JOURNAL OF BACTERIOLOGY, Aug. 2006, p. 5888–5895 Vol. 188, No. 160021-9193/06/$08.00�0 doi:10.1128/JB.00559-06Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Rippling Is a Predatory Behavior in Myxococcus xanthusJames E. Berleman, Tatiana Chumley, Patricia Cheung, and John R. Kirby*

School of Biology, Georgia Institute of Technology, Atlanta, Georgia 30332

Received 19 April 2006/Accepted 12 May 2006

Cells of Myxococcus xanthus will, at times, organize their movement such that macroscopic traveling waves,termed ripples, are formed as groups of cells glide together on a solid surface. The reason for this behavior haslong been a mystery, but we demonstrate here that rippling is a feeding behavior which occurs when M. xanthuscells make direct contact with either prey or large macromolecules. Rippling has been observed during twofundamentally distinct environmental conditions: (i) starvation-induced fruiting body development and (ii)predation of other organisms. Our results indicate that case (i) does not occur in all wild-type strains and isdependent on the intrinsic level of autolysis. Analysis of predatory rippling indicates that rippling behavior isinducible during predation on proteobacteria, gram-positive bacteria, yeast (such as Saccharomyces cerevi-siae), and phage. Predatory efficiency decreases under genetic and physiological conditions in which ripplingis inhibited. Rippling will also occur in the presence of purified macromolecules such as peptidoglycan, protein,and nucleic acid but does not occur in the presence of the respective monomeric components and also does notoccur when the macromolecules are physically separated from M. xanthus cells. We conclude that ripplingbehavior is a mechanism utilized to efficiently consume nondiffusing growth substrates and that developmentalrippling is a result of scavenging lysed cell debris.

Myxococcus xanthus is a predatory �-proteobacterium thatis able to lyse a variety of other bacteria and grow on thenutrients released (7, 21). However, it is best known for itsresponse to nutrient depletion in which growth halts andapproximately 105 cells aggregate into fruiting bodies, wherecells differentiate into metabolically quiescent spores (4).Predation and development reside on opposite ends of theM. xanthus life cycle, and there is little known about theconnection between the two processes, yet they share somecommon mechanistic ground. There is evidence for bothprocesses utilizing motility, intercellular communication,and of course, information about nutritional status (19).One striking commonality is that both predation and devel-opment have been observed to induce rippling motility be-havior—the organization of cells into moving groups thatresemble the movement of waves rippling on water (20).

The movement of M. xanthus cells on a solid surface istypically observed as a tangled collection of cells moving bothindividually and in streaming groups. Surface motility occursthrough the use of two complementary gliding motility systems.Type IV pilus-based motility is thought to pull cells in a man-ner similar to the twitching motility of Pseudomonas aeruginosa(14), while a poorly understood slime extrusion mechanism isthought to be able to propel cells forward (8). Both systemsrequire the chemotaxis-like frz pathway to modulate cell rever-sals and produce directed movement (17, 30). During rippling,cells are organized into nearly parallel lines of high cell density,with troughs of low cell density in between (24). Coordinatedmovement of cells perpendicular to the axis of the cell linecreates the appearance of a traveling wave. However, whencells of neighboring parallel waves come into contact, cell re-versals are induced such that the two waves reflect off each

other (26). Thus, although cells aggregate into waves duringrippling, there is no net cell displacement throughout theswarm, as each wave of cells oscillates between neighboringwaves. Directed movement of cells can also be observed duringfruiting body formation, but under these circumstances, cellstend to move in the direction of the stream, with net movementinto aggregation centers producing the early fruiting bodystructure (27).

