Energetics of Gliding Motility in Mycoplasma mobile

Mycoplasma mobile glides on surfaces at up to 7 µm/s byan unknown mechanism. We studied the energetics that power glidingby using a novel, growth medium-free system. We found that cellscould glide in defined media if the glass substrate is preconditionedby exposure to horse serum. The active component that potentiatesgliding is sensitive to proteinase K treatment. We used thedefined medium system to test the effect of various inhibitors,ionophores, and poisons on motility of M. mobile. Valinomycin,carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP),N,N'-dicyclohexylcarbodiimide, phenamil, amiloride, rifampin,and puromycin had no short-term effects on gliding. We alsoconfirmed that we were able to modulate the membrane potentialwith valinomycin and FCCP by using a potential-sensitive dye.Shifting the pH likewise had no effect on motility. These resultsrule out the use of conventional ion motive forces to powergliding. Arsenate had a dramatic inhibitory effect on gliding,and both the speed and the fraction of cells moving trackedATP levels. Sodium orthovanadate had a slight but significantinhibitory effect on gliding. Taken together, these resultssuggest that the motor system of M. mobile is likely an ATPaseor is directly coupled to an ATPase.

INTRODUCTION

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Introduction

While studies of locomotion in swimming bacteria are well advanced,investigations of gliding motility remain comparatively limited.Yet gliding motility, defined as a smooth translocation overa solid surface, is represented frequently throughout the eubacterialphylogenetic tree and in some instances has been associatedwith pathogenicity (25). Even several species of mycoplasmas,some of the simplest bacteria known in terms of size and genomiccontent, are known to perform gliding motility (14, 32).

The mycoplasmas are wall-less bacteria characterized by smallphysical dimensions and genome sizes (32). Among the mycoplasmas,the fish pathogen Mycoplasma mobile demonstrates extremely robustgliding motility (16, 34). M. mobile is one of the flask-shapedmycoplasmas (approximately 1.0 x 0.3 µm) and has a genomeof approximately 780 kbp (4). It has always been observed toglide in the direction of the "head" (corresponding to the taperedend of the cell) without reversals or pauses at speeds of upto 7 µm/s (34). It can tow an erythrocyte, roughly 10times its size, without significant loss in speed and has beenmeasured to exert up to 27 pN of force (28, 33). Some recentprogress at uncovering the molecular mechanism of gliding inM. mobile has been made, including localization of the glidingapparatus to the head region of its flask-like cell body andisolation of mutants with altered gliding phenotypes (29, 30,41). However, little is known about the prerequisites or energysource for gliding in M. mobile.

Flagellated bacteria use ion motive forces to power their flagellarmotors. For instance, Escherichia coli uses a proton motiveforce ( ${Delta}$ p_H), while various Vibrio species use a sodium motiveforce ( ${Delta}$ p_Na) to drive their polar (but not lateral) flagella(3, 18, 19, 24). The energy source for motility has been investigatedin some genera of gliding bacteria as well. In the case of theFlavobacteria, gliding motility appears to be powered by ${Delta}$ p_H,while the social motility system of the Myxobacteria relieson type IV pili and, therefore, ATP hydrolysis (25).

The mycoplasmas seem to lack any form of respiration and generateATP through fermentation of sugars and substrate-level phosphorylation(32). It is known that mycoplasmas can generate a transmembranepotential ( ${Delta}$ ${Psi}$ ) ranging from –28 to –48 mV (negativeinside the cell) and a ${Delta}$ p_H ranging from –52 to –72mV (37). Therefore, it would seem that mycoplasmas could useeither an ion motive force or direct utilization of ATP to powermotility. We set out to develop a medium-free system in whichwe could study gliding of M. mobile on glass and determine itsenergy source.

MATERIALS AND METHODS

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Materials and Methods

Reagents. Heart infusion broth and yeast extract were from Becton Dickinson(Sparks, Md.). 3,3'-dipropylthiadicarbocyanine iodide (DiSC₃[5])was from Molecular Probes (Eugene, Oreg.). The ENLITEN ATP measurementsystem was from Promega (Madison, Wis). All other reagents werefrom Sigma-Aldrich (St. Louis, Mo.). Water was 18 M ${Omega}$ deionized(dH₂O).

