Downloaded by guest on September 29, 2021 www.pnas.org/cgi/doi/10.1073/pnas.1621469114 Germany; Juelich, D-52425 a bacteria migrating Sabass of Benedikt groups by generation Force ocshv ihrvle hnclsaei groups. in are cells both during when and values generated characters, higher have forces complementary forces have the gliding that motion. and of show twitching direction the results in average, these on Together, push, protru- cells Advancing gliding groups. of in sions traction fivefold However, Pa. approximately 1 pro- amplified of order gliding is the isolated, on are traction average cells low When duces trans- adhesions. lateral substrate by driven of of is port timescales mechanism, Twitching on motility forces second pN. rapidly the with fluctuate 50 Gliding, but that of pN hotspots, order 100 traction the around produce traction on also local forces groups exert with bacterial cells hotspots individual small that in find type-IV we by retraction, powered twitching, pilus For We gliding. of microscopy. and mechanisms twitching force namely, motility traction distinct using two This here study motion. addressed bacte- collective how is during of forces question question coordinate the and to generate to regard ria devoted with been particular has study in such bacteria, little migration, has during gener- cells forces study eukaryotic ate how considerable of mechanisms Although biophysical the collectively. probed move cells often embry- living of and must groups healing organisms, multicellular wound (received in to 2017 development 23, bacteria onic May in Levine formation Herbert Member colony Board From Editorial by accepted and 2016) Germany, 30, Heidelberg, December University, review Heidelberg for Schwarz, S. Ulrich by Edited 08544 NJ University, Princeton omv na nemtetfradbcwr oin(,4 (Fig. 4) (3, motion uniquely (2) forward–backward 1 machineries and intermittent migration an characterized two in move uses well to It studies. is motility for organism suited This for- body (1). fruiting and mation predation, swarming, motion vegetative group including coherent coordination produce to the cells and unclear. multiple traction remain by surface forces prop- generated the of the However, and forces. of regulation, mechanical erties their generate to involved, cells con- ability single proteins made their how the has of decades particularly understanding few move, an past toward the the progress into over siderable Research microbes formation. of biofilm motility and aggregation, dation, M el r elgbe o xml,teda oc nacl moving cell a on these force for drag the coor- forces example, cells hydrodynamic For of negligible. and groups are Inertia cells measur- how any forces. and if produce these or cells dinate migration, individual during if force unclear able is it known, are the 1B). along (Fig. 17) sites (16, adhesion body Motion substrate cell 15). of (14, translation force through into converts occurs gradient periplasm proton and transmembrane membranes transducer the the gliding termed spans a that is Here, (13) 12) 11). motility. complex (2, (A), (10, system adventurous cells or motility to gliding, neighboring distinct, shown genetically on been second, polysaccharides A has also by retraction Pili triggered type- and 1A). and be of (Fig. adhesion, surface retraction (7–9) the cell–cell and motility to mediate adhere produces extension filaments retraction the extruded filament by whereby powered pili, is IV 6) (5, ity yoocsxanthus Myxococcus eateto ehncladArsaeEgneig rneo nvriy J08544; NJ University, Princeton Engineering, Aerospace and Mechanical of Department A yoocsxanthus Myxococcus lhuhmn ftemlclrdtiso hs w systems two these of details molecular the of many Although and ag rust aiiaesc ies hnmn spre- as phenomena diverse in such surfaces facilitate over to migrate groups to large ability the possess bacteria any .Frt wthn,smtmscle oil() motil- (S), social called sometimes twitching, First, B). a,b,1 | bacteria atisD Koch D. Matthias , c ei-ilrIsiuefrItgaieGnmc,PictnUiest,N 84;and 08544; NJ University, Princeton Genomics, Integrative for