008) Teichoic acids and related cell-wall glycopolymers in gram-positive physiology and host interactions. Nat Rev Microbiol 6: 276287.

FEMS Microbiol Lett ]] (2009) 18

 c

2009 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

B.L. James et al.

Supporting Information
Additional Supporting Information may be found in the online version of this article: Fig. S1. Tendril growth by Bacillus subtilis hag mutant DS64 on Mk medium is diverted by surfactin. Fig. S2. SPP1 phage transduction of the tendril-touching phenotype into a parental hag strain.

Appendix S1. Transposon mutagenesis. Please note: Wiley-Blackwell is not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.

 c

2009 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

FEMS Microbiol Lett ]] (2009) 18