The Mi­cro­scopic Flash Mob

by Melissa Wilks

Every day we see an­i­mals mi­grat­ing through the air, across plains, and in the oceans, in beau­ti­fully co­or­di­nated pat­terns; star­lings flock to­gether in the thou­sands while sar­dines swim to­gether in enor­mous shoals. These so­cial be­hav­iors are im­por­tant in al­low­ing an­i­mals to so­cial­ize, avoid preda­tors, and find refuge and food. But what about smaller or­gan­isms? Al­though more dif­fi­cult to vi­su­al­ize, mi­croor­gan­isms can per­form these co­or­di­nated be­hav­iors as well. As is true for birds and fish, fig­ur­ing out how and why bac­te­r­ial cells com­mu­ni­cate to or­ga­nize their move­ments will help un­der­stand the be­hav­ior of bac­te­ria in the en­vi­ron­ment.

Fig­ure 0. Rip­ples on the Ca­mas Prairie of north­ern Mon­tana. Source

Myx­o­coc­cus xan­thus is a soil dwelling mi­crobe that has at­tracted the at­ten­tion of mi­cro­bi­ol­o­gists in part due to its preda­tory ac­tiv­ity. To sur­vive in en­vi­ron­ments where nu­tri­ents are scarce, it kills and breaks open other bac­te­ria and feeds on the re­leased nu­tri­ents. Dur­ing times of star­va­tion, M. xan­thus cells self-or­ga­nizes into fruit­ing bod­ies, macro­scopic dome-shaped mounds that con­tain over 100,000 cells each. In these struc­tures, the cells dif­fer­en­ti­ate into meta­bol­i­cally in­ac­tive spores that re­main dor­mant and al­low sur­vival un­til times get bet­ter. Since the 1960s, M. xan­thus cells have also been known for or­ga­niz­ing their move­ments across a solid sur­face to pro­duce waves; this phe­nom­e­non, called rip­pling, re­sem­bles the waves pro­duced when you throw a rock into a pond, and can be seen in na­ture on a scale rang­ing from the mi­cro­scopic to the ge­o­logic.

Fig­ure 1. Myx­o­coc­cus xan­thus fruit­ing bod­ies. Im­age by Juer­gen Berger and Supriya Kadam. Source

Rip­pling pat­terns can be seen dur­ing two ma­jor stages of the M. xan­thus life cy­cle, de­vel­op­ment of the fruit­ing bod­ies and pre­da­tion. How do these sin­gle-celled or­gan­isms know to start rip­pling and why is it seen dur­ing both stages? It was orig­i­nally pro­posed that rip­pling dur­ing the de­vel­op­ment stage was due to the dis­play of a star­va­tion-in­duced sig­nal pro­tein CsgA on the sur­face of a cell. When the pole of one cell col­lides with the pole of an­other cell as they move through a medium, CsgA in­ter­acts with an un­known re­cep­tor on the neigh­bor­ing cell. This pole-to-pole ex­change al­lows cell sur­face in­for­ma­tion to be ex­changed as cells swim, herd-like, through the me­dia. As the lev­els of CsgA within the com­mu­nity rise, the first rip­ple is in­duced, lead­ing to ag­gre­ga­tion and fruit­ing body for­ma­tion. How­ever, to date no known re­cep­tor for CsgA has been found, nor does the ac­tion of CsgA ex­plain why rip­pling oc­curs dur­ing pre­da­tion.

A study by Berle­man et al. at­tempted to shed light on the cause of rip­pling be­hav­ior in M. xan­thus dur­ing both stages. Through pre­da­tion as­says, they demon­strated that rip­pling is as­so­ci­ated with the pres­ence of the prey and of macro­mol­e­c­u­lar sub­strates, such as poly­sac­cha­rides and pep­ti­do­gly­can, that are re­leased by the death of the tar­get cells. The move­ment of M. xan­thus cells across a solid sur­face such as agar is known to be mod­u­lated by the chemo­taxis-like frz path­way. These au­thors, along with oth­ers, noted that when M. xan­thus cells move through an en­vi­ron­ment that lacks prey or ly­sis de­bris, the pat­tern is tan­gled and un­or­ga­nized. But in the pres­ence of prey, the same frz path­way di­rects cells to or­ga­nize into nearly par­al­lel lines that move in a more co­or­di­nated fash­ion. Mu­ta­tions in the frz path­way caused M. xan­thus to aban­don its prey prior to com­plete ly­sis, sug­gest­ing that this or­gan­ism al­ters its motil­ity when in the pres­ence of prey or of macro­mol­e­cules re­lated to dy­ing prey.

Fig­ure 2. One as­pect of the ABM model, here used to il­lus­trate what's in­volved. Side-to-side con­tact sig­nal­ing in the ABM sim­u­la­tions. The side-to-side con­tact in the ABM sim­u­la­tions is de­fined by three pa­ra­me­ters: 1) the per­pen­dic­u­lar (to cell ori­en­ta­tion) dis­tance be­tween the cen­ter of the two agents (d⊥); 2) the par­al­lel dis­tance be­tween the cen­ter of the two agents (d∥); and 3) the an­gle formed by the two agents (Δθ in this fig­ure). L rep­re­sents the length of the cells and v rep­re­sents ve­loc­ity.

