2PS026 Helical Flow of Surface Protein Required for Bacterial Locomotion(The 50th Annual Meeting of the Biophysical Society of Japan)
2PS026 表面タンパク質のらせん流で動くバクテリア(日本生物物理学会第50回年会(2012年度))

Daisuke Nakane, Keiko Sato, Hirofumi Wada, Mark J. McBride, Koji Nakayama
2012 Seibutsu Butsuri  
TheBiophysicalSociety of Japan General IncorporatedAssociation Akihiro Tanaka], Daisuke Nakanei, Takayuki Nishizaka!, Makoto Miyatai (tGraduate Sehoot qf' Scienee, Osaka Cib, [,iiivers'io" 21[Ztc'uitv, of' Sc'ience, Gakushuin Uhiversity) filycoptasma mobite, a pathogenic bacterium forms membrane protrusion, the gliding rnachjnery, and g]ides on solid surface smoothly up to 4,S -mls and 27 pN in the direction of protrusion. M. mobile is thought to glide by repeated catch-pull-release of
more » ... release of sialylated oligosaccharide tixed on solid surface by hundreds of SO nm fiexible ]egs sticking out from the gliding rnachinery. Hewever, the directed disp]acement of ce]] is difficu]t to exp]ain, because the tegs are fiexible. To elucidate this mystery, we examined a starved cel] binding to sialylated oligosaccharide, by pulling forward and backward at a speed of 160 nmls, using a bead manipulated by a ]aser trap. Tension was generated at similar rate for forward and backwaTd, O.09 ± O.03 pNrnm. In these pTocesses, the tensionoccasiona[iyshifteddown,suggestjngternporaldetachmentof]egsftom sialyiated ol{gosaccharide fixed en solid surface. namely "stippage". The sLippage occurred at tension of aveTages, 22.5 ± 13.9 pN and 48.4 ± 28.0 pN for forward and backward, respectively. whereas no signifieant difference was found in the occasional decrease in tension between both directions. The tension at slippage did nol difreT significantly between directtons, when two types of non binding rnutants ]acking valid legs were artificially bound to glass, These results suggest that M. mobite binds to so]id suri'ace in a direction dependent manner, and this directed binding may cause the dirccted gliding, 2PS024 Leg movements suggested from inhibition of mycop]asma gliding by free sialylated oligosaccharid Taishi Kasait, Daisuke Nakanei, Hideharu [shida2, Hiromune Ando!'], Makoto Kiso2'3, Makoto Miyatai (iGraduate Schooi of Sc'ien('e, Osaka Ciu, U}iiversity, 2Fbuculcy, of'Alij)lied Bioiogieai Seienees, Gijb brniver.si4" ]iCems, K}'oto U}iiversit.v) Mycoplasmas. known as a pathogen ofhuman pneumonia. bind to solid suri'aces and g]ide to one directien. )n gliding, mycoptasrnas catch sia]ytated oligosaccharides,thecommontargetofmanypathogens.Inpreviousstudies,we analyzed the inhibitory efTects of synthesized sialylated oligosaccharide on binding of gliding rnycop]asmas and concludcd thc affinity. the cooperativity, and the recognized structure, In the present study, we analyzed the effects of those compounds to the gliding of M. mobile cel]s, to know the relationship betweensialylatedorigosaccharidebindingandtheglidingmechanism.Gliding speed was reduced by the additien of sialylated oligosaceharide with time, and the decrease appeared aceeleTated with time after the decrease became obvious. The decrease in gliding speed shou]d be caused by the decrease in the working legs in the addition of free sialylated compound. The acceleration in decrease may suggest that the binding inhibition ofa leg affkrct the aff'inity ofneighboring legs, The cells showed pivoting around the g]iding machinery befoTe the detach, The shortage of prope]ling force caused by the free sialylated o]igosaccharide may induce remaining of legs after stroke on glass, some of which 1'orm the center ef ce]] pivoting. AIL of these resu]ts support our assurnption that the leg after stroke should be removed by the continuous cell movement caused by other legs in the g]iding mechanism, 2pso26 xwheyJcofimsvfumTM<Jtofu7 A large group of bacteria belonging to the phylum Bacteroidetes show a eharaeteristic rapid surface movement known as gliding moti]ity, Bacteroidetes gtiding moti]ity is different from other motility systems, but its mechanism is unknown. Here, we visualized the dynamics ofcell adhesin SprB, and proposed "helica] loep track" to explain the motility. A soil