Faster than a Speed­ing Bolt: Myco­plasma Walk This Way

(With a nod to Aero­smith)

by by Daniel P. Haeusser

Many prokary­otes move ac­tively in liq­uid (swim) or on moist solid sur­faces (swarm and glide) to­ward or away from a stim­u­lus, such as a nu­tri­ent, light, or oxy­gen. Not sur­pris­ingly, prokary­otes have evolved nu­mer­ous means of lo­co­mo­tion built around dis­tinct mol­e­c­u­lar mech­a­nisms.

The motil­ity of all an­i­mals, such as a hu­man like Us­ain Bolt (A) or a gold­fish (B) is based on the actin and myosin pro­tein ma­chin­ery. Many bac­te­ria uti­lize comple­tely dif­fer­ent sys­tems to achieve motil­ity, such as the fla­gella in E. coli © or the unique 'walk­ing' of M. mo­bile (D). These dif­fer­ent motil­ity sys­tems are akin to var­i­ous ways to make sim­i­lar-look­ing boats move through the wa­ter, such as by the sails in Winslow Homer's Breez­ing Up (A Fair Wind) (E) or the in­ter­nal-com­­bus­tion en­gine of the Bat­boat (F).

How dis­tinct? A hu­man run­ning, a dog walk­ing, an ea­gle fly­ing, a fish swim­ming, a frog hop­ping, and a starfish crawl­ing are each unique, but all share a root mech­a­nism in the mol­e­c­u­lar prop­er­ties of actin and myosin in mus­cle cells. For prokary­otes how­ever, their move­ments don't just look dif­fer­ent; they of­ten evolved from dis­parate mo­lecular sys­tems or or­ganelles unique to a par­tic­u­lar gen­us or even species . Some of the more-stud­ied mecha­nisms of prokary­otic motil­ity are fla­gel­lar-based swim­ming and swarm­ing, type-IV pilus twitch­ing motil­ity, and the 'ad­ven­tur­ous' motil­ity of Myx­o­coc­cus xan­thus. This ad­ven­tur­ous motil­ity re­lies on both se­creted polysac­cha­ride and a he­li­cal mo­tor that pro­duces tread-like protru­sions along the cell sur­face.

Among the hand­ful of other known prokary­otic motil­ity mech­a­nisms are those em­ployed by the My­coplasma, a genus in the class Mol­li­cutes of the low G+C Gram-posi­­tive bac­te­ria (Fir­mi­cutes). (Video of My­coplasma motil­ity) The Mol­li­cutes share sev­eral defin­ing char­ac­ter­is­tics, most ob­vi­ously the lack of cell wall that gives them their name (mol­lis is Latin for 'soft' or 'pli­able'). There­fore, al­though they phy­lo­ge­net­i­cally fall in the Fir­mi­cutes, the Mol­li­cutes have no cell wall for the Gram stain and are not sus­cep­ti­ble to the an­tibi­otics that tar­get bac­te­r­ial cell wall syn­the­sis. In ad­di­tion, the Mol­li­cutes are some of the small­est cells known at 0.2 – 0.3 μm in length. Al­though they can be grown in­de­pen­dently, these bac­te­ria are of­ten closely as­so­ci­ated with host or­gan­isms, al­low­ing the bac­te­ria to at­tain dras­ti­cally re­duced genome sizes (The ~500,000 kilo­bases of M. gen­i­tal­ium is the lower limit for form­ing colonies on agar).

Re­cently fea­tured in the news and here for its role in the syn­thetic bi­ol­ogy re­search of the J. Craig Ven­ter In­sti­tute, My­coplasma also has played an im­por­tant role in patho­gen­e­sis in live­stock (M. my­coides) and in hu­mans (M. pneu­mo­niae and M. gen­i­tal­ium). They are also a fre­quent contami­nation of lab­o­ra­tory eu­kary­otic cell cul­tures, a sig­nif­i­cant risk for in­duc­ing data ar­ti­facts in cell cul­tures. To­gether, these traits make My­coplasma a fas­ci­nat­ing sub­ject for study in a va­ri­ety of fields. In par­tic­u­lar, the mys­te­ri­ous motil­ity ob­served for species of the M. pneu­mo­niae clus­ter has in­spired en­thralling re­search that be­gins to il­lu­mi­nate novel mech­a­nisms of prokary­otic motil­ity.