More re­cently, Zhang et al. have been fur­ther in­trigued by bac­te­r­ial so­cial de­vel­op­ment and by ad­vances in tech­nol­ogy that have per­mit­ted fur­ther quan­ti­ta­tive stud­ies of the rip­pling be­hav­ior. A reeval­u­a­tion of the pole-to-pole model was stim­u­lated by the dis­cov­ery that the frz path­way pro­teins FrzC and FrzD ap­pear to stim­u­late side-to-side con­tact dur­ing vig­or­ous pre­da­tion. These au­thors used Agent-Based Mod­el­ing (ABM) in con­junc­tion with flu­o­res­cence mi­croscopy to in­ves­ti­gate rip­pling mech­a­nisms dur­ing pre­da­tion. ABM is a com­puter pro­gram that al­lows mod­el­ing of how cells in­ter­act with one an­other and their en­vi­ron­ment based on ex­per­i­men­tal ob­ser­va­tions. This is not a sim­ple mat­ter and it's un­der­stand­able that it ap­peared in the jour­nal PLOS Com­pu­ta­tional Bi­ol­ogy. To give a fla­vor of what's in­volved, we in­clude the first of their fig­ures from the rather de­tailed Sup­port­ing In­for­ma­tion. This method re­vealed that M. xan­thus cells use a side-to-side con­tact-me­di­ated sig­nal to self-or­ga­nize. Sig­nally of this type uses re­cep­tors lo­cated along the axis of a cell to in­duce rip­pling, sim­i­lar to mech­a­nism pro­posed by Berle­man et al. of in­ter­cel­lu­lar sig­nal­ing in­duced by the pres­ence of prey macro­mol­e­cules.

With a bet­ter un­der­stand­ing of the mech­a­nism of rip­pling we can now ad­dress the phys­i­o­log­i­cal role of rip­pling dur­ing de­vel­op­ment and pre­da­tion. Zhang et al. de­vel­oped three pre­dic­tions based on their re­sult: 1) side-to-side sig­nal­ing in­creased the ef­fi­ciency and rate with which cells can spread over prey cell and ly­sis de­bris; 2) rip­pling causes in­di­vid­ual cells to bounce back and forth off of one an­other, pro­duc­ing an os­cil­lat­ing mo­tion that en­sures the en­tire com­mu­nity moves over an area con­tain­ing nu­tri­ents; 3) rip­pling al­lows M. xan­thus cells to re­main longer in an area con­tain­ing prey or macro­mol­e­cules for longer pe­ri­ods. They con­cluded that the over­all ef­fect of rip­pling is to more ef­fec­tively cover an area of a prey, more like a thick cel­lu­lar blan­ket than a dis­or­dered pile. This would cer­tainly pro­vide a com­pet­i­tive ad­van­tage over other cells for ac­cess to avail­able nu­tri­ents.

Fig­ure 3. Com­par­i­son of rip­ple ini­ti­a­tion in the ABM sim­u­la­tions (top pan­els) and ex­per­i­ments (bot­tom panels).The ini­tial time (0 hrs) cor­re­sponds to the ini­ti­a­tion of the sim­u­la­tion with a uni­form cell dis­tri­b­u­tion or the time M. xan­thus cells fully cover the prey in the field of view. The fields of view of both the ABM sim­u­la­tion im­ages and ex­per­i­men­tal im­ages have the same di­men­sions; the scale bar is 100 µm.

De­spite the many ef­forts to de­ter­mine why dif­fer­ent cel­lu­lar growth pat­terns are ob­served and how bac­te­ria com­mu­ni­cate to form them, there still re­main large gaps in our knowl­edge. Un­der­stand­ing cel­lu­lar com­mu­ni­ca­tion be­yond quo­rum sens­ing, as in the case of rip­pling, would help us un­der­stand how com­mu­nity be­hav­iors ben­e­fit the in­di­vid­ual cell. Ul­ti­mately, con­trol­ling com­mu­nity be­hav­ior could help us to har­ness im­por­tant bac­te­r­ial processes for in­dus­try or de­velop new strate­gies to pre­vent harm­ful biofilm col­o­niza­tion. Fur­ther­more, the au­thors state "This type of com­bined ap­proach is es­sen­tial to fur­ther our un­der­stand­ing of self-or­ga­ni­za­tion in more com­plex sys­tems such as de­vel­op­ment of mul­ti­cel­lu­lar or­gan­isms."

Ref­er­ence

Zhang H, Vaks­man Z, Litwin DB, Shi P, Ka­plan HB, Igoshin OA (2012). The mech­a­nis­tic ba­sis of Myx­o­coc­cus xan­thus rip­pling be­hav­ior and its phys­i­o­log­i­cal role dur­ing pre­da­tion. PLoS com­pu­ta­tional bi­ol­ogy, 8 (9). PMID 23028301

Melissa is a grad­u­ate stu­dent at the Uni­ver­sity of Akron where she has been men­tored by Hazel Bar­ton.