Strains. M. mobile strain 163K (ATCC 43663) was grown to an optical densityat 600 nm (OD₆₀₀) of 0.07 to 0.10 in plastic tissue cultureflasks at 22°C in Aluotto medium consisting of 10% inactivatedhorse serum, 2.1% beef heart infusion broth, and 0.56% yeastextract adjusted to pH 7.8 and supplemented with 50 mg of ampicillin/literand 250 mg of thallium acetate/liter (1).

Preparation of coverslips. Circular glass coverslips were subjected to the following sequenceof treatments (all at room temperature with gentle agitation):10 min in saturated ethanolic KOH, four 5-min changes in dH₂O,15 min in inactivated horse serum, and three 5-min changes indH₂O. The coverslips were then left to dry in a laminar flowhood and stored at room temperature until use, resulting ina preparation that was stable for at least 4 weeks. Note thatfetal bovine serum can also be used with equal effectiveness.

Protease treatment. Prepared coverslips were digested overnight with 20 mg of proteinaseK/ml (or dH₂O as a control) at 42°C in a humid environmentand washed with four 5-min changes in dH₂O.

Buffers. The following buffers were used: phosphate-buffered saline (PBS;150 mM NaCl, 50 mM sodium phosphate [pH 8.0]), PBS/G (PBS [pH8.0] plus 10 mM glucose), PBS-K/G (140 mM NaCl, 10 mM KCl, 50mM sodium phosphate, pH 8.0 [or other pH as specified], 10 mMglucose), ArBS-K/G (140 mM NaCl, 7.5 mM KCl, 47.5 mM sodiumarsenate, 2.5 mM potassium arsenate [pH 8.0], 10 mM glucose),and valinomycin buffer (100 mM NaCl, 50 mM KCl, 50 mM sodiumphosphate [pH 8.0], 10 mM glucose).

Motility assay. Comparisons were made of gliding speeds of cells in a givenbuffer and cells in the same buffer containing the compoundto be tested, referred to as control buffer and test buffer,respectively. Cells (diluted to an OD₆₀₀ of 0.025 in fresh medium[1 ml]) were centrifuged at room temperature for 10 min at 10,000x g, washed in 1 ml of control buffer or test buffer, and centrifugedagain in the same manner. The final pellet was suspended in50 µl of the control buffer or test buffer. Six microlitersof this suspension was pipetted onto a prepared coverslip andincubated at room temperature for 15 min in a humid environment.During that time, a flow chamber (5) was assembled with a sparecoverslip ringed with Apiezon-L grease (GEC-Alsthom Ltd., Manchester,United Kingdom) and equilibrated by continuous flow with controlor test buffer for at least 10 min. Coverslips with adheredcells were then ringed with grease and inverted onto the flowchamber. All observations were made at room temperature ( $~$ 22°C).Control buffer or test buffer was drawn into the flow chamberat the rate of 100 µl/min for 10 min to remove nonadherentcells, and then the motion of the remaining cells was recorded(with the flow left on). Cells were visualized with a NikonOptiphot microscope equipped with a 60x oil immersion phase-contrastobjective, a Zeiss optovar set to 1.25x, and a 10x projectionlens. Recordings were made with a charge-coupled device cameraconnected to a Mini-DV recorder (Sony GV-D1000, New York, N.Y.).In the standard assay, 10-s recordings were made every 10 minover the course of 30 min. Time zero was defined to occur atthe time of the first recording. Video was captured to a computerwith Adobe (San Jose, Calif.) Premier software at a rate of10 frames/s. The speed of gliding mycoplasmas was computed forframes 40 to 59 of each 10-s (100-frame) time point by usinga particle tracking program written by Darnton et al. (8). Slightmodifications were made to Darnton's program to output the properstatistics (the MATLAB scripts are available at http://www.rowland.org/labs/bacteria/index.html).Speeds of 30 to 50 cells were measured at each time point ineach experiment. The compound being tested was considered tohave a significant effect on motility only if a comparison ofthe test and control populations by a two-tailed Student's ttest had a P value of <0.05 at all time points studied.

pH shift. Cells were prepared in PBS-K/G (pH 8.0) and then shifted toPBS-K/G at the desired pH after the first recording was taken.The second recording was taken at t = 5 min.