Institute Lewis-Sigler xiiscmlxcletv behaviors, collective complex exhibits | rcinforce traction | c twitching una Liu Guannan , yoocsxanthus, Myxococcus | gliding . min. <1.5 c,d oadA Stone A. Howard , n salse rtcl 2,2,2) n lsi rprisare properties elastic and 23), follow- (see 22, prepared characterized (20, allow- are protocols gels pores, established PAA gel displacement. ing small gel the of into tracking embedded ing be fluo- can broad nanometer-sized elastic beads a Furthermore, rescent has used elasticity. which tunable widely (PAA), of A polyacrylamide range is traction. TFM of for calculation of substrate deformation allowing a gel, produce migration the cell during forces substrate 1073/pnas.1621469114/-/DCSupplemental at online information supporting contains article This Editorial the by invited editor guest a is 1 U.S.S. Submission. Direct PNAS Board. a is article This and control interest. of performed H.A.S., conflict G.L. no declare B.S., and authors M.D.K. The data; and analyzed article, the B.S. wrote experiments. tools; and results reagents/analytic discussed J.W.S. new contributed B.S. tatypical a at o F,clsaepae na lsi e n mgdfrom imaged and gel elastic an 1C on (Fig. below placed or are above cells TFM, For Results of gliding and xanthus twitching M. from resulting stress cell–substrate measurement the study, resolved of spatially this a using In questions such measurement. address we largely experimental remained mechani- direct date, the to to These have, are inaccessible migration groups? bacterial what back in of Finally, aspects the reorganization in cal from forward? force cells forward group of those move advancing timescales along cells the all pull push do to and Or of locally behind? front forces ranks advancing exert the the at that the within cells group distances “leader” the larger have span one or arise Might local, group? forces be balance Could interactions? this cell–cell understood. Would and a cell–substrate less between within balance even a bacteria from is of group migration for situation Collective translation contiguous the the (18). to if cells similar or friction, eukaryotic surface internal has the itself with machinery friction overcome to need of order uhrcnrbtos ..dsge eerh .. ... n ..promdresearch; performed G.L. and M.D.K., B.S., research; designed B.S. contributions: Author princeton.edu. owo orsodnemyb drse.Eal .aasf-ulc.eo shaevitz@ or [email protected] Email: addressed. be may correspondence whom To infiatyapie hnclsmv ngroups. in are move cells cells The individual when of traction. amplified gliding significantly or directional twitching of groups by produced in emergence forces gliding collective and for traction, of allows uncoordinated patterns local, Twitching distinct groups. to in to or leads individual lead an as mechanisms motion during gliding. these traction and that twitching find namely, We motility, of mechanisms mon a present xanthus We using traction migration. cell–substrate bacterial during of study measured first been yet surfaces not their and bacteria have Despite infection between force. forces host mechanical importance, of biomedical even generation the and on depend aggregation, migration, Bacterial Significance b nttt fCmlxSses2 oshnsetu J Forschungszentrum 2, Systems Complex of Institute 10 samdlorganism. model a as sn rcinfremcocp TM (18–21). (TFM) microscopy force traction using −2 Myxococcus a n ohaW Shaevitz W. Joshua and , ag rcinfre ilol cu fcells if occur only will forces traction Large pN. .Det h ekbceilforce, bacterial weak the to Due Appendix). SI irto pe of speed migration and d oehHnyLbrtre fPhysics, of Laboratories Henry Joseph i.S1 Fig. Appendix, SI . .xanthus M. www.pnas.org/lookup/suppl/doi:10. c,d,1 NSEryEdition Early PNAS xiistocom- two exhibits 1 i so the on is µm/min .Ltrlcell– Lateral ). Myxococcus ulich, ¨ | f6 of 1