Although rippling has been observed during predation,predatory rippling is rarely mentioned in the literature andrippling has primarily been studied in pure cultures duringstarvation-induced development. Developmental rippling hasbeen observed to occur spontaneously and sporadically instarving cultures of M. xanthus prior to and concurrent withfruiting body formation (6). Recent experimental and theoret-ical study of this process indicates that the rippling pattern canbe produced through a minimal requirement of cell-cell con-tacts and an internal biochemical oscillation system (1, 9, 10,26, 31). Developmental rippling has been proposed to rely onthe levels of the starvation-induced C-signal, a 17-kDa form ofthe CsgA protein (12, 27). The C-signal is thought to be pre-sented on the surface of one cell and to interact with anunidentified receptor on a neighboring cell to transmit thesignal (27). The CsgA protein is induced by starving conditions,with CsgA levels rising throughout the developmental processand proposed to induce first rippling and then aggregation andsporulation, all as a function of CsgA concentration (6, 11).

The C-signal model, however, does not sufficiently explainwhy rippling behavior is observed during predation. Underpredatory conditions, M. xanthus cells are digesting the avail-able prey cells and growing on the nutrients released, yet theystill demonstrate rippling behavior. It is unknown if predatoryrippling behavior requires the starvation-induced C-signal or ifit operates via an alternative mechanism. In this study, wedemonstrate that rippling behavior is a general mechanismrequired for efficient predation and that developmental rip-

* Corresponding author. Mailing address: School of Biology, Geor-gia Institute of Technology, Atlanta, GA 30332. Phone: (404) 894-8418. Fax: (404) 894-0519. E-mail: john.kirby@biology.gatech.edu.

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pling only occurs under conditions which mimic predation,indicating that rippling is solely a predatory behavior in M.xanthus. We conclude that rippling behavior serves to maxi-mize predation efficiency and nutrient scavenging.

MATERIALS AND METHODS

Strains and growth conditions. M. xanthus strains DZ2 and DK1622, bothcommonly referred to in the literature as wild type, were used in this study. StrainDZ4469 (DZ2 �pilA::tet) was kindly provided by D. Zusman (28). Escherichiacoli strain �2155 was used as prey. �2155 was used for quantitative predationassays, since it is a Kanr diaminopimelic acid (DAP) auxotroph and thus prey celldensities remain relatively constant in any media lacking DAP. For routineculturing, M. xanthus was grown in Casitone yeast extract (CYE) broth, whichcontains 10 mM morpholinepropanesulfonic acid (MOPS), pH 7.6, 10 g/literCasitone, 5 g/liter yeast extract, and 8 mM MgSO4. E. coli was cultured in LBbroth (23). Kanamycin was supplied when appropriate at 100 �g/ml and DAP at100 �g/ml. CF agar was utilized for analysis of M. xanthus cells under lownutrient conditions. CF contains 10 mM MOPS, pH 7.6, 1 mM KH2PO4, 8 mMMgSO4, 0.02% (NH4)2SO4, 0.2% citrate, 0.2% pyruvate, and 150 mg/liter Casi-tone. MMC buffer was used for harvesting and washing of cells and consists of 10mM MOPS, pH 7.6, 4 mM MgSO4, and 2 mM CaCl2. Saccharomyces cerevisiaestrain PJ69-4A (provided by P. James) and Bacillus subtilis strain OI1085 (pro-vided by G. Ordal) were cultured according to standard methods.

Predation assays. Cultures of M. xanthus and E. coli were harvested at mid-logphase and washed three times in MMC buffer. For qualitative assays, 30% Indiaink was added to M. xanthus cells for visualization, and M. xanthus and E. colicells were pipetted 1 mm apart in 2-�l aliquots containing �2 � 107 cells each.For quantitative analysis, cell suspensions were pipetted onto either CF or CYEmedia in 10-�l aliquots, with �107 M. xanthus cells plated first and allowed todry, followed by the addition of �109 E. coli cells in 10 �l and incubation at 32°C.To measure the number of E. coli survivors, cells were harvested from plates atthe time points indicated and resuspended in MMC buffer. The resuspension wasthen serially diluted and cells plated on appropriate media to determine thenumber of CFU. Addition of kanamycin to the media prevents the outgrowth ofM. xanthus cells present in the suspension. Three samples were harvested andquantified at each time point. The same procedure was used for the analysis ofcomponents which induce rippling, with each substrate that was tested resus-pended in both water and in melted agar to a final concentration of 1.5%immediately before plating. Substrates were tested at a concentration of 10mg/ml. Prey were tested at densities of 108 cells/ml for B. subtilis, 1010 PFU/ml forP1 phage, and 107 cells/ml for S. cerevisiae. Each substrate was tested in thepresence of at least three independent cultures.