Arsenate and ATP. Measurements of motility parameters and ATP levels were madein parallel by slightly modifying the standard motility assay.Eleven 1-ml aliquots of cells at an OD₆₀₀ of 0.025 were washedin PBS-K/G as described above. Cells in one tube were resuspendedin 50 µl of PBS-K/G and used for the flow chamber experimentswhere they were treated as described above, while the remaining10 tubes were washed an additional time in PBS-K/G to simulatethe wash experienced by the cells in the flow chamber. At timezero of the experiment, the buffer feeding the flow chamberwas either switched to ArBS-K/G or allowed to remain in PBS-K/Gin the case of the control. Concurrently, each of the remaining10 tubes was resuspended in 100 µl of ArBS-K/G or PBS-K/G,depending on the experiment. Ten-second recordings were madeevery 10 min as was done before, but the time course was extendedto 90 min because there was no preincubation with test buffer.At each time point, one tube of cells was extracted for ATPby adding 2 µl of trichloroacetic acid to the tube andvortexing and then adding 12 µl of 1.5 M Tris base toneutralize the acid (12). The sample was then diluted with 342µl of 5 mM Tris buffer (pH 7.75) and frozen at –20°Cuntil the ATP assay was performed. In some cases, further dilutionof the sample with 5 mM Tris (pH 7.75) was necessary to getthe sample within the range of the assay standard curve. Forthe ATP assay, 10 µl of each sample was added to a glassvial, and then 100 µl of ENLITEN rL/L reagent was addedto the tube and mixed by repeated pipetting. Measurements weretaken exactly 15 s after the addition of the rL/L reagent ina home-built luminometer consisting of a photomultiplier tubeconnected to a current-to-voltage converter and a chart recorder(27). Two measurements were made for each sample and were comparedto a standard curve of ATP constructed from duplicated measurementsof samples of ATP ranging from 31.3 to 500 fmol.

Membrane potential. Cells were grown as described above, and a sufficient amountof culture was centrifuged at 10,000 x g such that resuspensionin 2 ml of test buffer resulted in an OD₆₀₀ of 0.175 to 0.200.This suspension was placed in a cuvette, and DiS-C₃-[5] wasadded from an EtOH stock to a final concentration of 150 nM(final EtOH, 0.05%). Fluorescence was monitored at excitationand emission wavelengths of 625 and 667 nm, respectively, ina Fluoromax fluorimeter (Spex, Edison, N.J.) equipped with amagnetic stirring cell. The dye was allowed to partition intothe cells until a stable signal was obtained (4 to 5 min), andthen the test substance (valinomycin, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone[FCCP], or N,N'-dicyclohexylcarbodiimide [DCCD]) was added.Changes in the fluorescence signal were observed within a fewseconds of the addition of the test substance if changes wereobserved at all.

Isolation of proteins that bind to glass from horse serum. Approximately 100 µl of glass beads (425 to 600 µmin diameter) were prepared in exactly the same way as the coverslipsdescribed above. After drying, the beads were boiled in Laemmlisodium dodecyl sulfate-polyacrylamide gel electrophoresis buffer(36). The resulting sample was loaded to a sodium dodecyl sulfate-4to 12% polyacrylamide gel electrophoresis gradient gel (Invitrogen,Carlsbad, Calif.) and subjected to electrophoresis. Major bandswere excised and subjected to in-gel tryptic digestion and identificationby ion trap mass spectrometry (13).

RESULTS

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Results

Conditioning of glass. In pilot experiments for this project, we noticed that M. mobilecells would glide on glass surfaces if they were pelleted bycentrifugation and then simply resuspended in a pure buffertest medium (e.g., PBS/G). However, when an intervening washstep was included, cells would adhere to glass in aggregatesbut would not glide. Evidently, in the former case, an essentialcomponent from the medium that potentiates gliding was carriedover from the pellet, while in the latter case, the extra washstep eliminated the component. We tested the major componentsof the growth medium (horse serum, beef heart infusion, andyeast extract) by adding each one back to a washed culture andfound that only horse serum restored motility. We subsequentlyfound that a simple pretreatment of glass with fresh horse serumwas enough to potentiate the motility of washed cells. Therefore,the factor that potentiates gliding on glass surfaces is nativeto horse serum and is not a factor produced by mycoplasmas inculture. Subsequent to this discovery, we adopted the coverslippretreatment method described in Materials and Methods.