MICROBIOLOGY BIOPHYSICS AND COMPUTATIONAL BIOLOGY resentative results for the displacement field and traction maps A pilus-driven twitching C beads of that clearly demonstrate the presence of substrate forces below pili 2 colors and ahead of migrating bacteria. For wild-type cells, the alterna- tive modes of twitching and gliding lead to a high variability of traction in different experiments (SI Appendix, Fig. S2 A and B). A twitching- and gliding-deficient mutant ∆pilA-∆aglQ (13, 17) produces no measurable traction (SI Appendix, Fig. S3). B gliding motion Individual Twitching Cells Produce Small Hotspots of Traction. To isolate the different motility systems, we first probed twitching elastic gel substrate cells that lack the ability to glide due to a deletion of the aglQ adhesions gene (15). We observe localized areas of substrate deformation D immediately in front of twitching cells, yielding bead displace- substrate interaction of twitching wt cells ments on the order of 100 nm; see Fig. 2A, I–III and Movie 0 s 100 s 200 s S1. The corresponding calculated traction is concentrated in 2 i ii iii hotspots, which have an apparent size on the order of 1 µm due to resolution limitations. Time-lapse images in Fig. 2A, I–III demonstrate that the traction field is dynamic and changes motion on a timescale on the order of a minute. Among moving cells, not all show measurable traction at all times. If hotspots are present,

A 0 s 90 s 180 s gel displacement

200 nm I II III

gel displacement 300 nm traction I II III

5 m 0 22[Pa]

Fig. 1. Measurement of substrate traction resulting from the two migra- tion machineries of M. xanthus.(A) Type-IV pilus extension–retraction cycles allow bacterial motion referred to as twitching motility or S motility. Pili

can also mediate mechanical cell–cell coordination. (B) Gliding or A motil- traction ity results from lateral translation of transmembrane complexes along the bacterium. Steric interactions allow directional coordination of gliding cells. (C) Top view of the setup for TFM. Cells are placed on a gel containing fluo- 0 35[Pa] rescent marker beads of two colors. (D) Gel deformation and substrate trac- B 10 m C local force tion resulting from motion of individual wild-type bacteria that can twitch counts in hotspots [pN] and glide. White outlines show contours of bacteria. (i–iii) Red quivers show mean: 300 gel displacements. (I–III) Calculated traction magnitude. mean: 60 2.8 m 55 pN 250 50 200 it is necessary to use very soft gels with an elastic shear modu- 40 0 lus of G ' 121 Pa. We also use fluorescent marker beads of two 30 150 colors, which increases the spatial resolution of TFM to around 20 100 1 µm. To improve bacterial motility, gels are coated with chi- 50 tosan, which has been previously used for studying M. xanthus 10 (17, 24) (see SI Appendix). Time-lapse imaging of the fluorescent 0 1 2 3 4 5 6 7 8 9 10 11 12 13 beads allows measurement of a spatiotemporally varying defor- distance between hotspot and cell pole [ m] mation field relative to the first frame of a sequence. This defor- mation field is then used to calculate a continuous field of relative Fig. 2. Twitching of individual, gliding-deficient bacteria that move by traction that bacteria exert on to the substrate (20). The traction using their pili (∆aglQ). (A) Gel deformation and substrate traction at three calculated in this way is measured relative to the possibly pre- time points. White outlines show contours of bacteria. (i–iii) Quiver plot of stressed first frame of an imaging sequence. To obtain a reliable gel displacements. (I–III) Calculated traction magnitude. Note that hotspots estimate of forces applied by pili, we use an alternative method appear in front of bacteria. (B) Distances between hotspots and the nearest cell pole. (C) Magnitude of overall force in individual hotspots as estimated relying on the assumption that pili produce point forces (20, 25) by assuming point forces. Dots are individual measurements, box contains (see SI Appendix). To test if M. xanthus produces any measur- 25 to 75% of data around the median; diamond shows the mean. Data able substrate forces during migration, we investigated wild-type for B and C were collected from seven experiments with more than five cells with the ability to both twitch and glide. Fig. 1D shows rep- cells each.

