Proc. Natl. Acad. Sci. USAVol. 90, pp. 3378-3382, April 1993Microbiology

The two motility systems of Myxococcus xanthus show differentselective advantages on various surfaces

(gliding bacterium/swarming/fruiting/video microscopy)

WENYUAN SHI AND DAVID R. ZUSMANDepartment of Molecular and Cell Biology, 401 Barker Hall, University of California, Berkeley, CA 94720

Communicated by Horace A. Barker, January 8, 1993 (received for review August 26, 1992)

ABSTRACT Myxococcus xanthus, a bacterium that formsfruiting bodies, moves by gliding motility utilizing dual motilitysystems that differ both genetically and morphologically [sys-tem A, having at least 21 genetic loci and moving mainly singlecells, and system S, having at least 10 genetic loci and movinggroups (rafts) of cells] [Hodgkin, J. & Kaiser, D. (1979) Mol.Gen. Genet. 172, 177-191]. In this study, we found that A- andS-gliding-motility systems have different selective advantageson surfaces containing different concentrations of agar. Weobserved that colonies of A+S- cells (A-motile cells) swarmedbetter than A-S+ cells (S-motile cells) on relatively firm anddry surfaces (e.g., 1.5% agar). In contrast, colonies of A-S+cells swarmed much better than A+S- cells on soft and wetsurfaces (e.g., 0.3% agar). Individual A-motile cells moved ata rate of 2-4 jam/min on 1.5% agar but they barely moved on0.3% agar (<0.5 ,um/min); in contrast S-motile cells moved3-5 times faster on 0.3% agar than on 1.5% agar. Wild-typecells with both A- and S-motility systems were able to move wellover a wide range of surfaces. These results suggest that dualmotility systems enable the myxobacteria to adapt to a varietyof physiological and ecological environments and show simi-larities in function to the dual motility systems of flagellatedbacteria such as Vibrio spp.

Dual motility systems are common in the microbial world.Many flagellated bacteria (e.g., Vibrio and Proteus) are ableto produce two types of flagella under different conditions(1-6). Most of these bacteria inhabit very complex environ-ments and their multiple motility systems have differentselective advantages that enable them to adapt to a variety ofphysiological and ecological environments. One of the bestunderstood examples is Vibrio parahaemolyticus. When thebacterium is grown in liquid, it produces a single sheathedpolar flagellum that is used for swimming; when grown on asolidified medium, it produces numerous unsheathed lateralflagella that are responsible for swarming over the solidsurface (5, 7-9).Myxococcus xanthus is a gliding bacterium that can move

on a solid surface without flagella (for recent reviews, seerefs. 10 and 11). The mechanism of gliding motility is stilllargely unknown; however, genetic and morphological anal-yses suggest that M. xanthus also contains dual motilitysystems: (i) system A is required for the movement of singlecells or small groups of cells (Fig. lj) and has at least 21genetic loci; (ii) system S is mainly involved in the movementof cells in groups (Fig. lk) and has at least 10 genetic loci(12-14). Although the genes for A- and S-motility (12-17) andthe cell surface structure related to A- and S-motility (18-22)have been partially characterized, little is known about thephysiological differences of the two motility systems in M.xanthus.

In this paper, we report that A-motility and S-motility showdifferent selective advantages on different surfaces: A-mo-tility allows cells to move better than S-motility on relativelyfirm and dry surfaces, whereas S-motility allows cells tomove much better on relatively soft and wet surfaces. Theseresults show that, like flagellated bacteria, the dual motilitysystems in gliding bacteria allow cells to adapt to a variety ofphysiological and ecological environments.

MATERIALS AND METHODS

Strains and Culture Conditions. M. xanthus strains used inthis study are listed in Table 1. M. xanthus cells were grownin a medium consisting of casitone (10 g/liter), yeast extract(5 g/liter), and 8 mM MgSO4 in 10 mM Mops buffer (pH 7.6;CYE) (24) at 32°C on a rotary shaker at 225 rpm. Sometimesa less-rich growth medium (CMM) was used, which consistsof casitone (5 g/liter), 8 mM MgSO4, and 10mM Mops buffer(pH 7.6). CF medium, used for testing fruiting body forma-tion, was prepared as described (25). Different concentra-tions of agar (Difco), agarose (Sigma), and Gel-Gro (ICN)were added to the various media (CYE, CMM, or CF) tomake swarming or fruiting plates.Swarming and Fruiting. For swarming assays, 2 /,u of cells