Microscopy. Rippling behavior was observed using a Nikon SMZ10000 dis-secting microscope and a Nikon Eclipse E400 phase-contrast microscope. Imageswere captured using QImaging camera and software.

Autolysis assays. Mid-log-phase cultures of M. xanthus cultures were har-vested, washed in MMC buffer, and concentrated to �108 cells/ml equivalent.Replicate 1-ml samples of each cell suspension were added to 12-well plates andincubated at 32°C. To quantify the number of intact cells, the suspension wasaspirated, transferred to a 1.5-ml tube and sonicated at low power for 3 s todisperse large cell clumps. Direct cell counts were performed using a hemacy-tometer at the time points described in Results. Three independent cultures ofeach strain were examined, with three samples analyzed at each time point.

RESULTS

Predation-induced rippling behavior. When incubated onlow nutrient media at sufficiently high cell density, M. xanthuscells will glide into aggregates that become fruiting bodies.However, even on low nutrient media, not all of the cellscongregate into fruiting bodies. Many cells glide away from theinitial inoculum to colonize the agar surface. To analyze prey-induced rippling, �2 � 107 M. xanthus cells mixed with Indiaink were pipetted adjacent to �2 � 107 E. coli cells on low-nutrient CF media. As M. xanthus cells colonize the agar sur-face, the swarm expands in a thin, nonuniform layer of indi-viduals and groups that results in a rough, tangled appearance(Fig. 1A and B). Direct contact of M. xanthus cells with suitable

prey bacteria, such as E. coli, results in penetration of the preycolony by M. xanthus cells and subsequent lysis and digestion ofthe prey. Tangled behavior continues in the leading edge of M.xanthus cells as they migrate through the prey colony, butwithin 16 to 24 h of the initial contact with prey bacteria,rippling behavior is induced in trailing cells, resulting in aggre-gations of cells into nearly parallel waves which form perpen-dicular to the direction of the M. xanthus swarm migration(Fig. 1C and D). Under these conditions, rippling pattern for-mation is localized solely to the initial area occupied by theprey colony and continues for several days. Areas of the M.xanthus swarm adjacent to the prey locale, but outside of theprey colony, do not display the rippling phenotype at any time(Fig. 1E and F). Note that even as the swarm moves beyond theprey colony, and the nutrients contained therein, cells displaytangled rather than rippling behavior. Also, several fruitingbody aggregates can be observed forming near the initial M.xanthus inoculum; however, rippling behavior is not observedassociated with these fruiting bodies.

Analysis of predatory efficiency. The induction of ripplingduring predation indicates that rippling may be beneficial oreven necessary for the efficient digestion of available preysubstrates. To test whether rippling is required for predation,we incubated M. xanthus cells in the presence of E. coli on low-and high-nutrient medium. Preliminary results had indicatedthat rippling behavior is inhibited by excessive nutrients, al-though entry into the E. coli prey colony is not inhibited. Toexamine this process further, we used an E. coli DAP auxo-troph (�2155) as prey, since the viable prey cell levels remainrelatively constant in this strain on both CF and CYE medialacking DAP. Incubation of �109 cells of �2155 in the pres-ence of �107 cells of M. xanthus strain DZ2 on CF mediaresults in rapid lysis of prey, with 90% of the E. coli cells lysedin the first 24 h and the E. coli CFU dropping below detectablelevels by 48 h (Fig. 2). By comparison, predation occurs muchmore slowly when the cells are incubated on ripple-inhibitingnutrient rich medium, with �90% of prey cell lysis not ob-served until 96 h. It should be noted that while viable E. colicells are not detected after 48 h of incubation on CF in thepresence of DZ2, rippling is still observed for at least 72 hbeyond this point. This indicates that while live prey bacteriamay induce rippling, complete lysis of the available preybacteria does not correlate with the termination of ripplingbehavior.