Identity of conditioning factor. We subjected pretreated coverslips to overnight incubation witheither proteinase K or water. The coverslips were then washedwith water to eliminate the protease so that gliding could betested. We found that coverslips treated overnight with proteinaseK no longer facilitated gliding motility of a washed culture,whereas the control coverslips treated with water alone stillpotentiated gliding of mycoplasmas. However, the coverslipstreated with proteinase K still allowed adherence of cells tothe glass with approximately the same number of cells stuckas is found in the control. We therefore conclude that a proteinor combination of proteins either directly or indirectly mediatesthe ability of M. mobile cells to glide on glass surfaces.

As albumin is the most abundant protein known in serum, we pretreatedcoverslips as described above using a solution of bovine serumalbumin (1 mg/ml) in place of the horse serum; note that thepretreatment of coverslips with fetal bovine serum also potentiatedgliding. This type of pretreatment did not potentiate glidingof washed cells, so a protein or proteins other than albuminare probably involved in conditioning the glass surface. Weattempted to isolate this component(s) by identifying the majorspecies that bound to glass from horse serum. Mass spectrometrywas able to identify albumin (as expected), immunoglobulin,and casein as the top three proteins bound to glass beads. However,attempts to coat glass coverslips with solutions of these proteins(1 mg/ml each) did not potentiate motility. Rather, immunoglobulinand casein abolished cytadherence of the cells, while albumincaused clumps of nonmotile cells to adhere to the glass surface.The actions of these proteins in combination have not yet beentested.

Gliding of M. mobile in artificial media. The development of the pretreatment procedure for glass coverslipsallowed us to study motility in artificial media (e.g., simplebuffer systems). Movies of M. mobile gliding can be seen athttp://www.rowland.org/labs/bacteria/showmovie.php?mov=mycoplasma_mobile.A comparison of the basic buffers used in subsequent experimentsis shown in Fig. 1. While none of the artificial media usedresulted in gliding speeds as rapid as those in growth medium(Fig. 1a), most media supported motility of >80% of the cells(Fig. 1b). The omission of glucose from the medium caused asubstantial decrease in both speed and percentage of cells thatwere moving. The sole source of ATP generation in most mycoplasmasis through substrate-level phosphorylation during glycolysis,although some mycoplasmas can use arginine as an energy source(32). This indicates that ATP synthesis may play a key rolein the energetics of mycoplasma motility. We found that sucrosecould also be used in place of glucose as an energy source inmotility experiments (data not shown), indicating the abilityof M. mobile to metabolize sucrose.

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FIG. 1. Gliding in growth and artificial media. (a) Gliding speed of motile cells. Only cells moving at speeds greater than 0.25 µm/s are considered. Error bars represent one standard deviation. (b) Percentage of cells moving at speeds greater than 0.25 µm/s. Symbols: ${blacksquare}$ , growth medium; ${diamondsuit}$ , PBS-K/G; ${blacktriangleup}$ , PBS/G; •, PBS.

Gliding speeds of mycoplasma in medium containing Na⁺ and glucose(PBS/G) were consistently around 1 µm/s, but the cellsappeared thin and elongated. The inclusion of a small amountof K⁺ (10 mM KCl) in the medium caused a 33 to 50% increasein gliding speed, and the cells looked more like those in growthmedium. However, the addition of too much K⁺ (>100 mM) causedcells to swell and become coccus shaped, which inhibited motilitybut not glass binding (data not shown). Mycoplasmas are knownto selectively accumulate K⁺ to over 200 mM against a gradient(37). This fact, combined with these observations on glidingin artificial media of different ionic compositions, suggeststhat K⁺ plays an important role in osmoregulation and shapedetermination in M. mobile and, in turn, that osmoregulationand shape determination are important factors in gliding motility.

We also tested the ability of cells to glide in a medium inwhich almost all cations were completely impermeant to the membrane(200 mM choline chloride, 200 µM HEPES [pH 7.9], 10 mMglucose, and final Na⁺ of about 160 µM due to titrationwith NaOH). We found that >80% of cells glided in this medium,achieving speeds of up to 2.6 ± 0.46 µm/s. However,gliding cells tended to fall off of the glass surface aftera short period of observation (10 min) for reasons that arenot yet understood.