at a concentration of 1000 Klett units with a red filter (1 x 107cells) was added to the center of the swarming plates;alternatively, a wooden stick was used to inoculate bacteriafrom the CYE plates on the center ofthe swarming plates. Allswarming plates were incubated at 32°C for 3-4 days. Forfruiting, 20 ,ul of cells at 1000 Klett units (2 x 108 cells) wasadded to the fruiting plates and incubated at 32°C for 2-3days. The morphology ofthe colonies on swarming or fruitingplates was recorded by a video camera (COHU, model4815-2000) with a video macrolens. The morphology of theedges of colonies was recorded by the same video camerathrough a Zeiss microscope (model 47 60 05-9901). The videoimages were printed by a video printer (Hitachi VY-50).

Microscopic Observation of Gliding Motility. Gliding mo-tility on agar surfaces was observed with a Zeiss microscope.A designated medium (5 ml) with a designated concentrationof agar was added to a Falcon tissue culture dish (60 x 15 mm;Becton Dickinson). After the agar solidified, 10 ,ul ofbacterialcells was added to the center of the plate. After a 30-minincubation, the bacterial behavior was recorded by videomicroscopy for further analysis. Due to the slow movementof the M. xanthus cells, a time-lapse video cassette recorder(JVC, model BR-9000U) was used. Bacterial movementswere recorded at a 120 times slower rate and played back atnormal speed. The bacteria were maintained at ==22°C, unlessotherwise indicated.

13-Galactosidase Assay of Tn5-lac Transcriptional Fusionsto A- or S-Motility Genes. Cells were harvested directly fromthe swarming colonies on different surfaces and broken bysonication. The amounts of protein in these extracts weredetermined by the BCA protein assay (Pierce). Protein (80

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Proc. Natl. Acad. Sci. USA 90 (1993) 3379

A+ S A+ S A S+ A S

a b c d

I

e f g h

k

I

m n 0 p

FIG. 1. Swarming colonies and morphologies of the edges of colonies on different concentrations of agar. A+S+, DK1622; A+S-, DK1300;A-S+, DK1217; and A-S-, DZF4150. The medium used was CYE medium with 1.5% or 0.3% agar. The cells (2 Al) were inoculated onto theplates (the initial size of the colonies was around 0.2 cm) and incubated at 32°C for 3 days. The diameters of the swarming colonies (a-h) were1.6, 0.8, 0.6, 0.35, 2.1, 0.5, 1.7, and 0.35 cm, respectively. The approximate dimensions for the edges of colonies presented in the photos areas follows: i and m, 450 um x 340 ,um; j- and n-p, 110 pm x 90 Am.

ug) was assayed for /-galactosidase activity, as described byMiller (26).

RESULTS

M. xanthus Displays Different Swarming Behaviors on PlatesContaining Different Concentrations of Agar. As reported(12-15, 27, 28), when M. xanthus cells were placed on a solidagar plate (1.5% agar) containing abundant nutrients (such asCYE medium), the cells grew vegetatively and showedcooperative swarming movements. Wild-type (A+S+) cellsmoved away from the colony center to form a large swarmingcolony (Fig. la). Mutants defective in S-motility (A+S- cells)or mutants defective in A-motility (A-S+ cells) also swarmedon 1.5% agar to expand their colonies (Fig. 1 b and c);however, both ofthem had less swarming than wild-type cells(ref. 14 and Table 2) and A+S- cells swarmed better thanA-S+ cells (ref. 28 and Table 2). Nonmotile mutants that lackboth motility systems (A-S- cells) did not swarm and,therefore, formed small colonies (Fig. ld).When the same M. xanthus cells were inoculated onto