The differences observed in predation under high- and low-nutrient conditions may be due to other factors besides rip-pling, such as differences in production levels of lytic enzymes.Analysis of a DZ2 �pilA mutant (DZ4469), which is unable tomove by the type IV pilus motility system (32) and also unableto ripple (data not shown), indicates that it is defective inpredation at both nutrient levels tested. On CF there are �105

viable E. coli cells remaining after 48 h. After 96 h, E. colisurvivors are not detected, indicating that lytic enzymes are stillproduced in this strain and that the E. coli cells are eventuallylysed, but the rate is significantly slower than that of the parentstrain. In the presence of high nutrient levels, E. coli lysis bythe �pilA strain is indistinguishable from the basal rate of lysisof E. coli cells alone.

Rippling occurs in response to a variety of macromolecularsubstrates. The observation of rippling continuing beyond the

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completion of prey lysis indicates that the substrate(s) whichstimulate rippling may still be present. To determine whatcomponents of prey are capable of inducing rippling, we plated�107 M. xanthus cells in a 10-�l suspension adjacent to a 10-�laliquot of the test substrate on CF plates and allowed them todry. In this assay, heat-killed E. coli cells and the insolublefraction from lysed E. coli cells are both capable of inducingrippling behavior (Table 1). The soluble E. coli fraction wasunable to induce rippling. A previous study by Shimkets andKaiser demonstrated that M. xanthus rippling behavior is in-ducible by peptidoglycan extracts from M. xanthus as well asseveral other proteobacteria and gram-positive bacteria (24).Our data confirms this result, but further testing indicates that

several other substrates also serve to induce rippling. Wefound that in addition to E. coli, B. subtilis, S. cerevisiae and P1phage are all capable of inducing rippling. While B. subtilis hasa peptidoglycan structure similar to that of E. coli, P1 phageand the yeast S. cerevisiae are two biological entities that do notcontain peptidoglycan.

To determine if M. xanthus cells will ripple in the presence ofmolecules other than peptidoglycan, we incubated M. xanthuscells with bovine serum albumin and salmon testes chromo-somal DNA. These substrates were chosen based on their largesize and their representation of amino acid and nucleic acidcomposition. In both cases, rippling was observed, although thebehavior was not as prolonged as with live cells or peptidogly-

FIG. 1. Predatory behavior of M. xanthus. M. xanthus strain DZ2 cells mixed with India Ink (left) and E. coli cells (right) were pipetted ascolonies 1 mm apart on CF medium. The M. xanthus swarm expands from the initial spot in a tangled motility pattern. Lysis of E. coli prey cellsoccurs as M. xanthus cells make direct contact with the E. coli colony. Expansion of the swarm through the E. coli colony induces rippling motilitybehavior. Beyond the E. coli colony, the swarm resumes the tangled motility behavior. Pictures were taken at the following time points: 16 h (Aand B), 40 h (C and D), and 64 h (E and F). The panels on the left were captured at a magnification of �20. The panels on the right were capturedat a magnification of �200 and correspond to the regions marked with rectangles on the left.

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can. Examination of the monomeric components of these mac-romolecular substrates indicates that they fail to induce rip-pling under the conditions of this assay. Peptidoglycancomponents N-acetylglucosamine, diaminopimelic acid, Casi-tone (a mixture of amino acids and small peptides from hy-drolyzed casein protein), and deoxynucleoside triphosphateswere unable to induce rippling behavior. When physical sepa-ration of the M. xanthus cells from each substrate was per-formed through premixing of the substrate with melted agarprior to addition in the assay, the M. xanthus rippling responsewas not observed, indicating that direct cell-to-substrate orcell-to-prey contacts are required for this behavior.