2 of 6 | www.pnas.org/cgi/doi/10.1073/pnas.1621469114 Sabass et al. Downloaded by guest on September 29, 2021 Downloaded by guest on September 29, 2021 eu to up be aase al. et expected Sabass is as from group, traction cell The the group. toward the points of spots periphery outermost the the at spots in forces time. 3 observation (Fig. the during analysis slightly bac- TFM only of out clumps spread initial to substrate, tend a teria with on aligned deposited strongly When not other. are each cells individual where groups, nized twitching while of force groups examined ∆ distribute we cells Traction. motility, twitching collective Fluctuating performing of Local, groups Exert how Cells investigate Twitching of Groups environment individual the affects of hardly that gliding mechanically. process that low-friction a conclude is we cells Consequently, of individual of order cells. from front the ing traction on in Estimated is not the cells them. localized gliding deform beneath is but do Traction cells bacteria degree. the gliding some that to implies substrate bacteria with trac- of random tion colocalization precision, approximate However, measurement prominent. is the noise to close is are deformation displacements substrate Here, other. below each contacting without S6 move mutant Fig. but exper- twitching-deficient pili performed the use we using not mechanism, iments do gliding that complementary cells the individual by of motion Traction. the Little gate Very Exert Cells Gliding Individual optical by measured forces stall the tweezers. with consistent are hotspots reported been of has 2. retraction of It forces that tweezers. maximum 28 optical ref. from in data with corre- pared For hotspots than the (20). smaller around that traction to find average, localized on at we spond, for only bacteria, applied magnitude twitching is individual force force of the corresponding estimate that force (see assuming overall point by one the hotspot estimate each to to possible it makes those magnitude suppresses force the strongest. undersampling affect that of because at variations integration spatial level field, local applied high-frequency traction the from force the on estimated the be of balance addition, cannot force hotspots In seem- a individual difficult. are of bacteria they assessment individual ren- when allows detailed cells pili among even a links of mechanical other der range invisible These each long apart. the to far interac- where ingly that connect substrate model Note to the body. a bacteria cell from and the resistance pili with forces passive other tion against counteracting against pull where pulling pili model pili consis- from a do are result experiments between forces these distinguish counteracting However, not of dynamics. Appendix, tug-of-war presence (SI with the tent motion the and twitching experiments, S4) of our Fig. In character other. diffusive each random counteract pili different at (26). lengths pili for average, data pili on between that is, distance pole demonstrates The cell which closest tips. bodies, the their and cell at hotspots substrate the the to stretch engage not way one mostly do as mostly the cells little of of as front with in all Hotspots them, six. of as three many as to and two average on observe we u atrai uhsoe hntemxmmplsretrac- pilus maximum the than (see slower speed much tion is bacteria our 5 aglQ eetees h la oaiaino rcini hotspots in traction of localization clear the Nevertheless, forces where (27), motion tug-of-war to lead can Twitching eemaue tlwfre of forces low at measured were µm/s 0nm 20 el 1) ldn-ecetmtnsfr lgtydisorga- slightly form mutants Gliding-deficient (15). cells hw yia eut o atrata oeindividually move that bacteria for results typical shows ∼13 n eyltl vrl rcini bevd Because observed. is traction overall little very and , 5 pN ∼150 ,cnitn iheeto microscopy electron with consistent 2B), (Fig. µm .Ti prahyed nimproved an yields approach This Appendix). SI i.S4 Fig. Appendix, SI iu ercinsed fu to up of speeds retraction Pilus pN. ∼149 A .Teenmesmyb com- be may numbers These 2C). (Fig. and hw ihylclzdsubstrate localized highly shows B) hr lotalfre are forces all almost where pN, 50 .xanthus M. 10 ie mle hnfrtwitch- for than smaller times ,btfre esrdat measured forces but ), yeI iisal at stalls pili type-IV ∆pilA. oinof Motion pN. 15 onx investi- next To ∼3 IAppendix, SI u can but µm To eain ftato ea eyrpdyo ieclsof timescales on cor- rapidly The very signal. decay noise real traction the The of than cells. relations smaller from cell much away clearly beneath is occurring traction correlation noise between traction