CYE medium with 0.3% agar, the swarming behavior ofcolonies was dramatically changed. Wild-type (A+S+) cells

formed much larger swarming colonies on 0.3% agar (Fig.le). However, the swarming ofA+S- cells was lower on 0.3%agar plate (Fig. lf) than on 1.5% agar (Fig. lb). In contrast,A-S+ cells showed limited swarming on 1.5% agar (Fig. lc)but greatly expanded swarming on 0.3% agar (Fig. lg). As acontrol, A-S- cells did not swarm on 0.3% agar either (Fig.lh). Table 2 compares the colony spreading on 0.3% agar tothat on 1.5% agar and shows that A+S+ cells spread more on0.3% agar, A+S- cells spread less on 0.3% agar, and A-S+cells spread much more on 0.3% agar. These results illustratethat changing the agar concentration from 1.5% to 0.3%causes A-motility to be reduced whereas S-motility is in-

creased.Fig. 2 shows a detailed study on the effects of agar

concentration on A- and S-motility. At low concentrations ofagar (<0.7% agar), cells having only S-motility (A-S+ cells)moved much better than cells having only A-motility (A+S-cells); at high concentrations of agar (>1.0%), cells havingonly A-motility moved better than cells having only S-mo-tility. When agar concentrations were too low (<0.3%) or toohigh (>1.5%), neither motility system appeared to functionvery well (Fig. 2). Again, these results suggest that the twomotility systems of M. xanthus function with different effec-

1 .5% agar

0.3% agar

1.5% agar

0.3% agar

Microbiology: Shi and Zusman

i

I

3380 Microbiology: Shi and Zusman

Table 1. M. xanthus strains used in this study

Motility Ref. orStrain Relevant genotype phenotype source

DZ2 Wild type A+S+ 23DK1622 Wild type A+S+ 20DZF1 sglA (leaky) A+S- 24DK1300 sglGl A+S- 14DK1253 tgl-l A+S- 14MXH1651 Tn5-lac insertion in an

S-motility gene A+S- P. HartzellMXH1226 TnS-4ac insertion in an

S-motility gene A+S- P. HartzellDK1217 aglBI A-S+ 14DK1218 cglB2 A-S+ 14MXH1216 Tn5-ac insertion in an

A-motility gene A-S+ P. HartzellMXH1273 TriS-4ac insertion in an

A-motility gene A-S+ P. HartzellDZF4150 Mutations in A- and

S-motility genes A-S- This paper

P. Hartzell is at the University of California, Los Angeles.

tiveness on surfaces made by different concentrations ofagar. It is particularly interesting to note that wild-type cellswere able to move well over a wide range of surfaces(0.3-1.5%) with a combination of A- and S-motilities (Fig. 2)and the rate ofmovement ofwild-type cells was more than thesum of A- and S-motility alone (Fig. 1).What causes different swarming behavior on different

concentrations of agar? It could simply be due to the reducedfirmness of the surface, since the surface of 0.3% agar issofter and wetter than that of 1.5% agar; alternatively,changing the agar concentration from 1.5% to 0.3% couldreduce some "key" chemical contaminant in the agar thatcontrols A- or S-motility. We prepared plates with ultrapureelectrophoresis grade agarose or Gel-Gro (an agar substitutemade by ICN) and used these substrates as agar substitutes.At 1.5% agarose and 0.6% Gel-Gro, M. xanthus formedcolonies with morphologies similar to that found on 1.5%agar; whereas at 0.5% agarose and 0.1% Gel-Gro, M. xanthuscolony morphology was similar to cells on 0.3% agar (data not

4.0

3.0-

E0

N2.0-c

0

0

Table 2. Effect of agar concentration on swarming

Swarmingcolony size,

cm2 0.3%/1.5% agar

Strain Motility type 1.5% 0.3% size ratio

DZ2 A+S+ 2.0 3.8 1.9DK1622 A+S+ 1.8 2.8 1.6DZF1 A+S- (leaky) 1.0 0.7 0.7DK1300 A+S- 0.4 0.2 0.5DK1253 A+S- 0.3 0.2 0.7DK1218 A-S+ 0.2 1.4 7.0DK1217 A-S+ 0.3 2.0 7.0DZF4150 A-S- 0.07 0.07 1.0

All plates were made with CYE broth and 1.5% or 0.3% agar,inoculated with 5 x 106 cells initially (duplicates for each strain), andthen incubated at 32°C for 3 days. The swarming colony sizes werethe average of the newly colonized areas. The error is within 10%o.

shown). These results suggest that the firmness and wetnessof the substratum are responsible for the pattern of cellswarming.