Effects of external pH. If a proton motive force is involved in gliding, changing the ${Delta}$ pH might have an effect on motility. We prepared all cells inpH 8.0 buffer and then subsequently shifted them to a lowerpH in the flow chamber. Shifting the external pH of the mediumdid not have a significant effect on speed or percentage ofmotile cells (Fig. 2).

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FIG. 2. Effect of shifting pH on gliding motility. All samples were prepared in PBS-K/G (pH 8.0) and then shifted to the indicated pH at t = 1 min. (a) Gliding velocity; (b) fraction of cells moving. Symbols: ${blacksquare}$ , pH 8; ${diamondsuit}$ , pH 7; ${blacktriangleup}$ , pH 6.

Effects of ionophores, inhibitors, and poisons. We examined the effects of several compounds known to interferewith various cellular processes that have been shown to be involvedin the motility of other organisms. In all cases, motility andthe percentage of motile cells were observed over a 30-min periodand compared to a carefully matched buffer control that didnot contain the compound in question. Buffer formulations arelisted in Table 1.

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TABLE 1. Buffer formulations used in this study^a

The addition of the potassium ionophore valinomycin to cellsin the presence of high external K⁺ would be expected to clamp ${Delta}$ ${Psi}$ at a lower-than-normal value (37, 38). Valinomycin had no significanteffect on motility compared to the control (Fig. 3a and b),even in the presence of 50 mM external potassium. This resultwas interesting, considering that high amounts of potassiumcaused cells to become coccoid and stop gliding (see above).However, at the end of the 30-min observation period, nearlyall cells were still moving at a rate comparable to that ofthe matched control. Similarly, the protonophore FCCP did notcause a significant decrease in either gliding speed or percentageof cells that were gliding (Fig. 3c and d). FCCP is reportedto abolish ${Delta}$ pH in various mycoplasmas (37), and the inclusionof FCCP in the medium would be expected to short circuit anyproton-driven motor by providing an alternate conduction pathfor protons and driving ${Delta}$ p_H toward 0.

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FIG. 3. Effect of ionophores, inhibitors, and poisons on gliding. Various agents were added (Table 1), and gliding was compared to that of a matched control. The graphs on the left-hand side of each pair show the speed of the cells that were moving at speeds greater than 0.25 µm/s relative to the initial speed of the cells in the matched control. The graphs on the right-hand side of each pair show the fraction of cells moving. (a and b) 10 µM valinomycin; (c and d) 10 µM FCCP; (e and f) 10 mM arsenate (note that no 30' test time point is shown [e] because no cells were moving at speeds greater than 0.25 µm/s); (g and h) 10 µM DCCD; (i and j) 1 mM sodium orthovanadate; (k and l) 10 µM phenamil; (m and n) 10 µM amiloride; (o and p) 5 µg of rifampin/ml; (q and r) 20 µM puromycin (note that the 30-min test time point was not taken due to technical difficulties). Filled squares are matched control preparations. Open squares are test preparations. Error bars are omitted for clarity, but standard deviations in speeds were generally ±25%.

We studied the effect of valinomycin and FCCP on the membranepotential ( ${Delta}$ ${Psi}$ ) of M. mobile by using the positively charged fluorescentindicator DiSC₃[5]. This compound partitions between the celland the external medium at higher intracellular concentrationswhen the inside of the cell is more negative. At higher concentrations,the dye dimerizes or aggregates, quenching the fluorescence(44, 45). Thus, a decrease in fluorescence indicates membranehyperpolarization. Under the conditions of the gliding experiment,the addition of valinomycin to the cells caused a depolarizationof the membrane, while the addition of FCCP caused a slighthyperpolarization of the membrane (Fig. 4a). These shifts occurredwithin seconds after addition of either compound, so their effectsmust have been manifested during the gliding experiments.

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FIG. 4. Effect of selected substances on membrane potential. Cell suspensions were incubated with DiSC₃[5] for 4 to 5 min, and then the indicated compound was added. Data are shown from 200 to 400 s of the experiment and are normalized to the average of data from the first 50 s of observations in the time period shown. Downward deflections indicate hyperpolarization. Upward deflections indicate depolarization. (a) Effect of 3 µM valinomycin, 5 µM FCCP, or 20 µM DCCD in PBS-K/G (pH 8.0). The valinomycin preparation contained 50 mM KCl to match the gliding experiments. (b) Effect of FCCP at various pHs of PBS-K/G.