distinguish and we groups where shown are (see experiments both our traction in are real constant noise of and correlation zero-lag as traction the real from by away Sec- of far normalized cells. noise correlations the traction Finally, of of correlations cells. vicinity record the also in we ond, measured are correlations tion time at vector traction as autocorrelation magni- traction 3C of Fig. snapshots in The shown pili. tude the of action pulling a s. from 60 to 30 of rates frame at taken images 67 of overall an with experiments u et aafrcl rusin groups cell measurements. for individual with Data are test. compara- distributions sum Dots are different cells. edge significantly the the indicate below at Stars measured forces those groups, to In ble groups. vanishing than a hotspots Pa. have weaker 0.1 Tractions is width points: Bin locally. Data (F balance (roi). forces cell–substrate interest that of showing mean, region a in edge noise traction ponent (E, denote cells. lines without dotted regions Lower, in line). measured (black below mean data and denote lines groups denoted Upper cell traction. region of the fluctuations in temporal inside quantifies rectangle pattern white traction a the by of (C of hotspots. evolution showing traction displaying Calculated (B) visibility. clearer for (∆ 3. Fig. 1 2 3 C EF A traction componentt[Pa] 500 nm oc antd fhtpt.Snl wthn el rdc significantly produce cells twitching Single hotspots. of magnitude Force ) -15 e ipaeet.Ol vr orhmaueeti displayed is measurement fourth every Only displacements. Gel ( A) aglQ). counts orlto aafo ordfeetexperiments different four from data correlation 3D, Fig. In its calculate we quantitatively, dynamics traction the assess To R 0s10s150s 120s 90 s τ 0s60s 30s 0 s displacement ≡ -10 t t olciemgaino wthn atrata r gliding-deficient are that bacteria twitching of migration Collective || || R + mean: 0Pa eutn rmpoeto of projection from resulting 0 -5 τ /R 015 10 5 0 0 0 | cells R 0 τ Saebr 10 bar: (Scale B. o ogtimes, long For . eosrt yaia rcinpattern. traction dynamical a demonstrate h ≡ τ 0 || ˜ t m tpsto ihindex with position at D ,τ 300 100 150 200 250 and 0 50 D B in hotspots[pN] local force · Inset 0 0 1 2 1 ˜ t IAppendix). SI single E m correlation 0123456 cells 55 eecletdfo orseparate four from collected were t ,τ i h autocorrelation The (D) µm.) itiuino h rcincom- traction the of Distribution ) 0 ntemi omlai fan of axis normal main the on +τ R *** traction tatgroups 114 τ traction i lag timeτ[min.] groups m NSEryEdition Early PNAS prahsanonzero a approaches ,τ 0 where , mean: R p 116 < τ interior m group i 0 narank a in 0.001 is,trac- First, . ˜ t m Snapshots ) n.s. ,τ 112 ∼1 R 18 Pa 0Pa 0 | group edges 0 sthe is 0 f6 of 3 min, | cells R τ

MICROBIOLOGY BIOPHYSICS AND COMPUTATIONAL BIOLOGY which demonstrates the presence of rapidly fluctuating traction. A displacement B traction These fluctuations can also be observed in the raw data images, 10 Pa where rapid, local displacements of marker beads occur (Movies S2 and S3). The movies also demonstrate that individual cells move rapidly in a seemingly random fashion, whereas the group edge does not move much. We next assess the traction orientation to see if cells at the group edge produce a net traction that pulls or pushes the group forward. As shown in Fig. 3E, Inset, we manually select regions of interest around the edges and record the traction components t|| that are aligned with a main axis normal to the edge. Trac- 0Pa tion that pushes away from the group edge yields t|| > 0. We 500 nm find a symmetric distribution of t|| with a mean value of 0 Pa. C D Thus, groups of twitching ∆aglQ cells do not exert coordinated correlation R traction. τ 1 Typical numbers of pili per M. xanthus bacterium have been traction ti at groups reported to be around 4 to 10 (26), where, in some cells, up to 50 pili were observed. Given the large number of potentially active 0s 30s 60s 1 pili in groups, it is not obvious that forces should be concen- 2 trated in the observed hotspots. However, if concentrated, the large number of available pili can produce strong forces on the order of nanonewtons (29), which is comparable to forces pro- 0 90 s 120 s 150 s 0123456 duced by much larger eukaryotes (30). Moreover, engaging the lag time τ [min.] substrate with many pili simultaneously could potentially lead to E F very slow dynamics because motion would require detachment counts traction (t² +t² )¹/ ² [Pa] of many pili. To clarify this issue, we compare the absolute force [10³] mean: +1 Pa x y magnitude of traction hotspots at groups with the magnitude of *** 6 *** hotspots at individual