Gliding Motility on Plates with Different Concentrations ofAgar. To study the changed swarming behavior on differentconcentrations of agar, we examined the effect of agarconcentration on gliding motility. Gliding motility on 1.5%agar has been studied by examination of the morphology ofthe edges of colonies (12-15, 27, 28). A nonmotile strain(A-S- cells) gave an unorganized smooth colony edge (Fig.1 1 and p). A+S- cells had a rough colony edge with manysingle cells moving out of the colony (Fig. lj) and A-S+ cellshad a rough colony edge with many rafts of cells moving asflares out of the colony (Fig. lk). Wild-type (A+S+) cells on1.5% agar had a rough edge with both single cells and rafts ofcells (Fig. ii). We also examined the morphology ofthe edgesof swarming colonies on 0.3% agar and found that A+S- cellsgave a smooth edge (indicating little A-motility, Fig. ln)whereas A-S+ cells had a rough edge with highly extendedflares (indicating activated S-motility, Fig. lo). These dataare consistent with the colony-spreading behavior. It isinteresting to note that wild-type cells formed a highlyorganized edge (Fig. lm): under the microscope, we ob-

Agar concentration (%)

FIG. 2. Effect of agar concentration on A-motility and S-motility (see Table 2 for detailed explanations of the experiment). A, Wild-type cells(DK1622); O, A+S- cells (DK1300); *, A-S+ cells (DK1217).

Proc. Natl. Acad. Sci. USA 90 (1993)

Proc. Natl. Acad. Sci. USA 90 (1993) 3381

served many A+S+ cells aligned with each other in largegroups moving outward away from the colony center; singlecells later filled the gaps in between.We also used time-lapse video microscopy to more directly

study gliding motility of M. xanthus. Consistent with theswarming data and morphology of the edges of coloniespresented above, we found that A+S- cells moved at a rateof 2-4 Am/min on 1.5% agar but barely moved on 0.3% agar(the moving speed was <0.5 ,um/min and many cells ap-peared to vibrate in place) (Table 3). On the other hand, A-S+cells did not exhibit much movement when initially platedonto 1.5% agar; however, motility became better after 1.0 or1.5 h. When S-motile cells were plated on 0.3% agar, theywere motile immediately. In addition, the speed of cellmovement was 3-5 times faster on 0.3% agar than on 1.5%agar, based on analysis of the video images (Table 3). Suchmovement could often be observed in real time and themaximum rate of movement we observed was >20 ,um/minat 22°C. We also observed discontinuous gliding movementsof the bacteria on 0.3% agar. S-motile cells on 0.3% agarmoved forward with an accelerating speed, then paused, andthen moved forward again or reversed their direction. Allreversals of direction were observed after pauses. Further-more, S-motile cells were observed to interconnect and glideover each other to quickly fill gaps so that the distribution ofcells became more or less uniform (data not shown).

Effect of Cell Cohesion on Swarming. What causes S-mo-tility to be activated and A-motility to be inhibited on 0.3%agar? Previous studies showed (10) that S-motile cells (bothA+S+ and A-S+ cells) can agglutinate in liquid medium byusing cell surface structures such as pili and fibrils, whereasA+S- cells remain suspended mainly as single cells. Thefollowing data suggest that cellular cohesiveness may play arole in colony swarming on 0.3% agar. We found the cohe-siveness of S-motile cells growing on 0.3% CYE agar to beextraordinarily strong: the colonies remained intact afterbeing transferred from 0.3% agar to liquid solution and thenvortex mixed at maximum speed for 1 min. In contrast, thecolonies of A+S- strains were very easy to disperse. Somechemicals such as EDTA and ethanol were found to inhibitthe agglutination of S-motile cells (29). We found that in thepresence of these chemicals (1.5 mM EDTA or 2% ethanol),S-motile cells failed to swarm on 0.3% CYE agar (data notshown). We have also isolated many mutants from A+S-

Table 3. Effect of agar concentration on motilityMotility of individual cells

CYE CF

Strain Motility type 1.5% 0.4% 1.5% 0.4%DZ2 A+S+ + ++ + ++DK1622 A+S+ + ++ + ++DZF1 A+S- (leaky) + + + +DK1300 A+S- + + + +DK1253* A+S- - - + +DK1217 A-S+ + + + ++DK1218 A-S+ ± + + ++DZF4150 A-S- - - - -