Developmental rippling is strain dependent. Our analysis ofrippling behavior during predation in M. xanthus strain DZ2indicated that while rippling is readily induced by the presenceof prey or prey components, it was not observed at all in ourcontrol samples lacking prey (Fig. 3). Other groups have re-ported that rippling will occur spontaneously on low-nutrientmedia in the absence of prey; in fact, this “developmental”rippling has been the focus of study on rippling behavior for anumber of years (27). The apparent discrepancy may be due tothe M. xanthus strain used, as DK1622 is most commonly usedduring experimental analysis of rippling behavior. The differ-ences between the DK1622 and DZ2 have not been specificallycharacterized, but the DK1622 strain has undergone significantgenetic modification, including the loss and subsequent resto-ration of both motility systems (2, 3, 29). The DZ2 strain hasnot been modified from the original isolate other than throughculturing for strain propagation (D. Zusman, personal com-munication). Figure 3 shows that both DZ2 and DK1622 ripplewhen preying on E. coli, but only DK1622 was observed toripple in pure culture. Rippling in DK1622 pure cultures isnonuniform, and the direction of wave propagation is unpre-dictable. The rippling pattern displayed during predation oc-curs uniformly and predictably for both strains, with the rip-pling pattern propagating out from the initial M. xanthus

inoculum in concentric waves. However, there is still a consis-tent difference in the rippling pattern displayed by the twostrains with DK1622 showing a shorter wavelength than DZ2.The basis for this difference is unknown.

The role of cell autolysis in developmental rippling. Sincerippling pattern formation occurs similarly when M. xanthuscells are in the presence of dead prey as it does with live prey,we felt that it was prudent to examine the role of developmen-tal autolysis in connection with developmental rippling. Thereis contradictory evidence as to whether or not cell lysis is anessential component of the developmental program, and weconsidered that part of this discrepancy could be strain depen-dent (18, 22). We analyzed the autolytic rates of DZ2 andDK1622 cells that had been resuspended in MMC buffer andincubated in 12-well plates at 32°C. Figure 4 shows thatDK1622 undergoes cell lysis at a faster rate than DZ2 after theonset of starvation, with �90% of the cells dying over thecourse of this assay. In strain DZ2, lysis is still observed but ata much slower rate. We hypothesize that the differences ob-

FIG. 2. Rippling behavior is essential for efficient predation. Rep-licate 10-�l aliquots containing �109 E. coli prey cells were pipettedonto dried 10 �l spots of �107 M. xanthus cells on either CF or CYEagar. At the times shown, E. coli cells were harvested and the numberof E. coli survivors was measured as CFU. The lines represent E. colionly on CF (■) and CYE (�), E. coli in the presence of DZ2 on CF(F) and CYE (E), and E. coli in the presence of DZ2 �pilA mutant onCF (Œ) and CYE (‚).

TABLE 1. Analysis of M. xanthus responses to preycell componentsa

SubstrateResult for suspension

Water 1.5% agar

E. coliLive �� Heat-killed �� Soluble fraction Insoluble fraction ��

B. subtilisLive �� Heat-killed �� Soluble fraction Insoluble fraction ��

S. cerevisiaeLive �� Heat-killed �� Soluble fraction Insoluble fraction ��

P1 phageLive � Heat-killed �

Peptidoglycan extract �� N-Acetylglucosamine DAP Bovine serum albumin � Amino acids Chromosomal DNA � Nucleic acids

a The substrates were resuspended in either water or 1.5% agar and thenpipetted (10-�l aliquots) onto CF agar plates and allowed to dry. Approximately107 M. xanthus DZ2 cells were pipetted in a 1-�l aliquot in the center of each testsubstrate. Concentrations were as follows: E. coli and B. subtilis, �108 cells/ml;S. cerevisiae, �107 cells/ml; P1 phage, �109 PFU/ml. The concentration for eachcompound tested was 10 mg/ml. The amino acid mixture used was a commercialpreparation of hydrolyzed casein (Casitone); the chromosomal DNA used was acommercial extract of salmon sperm DNA. For nucleic acids, an equimolarmixture of deoxynucleoside triphosphates was used. ��, prolonged ripplingresponse; �, weaker rippling response; , rippling was not observed within3 days.