cells (Fig. 3F). Although hotspots at indi- 8 5 *** vidual cells have a magnitude of ∼50 pN, we find, for hotspots 6 4 median: 1.8 at groups, a mean force of 114 pN with an uncertainty approxi- 3 4 t mately as large as the mean. Thus, measured forces are ampli- || + 2 0.3 fied in groups by about a factor of 2. Because these ∼100 pN are 2 0.18 skew: 1.1 1 smaller than the maximum stall force of ∼149 pN (28), our result 0 0 are still compatible with the notion that each traction hotspot -5 0 5 10 15 noise individ. groups in groups is caused only by one or a few pili. Furthermore, if traction component t|| [Pa] cells many pili cooperated to produce one traction hotspot, weaker forces would be expected for the edge of groups where fewer pili Fig. 4. Collective migration of twitching-deficient strains (∆PilA). (A) Groups are present. However, a comparison of force magnitude shows move with a finger-forming spreading pattern. Red quivers are gel displace- no significant differences for hotspots at group edges or below ments. Only every fourth measurement is displayed, for clearer visibility. the group interior. Together, we find that, although groups of (B) Calculated traction. Note that the traction points, on average, in the direc- tion of motion. (C) Snapshots displaying evolution of of the traction pat- M. xanthus likely only use relatively few pili simultaneously to tern inside the region denoted by a white rectangle in B. (Scale bar: 10 µm.) exert substrate forces, measured forces are considerably stronger (D) Traction autocorrelation Rτ . Upper lines denote data from four cell groups in groups compared with single cells. with mean (black line). Lower, dotted lines denote traction noise measured in regions without cells. (E, Inset) Distribution of the traction component Gliding Groups Can Exert Persistent, Coordinated Force. To next resulting from projection onto the average orientation of a fingering struc- assess the collective mechanics of bacterial gliding, we probed ture. Data points: Histogram bin width is 0.126 Pa. Data were recorded from groups of gliding ∆pilA cells. When placed on the imaging sub- areas where bacteria form a fingering structure. Positive values of the mean strate, clumps of bacteria present at the start of the experiment traction ht||i ' 1.1 Pa and the skewness demonstrate that twitching-deficient spread in a fingering fashion, where the fingers consist of closely mutants in a growing finger tend to push the substrate in the direction of motion. ( ) Bar plots of traction magnitude k k comparing noise away from packed bacteria that move parallel to each other. We find that, F t cells, individual gliding ∆PilA cells, and compact groups of ∆PilA cells. Stars although gliding of individual cells does not produce much trac- indicate significantly different distributions with p < 0.001 in a rank sum tion, gliding motion in groups leads to measurable forces (Fig. 4 test. Data for individual cells were recorded from overall 28 cells in four exper- A and B). Here, traction is distributed in diffuse patches under- iments. Data for cell groups in D and E were collected from four experiments neath the moving group, and the traction magnitude is lower with ∼25 image frames each. than in the presence of pili. Furthermore, the cell–substrate trac- tion in the shown protrusion appears rather coordinated because the traction points in the direction of the advancing cells. causes traction variations that are slower than those resulting The snapshots of traction magnitude shown in Fig. 4C illus- from pili. trate that traction is dynamic, but changes appear less abrupt Because the traction images of gliding groups in Fig. 4B and than for twitching cells. To assess the traction dynamics quantita- Fig. S7 suggest a “pushing” nature of the forces under advanc- tively, we calculate the correlation measure Rτ for the twitching- ing fingers, we quantitatively assess the directionality of forces deficient mutants (Fig. 4C). Again, the correlation measure is in Fig. 4E. We manually select regions of interest around pro- normalized by the zero-lag autocorrelation below the cell groups truding fingers and record the traction components t|| that are 0 in each movie R 0|cells. The contribution of measurement noise is aligned with the protrusion direction. In contrast to the results here evidently stronger than in the case of twitching motion, due from twitching cells, the distribution of t|| is here asymmetric to the lower force magnitude. We find that the traction correla- and heavy on the positive side, as quantified by a positive skew of tion does not show a rapid decay on short timescales as in Fig. 1.1. The pushing nature of cell–substrate traction below advanc- 3D; instead, it decays over many minutes. Thus, gliding of groups ing fingers is corroborated by a positive distribution median of