(A) When treated with Tri­ton X‑100 deter­gent to per­me­abi­lize the cell mem­brane, M. mo­bile leaves be­hind a 'ghost cyto­skeleton vi­su­al­ized by neg­a­tive-stain elec­tron mi­croscopy. It re­sem­bles a 'jel­ly­fish' com­posed of two dis­tinct re­gions, a 'bell' and 'ten­ta­cles'. Scale bar = 200 nm. (B) Av­er­aged com­pos­ite im­age of 'jel­ly­fish' par­ti­cles. Scale bar = 10 nm. (C) Car­toon of the dif­fer­ent cy­toskele­tal struc­tures be­tween the 'bell' and 'ten­ta­cles'. Source: Im­age with mod­i­fi­ca­tion.

It's first worth get­ting a sense of how fast these mi­crobes are mov­ing (with a nod to the won­der­ful se­ries of "Mi­cro­bial Olympics" es­says to which our own Merry Youle con­tributed. If you've witnes­sed bac­te­r­ial motil­ity un­der a mi­cro­scope, you're per­haps most fa­mil­iar with fla­gel­lar-me­di­ated E. coli swim­ming, which gen­er­ates speeds of ~10 – 35 mm/sec. This trans­lates to a top burst speed of ~17.5 'body' lengths (bl)/sec. To put this into per­spec­tive (based on num­bers here), the world's fastest fish, the sun­fish, clocks in at a stag­ger­ing speed of 90 bl/sec, a gold­fish man­ages a healthy 4.5 bl/sec, and US Olympian Michael Phelps achieves a mea­ger 1 bl/sec. Of the motile Mycoplas­ma, the swiftest known is the aptly named M. mo­bile, a pre­sumed path­o­genic res­i­dent of gills in fresh­wa­ter fish. Dis­cov­ered in the late 1970's, M. mo­bile has been stud­ied as a po­ten­tial bio­mo­tor with glid­ing speeds of 2 – 7 mm/sec. Con­sid­er­ing their small size (~0.7 mm) this gives a top burst speed of ~10 bl/sec. This M. mo­bile glid­ing is about half as fast as E. coli swim­ming, but still quite re­spectable. In­deed, it is dou­ble the sprint­ing speed of world-record-hold­ing, Ja­maican Olympian Us­ain Bolt (5.3 bl/sec.)!

So then, M. mo­bile gets around. How does it do it? Myco­plasma species lack fla­gella, pili, or ho­mologs to any known motil­ity sys­tem, prokary­otic or eu­kary­otic. It turns out that their motil­ity is closely re­lated to their shape – again, form fits func­tion. Species in the M. pneu­mo­niae clus­ter (includ­ing M. mo­bile) have a light-bulb shaped mor­phol­ogy con­sisting of a body and a po­lar mem­brane pro­tru­sion (head) called the at­tach­ment or ter­mi­nal or­ganelle. The cells glide uni­di­rec­tion­ally, lead­ing with this head in an asym­me­try that al­lows at­tach­ment to sur­faces by the ter­mi­nal organ­elle and that has im­pli­ca­tions in di­vi­sion.

Be­neath this bulb-shaped mor­phol­ogy lies a com­plex cyto­skeleton. When ex­posed to the de­ter­gent Tri­ton X‑100 to per­me­abi­lize the cell mem­branes, M. motile cells leave be­hind a 'ghost' con­sist­ing of an in­sol­u­ble cy­toskele­ton that re­sem­bles a 'jel­ly­fish', with a lat­tice-like 'bell' in the head and 'ten­ta­cles' that ex­tend from the base ('neck') of the ter­mi­nal or­ganelle into the cell body. In­ter­est­ingly, glid­ing de­fi­cient mu­tants had dis­con­tin­u­ous and sig­nif­i­cantly re­duced ten­ta­cles com­pared to wild type cells. So what is this cy­toskele­ton made of? While some My­coplasma en­code known prokary­otic cy­toskele­tal el­e­ments such as FtsZ and MreB, these are ab­sent in M. mo­bile. In­stead, re­searchers iden­ti­fied a set of at least ten unique pro­teins as mak­ing up the M. motile cy­toskele­ton struc­ture.

In the cen­tipede model of M. mo­bile motil­ity, the glid­ing pro­teins ex­tend from the in­side of the cell, where they are linked to the 'ten­ta­cles' of the cy­toskele­ton, to those on the cell sur­face. ATP hy­drol­y­sis by P42 ini­ti­ates a flow of con­for­ma­tional changes in glid­ing pro­teins (i–iv) that trans­lates into a step-wise cel­lu­lar 'walk­ing' through re­peated binding/release of sur­face sub­strate by the 'legs/ feet'. Source: Im­age with mod­i­fi­ca­tion.