Motility was observed on 1.5% and 0.4% agar. +, Cells moving ata rate of 2-4 ,um/min; ±, cells moving at a rate of <2 ,Lm/min orjustvibrating in place; + +, cells moving at a rate of >4 Zm/min; -, nocell movement. Due to the softness of 0.3% agar, the video imagesof the cells were very sensitive to vibration. To obtain more stablevideo pictures, we often used 0.4% or 0.5% agar instead of0.3% agarfor these studies; control experiments showed that these media gaveresults similar to cells on 0.3% agar.*Usually, A- or S-motile cells move better on CMM or CF mediumthan on CYE medium. DK1253 was basically nonmotile when itwas plated on CYE medium with 1.5% agar; however, it moved wellon CF medium with 1.5% agar.

strains that have acquired the ability to swarm on 0.3% agarsurface (6 from DZF1, 12 from DK1300, and 10 fromMXH1226) and found that the vast majority (96%) of thesemutants (or revertants) agglutinated (S. Tavazoie, W.S., andD.R.Z., unpublished data).

Expression of A- and S-Motility Genes on Different AgarSurfaces. One possible hypothesis to explain what makesA+S- and A-S+ motile cells behave differently on 1.5% and0.3% agar surfaces is that different agar surfaces affect thegene expression of A- and S-motility genes. However, wewere unable to obtain data to support this hypothesis. Forexample, we put A+S- cells grown in liquid medium onto1.5% and 0.3% agar surfaces at the same time and found thatthe cells on 1.5% agar had good motility without a lag whereasthe same cells on 0.3% agar had much reduced motility. Sincethese cells were preadapted to a moist environment, theyshould have been more motile on a moist surface than the drysurface if surface moisture were responsible for the inductionof motility genes. Clearly that was not the case. In a secondapproach, we tested two strains containing TnS::lacZ inser-tions in A-motility genes to determine whether expression ofthe reporter genes was affected by the different agar surfaces.We found that the expression of the 3-galactosidase for thesefusion strains was about the same on both 1.5% and 0.3% agarplates (Table 4). These results suggest that cells on 0.3% agarmay have a potentially functional A-motility apparatus, butsomehow the soft and wet surface of 0.3% agar prevents themotility apparatus from functioning normally. Two strainswith TnS::lacZ insertions in S-motility genes also failed toshow variations in /3-galactosidase production when placedon different substrates (Table 4).

Effect of Surface Firmness on Fruiting Body Formation.When myxobacteria are starved, they will aggregate to formfruiting bodies. Although some of the A+S- cells failed tofruit, S-motility is not absolutely required for fruiting (14).The A+S- strain (DK1253) and the A-S+ strain (DK1217)showed a certain degree of aggregation and fruiting on CFplates with 1.5% agar (Fig. 3 a and b). When the same cellswere put onto the CF plates with 0.3% agar, the A+S- strain(DK1253) failed to show any aggregation (Fig. 3c); the A-S+strain (DK1217) formed fruiting bodies on a CF plate with0.3% agar (Fig. 3d). Similar results were obtained withseveral other A+S- and A-S+ strains (data not shown). Thusthe ability of A+S- cells to form fruiting bodies on CFmedium appears to be very much affected by the inhibition ofA-motility on 0.3% agar (Table 3).

Role of Nutrients and Cell Density in Cell Swarming. Richmedium seemed to be very important for swarming on 0.3%agar. When we replaced CYE medium with less-rich mediumsuch as CMM or CF medium, the swarming on 0.3% agar wasreduced (data not shown), even though poor nutrient mediumdid not inhibit S-motility (see examples in Table 3). High celldensity promoted swarming on 0.3% agar. A high cell inoc-ulum (4 x 109 cells per ml) caused swarming to beginimmediately after plating on 0.3% agar, whereas a small

Table 4. Effect of agar concentration on expression of A- andS-motility genes

/-Galactosidase activity, unit permg of protein per min

CYE CF

Strain Motility type 1.5% 0.3% 1.5% 0.3%MXH1216 S+, A::lacZ 152 153 180 164MXH1273 S+, A::lacZ 113 116 144 141MXH1651 A+, S::lacZ 113 102 137 115MXH1226 A+, S::lacZ 88 76 90 88

/-Galactosidase activities were assayed. The numbers listed arethe average of duplicate samples. The error is within 10%.