Mycoplasmas are expected to have an internal K⁺ of about 200mM (37). The addition of valinomycin in the presence of 50 mMexternal K⁺ (the concentration used in the gliding experiment)would be expected to clamp ${Delta}$ ${Psi}$ at RT/F ln (50 mM K⁺_ext/200 mM K⁺_int)= –35 mV, where R is the gas constant, T is the absolutetemperature, and F is the Faraday. We believe that the normal ${Delta}$ ${Psi}$ of M. mobile is greater than –76 mV because an analogousexperiment that we performed with 10 mM external K⁺ still induceddepolarization upon the addition of valinomycin. Thus, in thegliding experiment, ${Delta}$ ${Psi}$ was reduced by more than a factor of two.

The experiment also shows that FCCP affects ${Delta}$ ${Psi}$ as well as ${Delta}$ pH (37).The sign and magnitude of the shift in ${Delta}$ ${Psi}$ depends on the externalpH. At a higher pH (pH 7.0 or 8.0), FCCP caused a hyperpolarization,while at a lower pH (pH 6.0), FCCP caused a depolarization (Fig.4b). Gliding motility was not affected by external pH in thisrange (Fig. 2). Therefore, by employing valinomycin and FCCP,we were able to collapse both ${Delta}$ ${Psi}$ and ${Delta}$ pH (and therefore modulate ${Delta}$ p_H) without significant effect on gliding motility. Thus, theproton motive force does not power gliding in M. mobile.

Arsenate ions compete with phosphate ions in substrate-levelphosphorylation of ADP, and the resulting ADP-As molecule rapidlyhydrolyzes (43). Therefore, we replaced the phosphate in ourbuffer with arsenate to study its effect. Arsenate caused arapid and statistically significant drop in both the speed ofgliding and the percentage of cells moving in the experiment(Fig. 3e and f). The drop in speed and fraction of cells movingis likely due to a specific effect of arsenate rather than simplythe removal of phosphate since we were able to observe motilityfor extended periods of time in Tris- or TES [N-tris(hydroxymethyl)methyl-2-aminoethanesulfonicacid]-based buffers (data not shown). Presumably, the cellshave a phosphate or ATP reserve that is rapidly abolished inthe presence of arsenate. To further examine the effect of arsenate,we measured both ATP levels and gliding parameters from theonset of arsenate addition over an extended period of time.We found that both gliding speed and fraction of cells movingtracked the levels of ATP present in the cell (correlation coefficient,r² = 0.94 and 0.88, respectively) (Fig. 5). Thus, ATP must bean important energy source for gliding motility in M. mobile.

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FIG. 5. Effect of arsenate on gliding motility and ATP levels. Parallel preparations were assayed for motility and ATP. (a) PBS-K/G (pH 8.0); (b) ArBS-K/G (50 mM arsenate). Filled squares indicate the fraction of cells moving (right axis), gray diamonds indicate gliding speed (left axis), and open triangles indicate the fraction of ATP remaining compared to the value at t = 0 (right axis). Note that error bars have been omitted for clarity. Standard deviations in speeds were generally ±20%, while standard deviations for ATP values were <5%.

Given the demonstrated importance of ATP for motility, we testedseveral ATPase inhibitors to see whether they might be involvedin gliding motility. DCCD is an inhibitor of the F₀F₁-type protontranslocating ATPase and is also reported to collapse ${Delta}$ p_H insome mycoplasmas (21, 22, 37). This compound had no effect ongliding motility (Fig. 3g and h) and lends further evidencethat the proton motive force does not drive motility. The P-typeNa⁺ translocating ATPase inhibitor sodium orthovanadate causeda small but statistically significant decrease in gliding speedbut did not reduce the percentage of cells gliding in a meaningfulway (Fig. 3i and j) (39). To further examine the role of Na⁺gradients in gliding motility, we tested the sodium channelblockers phenamil and amiloride. Both of these compounds canabolish swimming motility in Vibrio species that utilize a sodiummotive force to power flagellar rotation (2, 18). Neither ofthese compounds affected gliding motility in M. mobile (Fig.3k to n), and it is therefore also unlikely that a sodium motiveforce ( ${Delta}$ p_Na) drives motility in M. mobile in the same mannerthat Vibrio species utilize ${Delta}$ p_Na.