To iden­tify the pro­teins in­volved in glid­ing, re­searchers com­pared the pro­tein ex­pres­sion pro­files of wild-type cells and glid­ing-de­fi­cient mu­tants. They also made an­ti­bod­ies that specif­i­cally inhib­ited M. mo­bile glid­ing and used these to lo­cal­ize the tar­get pro­teins. Three of the four iden­ti­fied pro­teins are found in the neck por­tion of the cell sur­face, cor­re­spond­ing to the re­gion where the ten­ta­cle por­tion of the in­ter­nal cy­toskele­ton re­sides. An­ti­body-me­di­ated in­hi­bi­tion of sin­gle glid­ing pro­teins cou­pled with struc­tural stud­ies us­ing con­ven­tional and atomic force elec­tron mi­croscopy led to a model where the glid­ing pro­teins form a leg-like struc­ture ex­tend­ing from in­side the cell (as­so­ci­ated in some way with the cy­toskele­ton com­po­nents) to a 'foot' that in­ter­acts with sur­face sub­strates. In this way, M. mo­bile is able to 'walk' across sur­faces and man­i­fest its vis­i­ble glid­ing motil­ity.

At this point you may won­der how this walk­ing is pow­ered. Re­call that three of the four iden­ti­fied glid­ing pro­teins lo­cal­ize to the neck of M. motile. The fourth glid­ing pro­tein, P42, has not been lo­cal­ized, but it is a puta­tive AT­Pase that has ATP hy­drol­y­sis ac­tiv­ity when ex­pressed in re­com­bi­nant E. coli. Fur­thermore, ATP is re­quired for M. mo­bile mo­tility and ad­di­tion of ATP amaz­ingly re­stored motil­ity to the Tri­ton X‑100 in­sol­u­ble M. mo­tile 'ghosts' (video here). This, and fur­ther da­­ta, have led to the 'cen­tipede' model, where cy­cles of ATP bind­ing, hy­drol­y­sis, and re­lease by P42 lead to con­for­ma­tional chan­ges in the three struc­tural glid­ing pro­teins and sub­se­quent bind­ing and re­lease from sur­face sub­strate. A re­cent pub­li­ca­tion fur­ther char­ac­ter­ized the physics of these mo­vements us­ing cells elon­gated by low de­ter­gent con­cen­tra­tions.

Schematic of the motil­ity sys­tem compo­nents within the 'head' (at­tach­ment or­ganelle) of M. pneu­mo­niae . Let­ters re­fer to pan­els in orig­i­nal source. In the 'inch­worm' model of motil­ity, con­for­ma­tional changes in the elec­tron dense core of the cy­to­plasm (xtend­ing from C–K) are trans­­mitted through a cy­to­plas­mic an­chor (B) to sur­face ad­he­sion pro­teins (A) to achi­eve for­ward move­ment. Source

Sur­pris­ingly, this mol­e­c­u­lar sys­tem of motil­ity ap­pears spe­cific for M. mo­bile. The glid­ing pro­teins of M. mo­bile are found in the closely re­lated M. pul­mo­nis, but no­where else known. Re­searchers have stud­ied the motil­ity of M. pneu­mo­niae for decades longer than M. motile and, while the over­all cell mor­phol­ogy and means of lo­co­mo­tion ap­pear con­served be­tween the two species, the cy­toskele­tal struc­ture of M. pneu­mo­niae (and that of the re­lated M. gal­lisep­ticum) is dis­tinct from that of M. mo­bile, with a com­pletely dif­fer­ent set of glid­ing pro­teins that has led to the 'inch­worm' model of My­coplasma mo­tility. This sug­gests sep­a­rate evo­lu­tion of dis­tinct motil­ity sys­tems ('cen­tipede' vs. 'inch­worm' mod­els) in dif­fer­ent My­coplasma species (M. motile vs. M. pneu­mo­niae, re­spec­tively).

Con­sid­er­ing their close as­so­ci­a­tion (and at times de­pen­dence) on host species and their re­duced genomes did the an­ces­tral My­coplasma lose their motil­ity only to re­gain sim­i­lar sys­tems indepen­dently? Were they im­motile bac­te­ria that found a need for motil­ity once be­holden to host? Do the cy­toskele­tal and glid­ing pro­teins of these species per­form other func­tions that have been coopt­ed? How are these cy­toskele­tal and motil­ity sys­tems kept or­ga­nized through the process of cell di­vi­sion? A host of ques­tions re­mains for these fas­ci­nat­ing and de­cep­tively sim­ple or­gan­isms sprint­ing about their hosts.

Note: Go here for ad­di­tional videos of My­coplasma motil­ity from the Miy­ata Lab.