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(Movies is groups 5 in Fig. traction in groups, in seen larger substantially as is speed instantaneous the that find aase al. et Sabass groups. twitching in or both individually For move that bacteria track Groups. we in generation, Faster Is Motion Cell a from (28). or mechanism either result tion machinery that the gliding forces that of the transmission suggest for from pili does use still hotspots can dynamic strain of twitching presence the the for than of weaker force icantly average an groups, with hotspots In traction individually. moving when Sim- gliding S9). the Fig. to Appendix, ilar (SI machinery retraction pilus shown impaired as times cells, 5 individual 4F Fig. than below in traction more median is the groups than gliding median higher below The measured cells. individual traction than of traction higher median much a produce with above weak, significantly quite nevertheless below is but that threshold, traction noise that the exert find traction cells We recorded. magnitude, gliding also noise are individual cells the without of areas in estimate magnitudes an obtain packed To densely beneath groups. or cells individual beneath directly either rus aawr bandb aultakn 5 o10clsi each kind. each in each of cells cells of individual groups 170 for for experiments to experiments 13 150 seven and from kind tracking obtained manual were by Data condition. obtained were Data groups. 5. Fig. of distribution calcu- the to record magnitude forces we point traction Instead, values. discrete force of bacteria, absolute assumption the late the below use distributed cannot is we gliding other. each from touching traction not are Because that cells individual from forces with pushing balances finger protruding tip. the the of at end forces rear the compression at where cells balance, of load long-range require necessarily rejected is median vanishing a with of hypothesis the where Pa, +0.62 aglQ ial,w opr h ocspoue ygiiggroups gliding by produced forces the compare we Finally, 0 1 2 3 4 5 6 '100% nxetdy efidta ruso ldn cells gliding of groups that find we Unexpectedly, Pa. 1 atraehbttems admmto atrs iha with patterns, motion random most the exhibit bacteria ntnaeu pe fticigadgiigsrisi ihrin higher is strains gliding and twitching of speed Instantaneous twitching individ. sacnrl eas td a study also we control, a As . cells ofiec yasg et hs uhn forces pushing These test. sign a by confidence 1.3 1 ∆pilA mean ∆aglQ ||t m cells in groups |≡ || tan hssri rdcslwtraction low produces strain this strain, tan n gliding and strains aglQ p and t ocmaemtlt otraction to motility compare To x 2 ,m .Tu,hge measured higher Thus, S5). + individ. ∆aglQ gliding ∆pilT t pilT ∆ y 2 cells ,m pilT hc ssignif- is which pN, 60 idpnetretrac- -independent teeyposition every at . 1.3 0.9 uatta a an has that mutant uat However, mutant. mean ∆ uat produce mutants pilA cells in groups tan,we strains, pilA ∆ pilT m tcahso ie.Acneuneo nesmln fthe of undersampling of eukary- consequence at TFM A of sites. context the problem adhesion dealing in a otic then is encountered which routinely are field, is We traction that scale. undersampled to, spatially measurement comparable a lengthscale our with a than, trac- on shorter real varies the or bacteria that by expect exerted we tion be Therefore, thickness. can bacterial deformation the substrate Using the every colors, by deformation. approximately two limited measured substrate of is the beads results of measure- fluorescent TFM force measurements local of of a resolution density allows spatial and The is migration assay ment. to that complementary barriers Our In of pillars. free (29). between voids substrate the a bridging to as up forces micropillars work, deformable using by group allowing rearrangement. (38). while load cells directional integrative other maintaining innately for an on mechanism produces forces variation stalling velocity picture, to this leads In cells individual rever- of motion sal determined biochemically cell– because mechanical contact-dependent interaction a cell from from emerge result or trac- (37) could amplified mechanisms biochemical groups The gliding group. under a must tion within the cells force in traction, pushing compressive push this transmit can produce To that groups, motion. traction In of trac- (36). direction measurable apicomplexa exert rear-end of bacteria Such gliding gliding