Microbiology: Shi and Zusman

3382 Microbiology: Shi and Zusman

A+ S (DK1253)

1.5% agar

CF

0.3% agar

CF

a

c

0)

a

FIG. 3. Effect of agar concentration on fruiting body formation ofA+S- and A-S+ cells. All CF plates were inoculated with 2 x 108cells and incubated at 32°C for 2 days. Strains used are indicated inthe figure. The bright spots in the colonies are fruiting bodies; theyare 0.1-0.2 mm in diameter.

inoculum (5 x 107 cells per ml) delayed swarming until afterthe cells had grown to a higher density (data not shown).

DISCUSSIONIn this study, we demonstrated that A- and S-motility systemsin M. xanthus are different not only genetically and morpho-logically but also functionally: cells that show only A-motility(A+S- cells) move better than cells that have only S-motility(A-S+ cells) on relatively firm and dry surfaces like 1.5% agar,

whereas cells that show only S-motility move much better thancells that show only A-motility on relatively soft and wetsurfaces like 0.3% agar. We do not know what differencesbetween 1.5% and 0.3% agar cause the changed behavior.Since these observations were also made on two agar substi-tutes (agarose and Gel-Gro), we conclude that the firmness andwetness of the substrate are probably responsible for thedifferent properties of A- and S-motility. M. xanthus naturallylives in the soil or on animal dung that changes its firmness andwetness day by day. Having two different motility systemswith different selective advantages should enable M. xanthusto adapt to a variety of physiological and ecological environ-ments in a similar way to the dual motility systems of flagel-lated bacteria (see Introduction). Similar multiple motilitysystems with selective advantages may also exist in some

other species of gliding bacteria, since many of them exhibitswarming patterns similar to A- and S-motility (30).What makes A- and S-motility function differently on

different agar surfaces? We do not yet know the answer tothat question. We think that it is unlikely to involve theregulation of gene expression of the motility genes sinceTnS-lac transcriptional fusions in two A- and two S-motilitygenes did not show different levels of f-galactosidase activitywhen they were placed on 1.5% or 0.3% agar. We found thatgliding motility of single cells (A-motility) requires a firm anddry surface. We hypothesize then that the motor for A-mo-tility must physically interact with the surface and pushagainst the surface to move the cells forward. A+S- cells

vibrated in place on 0.3% agar; it appears that the softnessand wetness of this surface fail to provide sufficient support

for the A-motility motor. Since we found that cellular cohe-siveness was correlated with swarming on 0.3% agar, it ispossible that the S-motility apparatus permits cells to inter-

connect and glide over each other, thereby creating transientsurfaces from cell-cell contact that enable the myxobacteriato overcome the softness and move forward on 0.3% agar.The cohesiveness of the S-motility apparatus is caused by cellsurface pili or fibrils that enable cells to contact each other.However, these very same phli or fibrils may also providecohesiveness to firm surfaces to slow down cell movementthat may be responsible for reduced S-motility on 1.5% agar.

In flagellated bacteria with dual motility systems, such asV. parahaemolyticus, the two flagellar organelles, whichconsist of independent motor-propeller structures, are di-rected by a common chemosensory control system (31). Wefound that frz genes (11), homologs to chemotaxis genes inenteric bacteria, affected the frequency of reversal of thedirection of movement of both A- and S-motility systems(unpublished data). It is therefore likely that the frz genescontrol both A- and S-motility movements in a pattern similarto that found in flagellated bacteria.We thank Drs. Dale Kaiser and Patricia Hartzell for strains. We

thank Drs. Gonzalo Acuna, Karen Smith, Mark McBride, and Mr.Sohail Tavazoie for their contributions to this work. We thank Drs.Julius Adler, Kathy O'Connor, Thilo Kohler, and Eldie Berger forvery helpful discussions. This work is supported by Public HealthScience Grant GM 20509 from the National Institutes of Health.

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Proc. Natl. Acad. Sci. USA 90 (1993)

A- S + (DK1 21 7)