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served in autolytic rates account for the spontaneous develop-mental rippling observed in DK1622.

To test if increased cell death could induce developmentalrippling in DZ2, we harvested and washed a culture of mid-log-phase DZ2 cells and split the cell suspension into twoparts: one of which was left untreated and the other exposed toheat at 95°C for 10 min. We then mixed the untreated DZ2cells with the heat-killed DZ2 cells in 10% increments rangingfrom 0 to 100% live cells and added the cell mixtures to CFplates to allow development. Under these conditions, we ob-served rippling to occur in all samples that contained 20% ormore dead cell material (Fig. 5). Interestingly, rippling is onlyobservable in the initial spot, indicating that live cells whichleave the area containing the dead cell material are not in-duced to ripple.

In an accompanying experiment, aliquots of heat-killed DZ2cells were mixed with water instead of live cells to yield incre-ments of dead cell material as above. These preparations werethen pipetted adjacent to 10-�l colonies of live M. xanthus DZ2cells on CF. Under these circumstances, physical separation ofdevelopmental rippling and fruiting body formation can beobserved, with M. xanthus cells located in the initial spot ag-gregating into fruiting bodies, while cells migrating into theregion containing the dead cell material are induced to ripple.

FIG. 3. Rippling differences observed in wild-type strains DK1622 and DZ2. M. xanthus cells from strains DK1622 and DZ2 were pipetted inthe presence and absence of E. coli prey on CF agar and incubated at 32°C for 72 h. (A) DK1622 alone; (B) DZ2 alone; (C) DK1622 with E. coli;(D) DZ2 with E. coli. Although rippling is observed in both M. xanthus strains in the presence of prey E. coli cells, rippling was only observed inthe pure cultures of strain DK1622.

FIG. 4. Lytic differences observed in wild-type strains DK1622 andDZ2. Log-phase cultures of strains DK1622 (}) and DZ2 (F) wereharvested and washed in MMC buffer. One-milliliter cell suspensionscontaining �108 cells/ml were added to 12-well plates and incubated at32°C. Cells were counted directly in a hemacytometer at the timesindicated. Due to different levels of cell lysis, �108 cells were added towells, and the viability was measured through direct cell counts at thetimes indicated.

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Rippling was observed in all samples containing the equivalentof 20% or more dead cell material.

DISCUSSION

When the phenomenon of rippling was first reported byReichenbach in the 1960s, he noted that it occurred duringboth predation and development (20). In the early 1980s,Shimkets and Kaiser showed that rippling was inducible bypeptidoglycan extracted from a variety of proteobacteria andgram-positive bacteria, including M. xanthus, raising the pos-sibility that developmental rippling occurs in response to thepeptidoglycan released from lysing M. xanthus cells (24). In thiswork, we demonstrate that rippling occurs as a general re-sponse to the presence of a variety of prey or macromolecularsubstrates. Physical separation from the macromolecular sub-strate or incubation in the presence of the monomeric subunitsof these macromolecules does not induce rippling behavior.This result indicates that M. xanthus cells are capable of sens-ing a variety of macromolecules directly, rather than throughrelease of extracellular digestive enzymes and subsequent sens-

ing of the monomeric components. M. xanthus cells move at arate which is slower than the diffusion of most water-solublemolecules, so it seems logical that their cell movements wouldbe attuned to substrates that move more slowly then they do.Determining the mechanism for sensing these large moleculesmay be challenging, but there are other reported instances ofbacteria utilizing mechanisms specifically designed for dealingwith macromolecules, such as the cell-associated degradationof polysaccharides in Bacteroides spp. (25). Related predatorsof the Bdellovibrio genera (�-proteobacteria) require direct cellcontact and entry into the periplasm of their prey for predationto occur (13), so it is possible that M. xanthus predation func-tions best when in direct contact with suitable prey.