unrelated cells. was the and gliding motion for limits of found deadhesion of mechanical ends build-up that indicates rear a tion observe the and rarely low at we twitching. very traction Also, for is measurable. those cells hardly of migrating glid- often individually reverse for by very results exerted the Our substrate Traction almost 35). the (17, are pole to mutants rear respect ing the with at stationary disassembled are and that sites adhesion mechanical their probe to that cells (34). role surroundings allowing sensory in a to migration plays about anchoring retraction speculate collective pilus might provide gen- one for clearly and forces efficiently pili substrate, the the However, used that the experiments. not conclude over our We are move in min. pili hardly a 20 by whole to of erated 10 a timescale of as the course both groups on time are the groups decay in whereas correlations velocity collec- minute, and cell the and dynamic, and Traction (28), highly higher pili. motors of thereby retraction action and the tive resistance cellu- in higher including generation to mechanisms, force leads physical that groups by jamming in caused lar forces bio- be higher from also However, col- originate could 33). This may (32, cells. activity regulation of mechanical chemical twitching in erratic increase faster, lective measured by amplified accompanied for are significantly observe around a we magnitude with what force albeit to similar cells, dis- with traction, individual observe pattern of instead, traction hotspots we, twitch- organized continuous is However, in rather area pili directionality. a many substrate coordinated the produce per that of groups pili expect ing order might available one of the Therefore, number high. on the magnitude groups, rial force hotspots in in a results concentrated cells are with individual that forces of substrate twitching of counteracting pilus-driven pat- migration that distinct the find very during We organization two force for of evidence terns provide data presented The Discussion random limited superdiffusive S4D at Figs. a hints Appendix, with (SI which walk consistent entirely, motion, zero gliding of to of persistence decay autocorrelation not velocity does the bacteria to contrast, timescale In minute a zero. within decays that autocorrelation velocity atra elsbtaefre aebe esrdpreviously measured been have forces cell–substrate Bacterial of Gliding .xanthus M. 1 Nrsl rmprle cino aypili many of action parallel from result nN h ihrfre ngroups in forces higher The pN. 100 spwrdb lsial connected elastically by powered is n S5B). and 0. 5 hc scmaal to comparable is which µm, NSEryEdition Early PNAS nbacte- In pN. ∼50 .xanthus. M. | ∆ f6 of 5 pilA

MICROBIOLOGY BIOPHYSICS AND COMPUTATIONAL BIOLOGY displacement field is that the absolute traction magnitude is usu- Many facets of bacterial surface mechanics are yet poorly ally underestimated (20). understood, for instance, substrate dependence of migration (39) A number of challenging refinements of our methodology are or surface-dependent virulence of P. aeruginosa (34, 40). We desirable. First, accurate 3D tracking of beads in the substrate have shown that the combination of traction measurement with would possibly allow the assessment of vertical forces and allow genetic perturbations provides a viable tool for studying the for precise determination of the vertical distance between the emerging world of bacterial mechanics. beads and the bacteria. Such analysis was precluded, in our stud- ies, by bacterial photodamage from the fluorescence excitation Note Added in Proof. We would like to point interested readers light. Second, the gel displacements are measured with respect to a recent preprint article on substrate forces during bacterial to a prestressed state, because it proved difficult to recover microcolony morphogenesis (41). the fully relaxed state after removal of bacteria. Although not essential for this study, it is generally desirable to obtain the ACKNOWLEDGMENTS. We thank S. Thutupalli for discussions and invaluable relaxed state of the substrate. Third, comparison of TFM results advice. We thank G. Laevsky and A. Perazzo for technical guidance. Tamˆ Mignot is thanked for providing bacterial strains. Work was supported by with other migration assays would be facilitated if sufficiently National Science Foundation Grants PHY-1401506 and MCB-1330288, Ger- soft and well-characterized agar-based TFM substrates were man Research Foundation (DFG) Award Ko5239/1-1, and a German Aca- available. demic Exchange Service (DAAD) fellowship.

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