bacterium, Fiavobacterium .iohnsoniae has a 669 kDa ce]]-surface adhesin, SprB, which is responsible for the gliding rnotility and cei] adhesion. Electron microscopic experiments revealed that SpTB fonmed 150 nm long filaments, and dynamic movements of SprB were observed by fluerescence microscopy, SprB was located dispersed]y on a long pitch of helical loop along the membrane, and moved on the cell surface along an apparent right-harided helical closed loop. When the apparent velocities of SprB signals were determined with respect to the gtass substratum, about halfofthe SprB sigrials meved with ve]ocities of3.4 pmls and the other halfmoved with velocities of -O.5 pmis. Taking the velocity of cells (1.9 -mls) into consideration, SprB signals migTating in opposite directions appeared to move at the similar speeds with respcct te the ce]]. The resu]ts suggest a modeL for Flavobacterium gliding, supportcd by mathematical analyses, in which adhes{ns always moved on an endtess right-handed hetical track at a constant speed, while some SprB tightly bound to the so]id surface, generating rotation and translation of the cell. 2PS027 Gene manipulation of gLiding bacterium, Alycoplasma mobile Isil Tulum, Atsuko Ucnoyama, Makote Miyata CGraduate Sthool ofScience, O.saka Cin. , Univer.tiO,) op'c'tiplasma mobiie, a pathogenic bacterium, glides on solld surflaces with a unique mechanism. not related to known bacterial motMty systems or conventiona] motor proteins. The gliding machinery is composed ofthree huge surface proteins and more than ten interna] proteins, To elueidate the gliding rnechanism and the roles oi' component proteins, the genetic manipulation including homologous recornbination shDuld be a powerfu1 tool. HoweveT, this organism has not been transformed so far by standard procedures used for other ndycopiasma species. In this study, we examined and improved the steps of transformation as fo11ows. (D The electroporation condjtions have been adjusted to fit to the M. mobile ce]]s which are much tougher than the other species. (ii} The recovery time after electroporation was elongated from 2 h to 12 h. (iii) The growth medium was modified to get clearer co]ony shapes, Then, we achieved the transformation of M mobile wlth eenciencies ranging 10'S to 10'7 per competent cells by 1O pg ofvector DNA, and also fluorescence labeling ofeells by using cnhanced yel]ow fluoreseent protein (EYFP). We are constmcting specia] vectors atlowing us gene expression and horno]ogous recombination. Swarming is an important step for bacterial celonization and spreading. We investigate the swarming dynamics of the dual flagellated bacterium. Vibrio alginolyticus, in different growing stages and externa] conditions, While growing on agar surfhce, Vibrio alginolyticus elongate and synthesize lateral flagella that can move rap3dly in the thin liquid layer above agaT, The ce]] motion is eonstrained by the cell to eell interaction and hydro-dynamical intersction with the ]iquid boundary conditions. We measured the colony spreading speed and the single cell moti]ity in different growing stages and agar concentrations. The ce]ls lengths are widely distributed, The cell density is higher in the colony cdge. Edge-cells show paralle] alignments whilc intcr;orcells show swirling motion with different time and ]ength scale. We found the edge-cel]s shows waving whi]e the interior-cel[s shows turbuient like coliective motion. Far behind the spreading edge, celt density reduced down to single-cell layer. We exam the collective motion and ctuster size distribution in this conditien. Vibrio atginolyticus shows different motility dynamics in different growing stage. We aim to build up a simply model to explain the co]lective motionofVibrioalgino]yticus, 2PS028 Akihiro Kawamotoi Hughes],Toshio qffrontierBiosci. The bacterial functions as a rotary motor and a [ong helical fi]ament that works as a prope]ler, Most of flagellar axial proteins are translocated into the central ehannel of the growing flagellurn for selrassembly at the distal end by the flagellar protein export apparatus,
doi:10.2142/biophys.52.s114_3 fatcat:3rq2x7wxdbdgtawlnntgmpgjuy