A recent study has shown that colliding rippling waves of M.xanthus cells reflect off each other, causing individual cells toreverse after contact such that any given wave of cells istrapped in an oscillation between neighboring waves. The re-sult is that the entire swarm moves in a coordinated fashionthat ensures multiple passages over a given area. The fact thatoscillating behavior is induced when M. xanthus cells are lo-cated in the area of prey implies that rippling behavior is

FIG. 5. Effect of dead cells on developmental rippling. Ten-microliter aliquots of M. xanthus cells were pipetted onto CF media as mixtures oflive and dead (heat-killed) cells and photographed after 24 h of incubation at 32°C. (A) 10% dead cells; (B) 80% dead cells. Ten-microliter aliquotsof live cells were also pipetted separate from but adjacent to the same quantity of dead cell material. (C) 10% dead cells; (D) 80% dead cells. Thevolume of the dead cell material in each of these aliquots was raised to 10 �l with water.

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beneficial for predation. This is supported by the fact thatgenetic and physiological conditions which inhibit rippling alsoinhibit efficient predation. We propose a model in which thedetection of macromolecules by M. xanthus stimulates the os-cillatory behavior of moving cells as a method to ensure that allavailable growth substrate is consumed.

M. xanthus is a very slow moving bacterium confined togliding along surfaces, and thus, it has been difficult to dem-onstrate chemotaxis by M. xanthus cells within a gradient ofdiffusible chemicals in a manner similar to that displayed by E.coli (5). However, directed movement of M. xanthus cells re-quires the che1 (frz) chemotaxis-like pathway, and cells carry-ing mutations in the frz pathway have been shown to exit E. coliprey microcolonies prior to complete lysis of prey (15, 16). Thisindicates that the Frz chemotaxis homologs are part of themolecular pathway necessary for M. xanthus to properly rec-ognize and modulate cell movement in response to prey. Ourobservations of M. xanthus predatory behavior indicate thatwhile M. xanthus cells do not appear to be specifically attractedto areas containing prey, cell movements are clearly altered inthe presence of prey. It is possible that the ability of a givensubstance to induce rippling may be the best indication of itssuitability as a chemoattractant for this species. Chemotacticmovement allows E. coli cells to accumulate in locations sup-porting optimal growth. For a bacterium that is constrained, asM. xanthus is, to two-dimensional movement and adapted tothe digestion of large molecules or prey cells, there is littleadvantage to a mass accumulation of cells when the growthsubstrate is nondiffusing. Cells which cannot make direct con-tact with the growth substrate may not gain any benefit. Thus,the induction of rippling may serve to trap some cells in anutrient-rich environment while also serving to limit competi-tion. Further examination of the process of rippling will berequired to determine how it relates to typical chemotacticmechanisms.

Finally, we also demonstrate that the wild-type M. xanthusstrain analyzed is critical for the interpretation of ripplingbehavior. Although DK1622 and DZ2 are both induced toripple in the presence of prey, only DK1622 was observed toripple in pure culture, a response that appears to be depen-dent on the elevated level of cell lysis detected in this cellline. Even in the presence of prey, rippling features such aswavelength are markedly different between the two strains.The observation of rippling behavior in the presence ofnutrients released during predation challenges the idea thatrippling behavior is stimulated purely by cell-cell transmis-sion of the starvation-induced C signal. Also, the ability ofdead cell material to stimulate rippling in developing colo-nies of DZ2 indicates that, while it is possible that C signal-ing is utilized to mediate the response to prey, it seems thatboth developmental and predatory rippling also require thepresence of macromolecular growth substrates. Thus, a deeperunderstanding of rippling behavior and its requirements de-pends on a better understanding of the differences between thetwo M. xanthus wild-type strains and a careful dissection of theresponses of this bacterium to the separate stimuli of starva-tion and predation.

ACKNOWLEDGMENTS

We thank D. Bodenmiller for editorial comments. We also thankmembers of the Kirby lab for valuable comments and members of theZusman lab for valuable communication and strains used in this work.

This work was supported by Georgia Tech start up funds and grantAI059682 from the National Institutes of Health to J.R.K.

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