Finally, we tested to see whether short-term biosynthesis ofprotein was required for motility. We thought that this mightbe a reasonable possibility given that some microorganisms arereported to move by continuous extrusion of biomolecules orby continuous shedding of a protein trail during gliding motility(11, 40). We attempted to shut off transcription or translationin separate experiments. The transcriptional inhibitor rifampinprevents RNA synthesis, while the protein synthesis inhibitorpuromycin mimics aminoacyl-tRNA and aborts translation (23).These compounds had no effect on motility over the 30-min spanof the experiment (Fig. 3o to r).

DISCUSSION

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Discussion

While there have been many remarkable observations of glidingmotility of M. mobile, the source of energy for this phenomenonhas not been convincingly demonstrated (15, 16, 28-30, 33-35).One study did examine the effects of various substances on M.mobile gliding (including some which overlap those discussedhere), but that effort was directed at longer-term effects anddid not specifically address the issue of energetics (31). Westudied the energy source by employing techniques and substancesthat were useful for investigating motility in many swimmingbacteria. We hoped that elucidation of the energy source couldguide further experimentation as to the mechanism of glidingmotility.

We surmised that the energy for gliding motility in M. mobilemight by supplied by an ion motive force (such as ${Delta}$ p_H or ${Delta}$ p_Na)or that it could come directly from the hydrolysis of ATP. Manyswimming bacteria (such as E. coli) utilize ${Delta}$ p_H as the sourceof power for motility, although some (such as Vibrio alginolyticus)use ${Delta}$ p_Na (24, 26). While mycoplasmas are certainly capable ofand in fact do generate a proton motive force, it does not seemto supply the energy for gliding motility in this case. Rather,our observations suggest that motility is directly powered bythe hydrolysis of ATP. Of the various compounds that we studied,arsenate was the only chemical that we found that had a dramaticallysignificant effect on motility. We were able to successfullymanipulate ${Delta}$ ${Psi}$ and ${Delta}$ pH of the cell but without effect on motility.However, we did observe a strong real-time correlation betweenATP levels, gliding speed, and percentage of cells moving. Wenote that changes in ATP levels should eventually affect ${Delta}$ ${Psi}$ and ${Delta}$ pH; however, this effect would occur on a time scale much longerthan that for the changes induced by the addition of valinomycinor FCCP (compare Fig. 5b and 4a). We propose that the motorapparatus in M. mobile, which has yet to be identified, is mostlikely an ATPase or is directly coupled to an ATPase. This ideais strengthened by our observation that sodium orthovanadatecaused a small but significant decrease in gliding speed. Vanadyl(VO₄^3–) ions are well-known inhibitors of P-type Na⁺/K⁺translocating ATPases from a variety of organisms and are usuallyeffective at concentrations 10- to 100-fold lower than thoseused here (6, 7). The ATPase motor system of M. mobile mightbe marginally sensitive to vanadate or might be derived froma P-type ATPase. This proposal may be helpful in targeting proteinclasses in M. mobile for further study with respect to gliding.Alternatively, it has been demonstrated that some sugar transportABC-type ATPases are sensitive to vanadate, and the effect ofvanadate in this experiment might be simply to limit the supplyof fermentable sugar inside the cell (20).

M. mobile is also remarkable for its robust gliding motilitycompared to that in other mycoplasmas (14). Here, we show thatmotility in M. mobile is facilitated in part by protein componentsfrom the growth medium that condition glass surfaces to enablegliding. While our attempts to specifically identify the proteincomponent by mass spectrometry were unsuccessful, we suspectthat the essential protein(s) might act as a surfactant, allowingtransient cytadherence that would be necessary for gliding motility.Lipids might also be involved.

The mechanism of gliding motility in bacteria remains a mystery,and it is likely that several different schemes are employedin different genera. Variations on the basic mechanism mightexist even within the closely grouped mycoplasmas. For instance,several large proteins implicated in surface adherence and motilityin M. mobile have been identified (41). Mycoplasma pneumoniae,another gliding mycoplasma with a similar flask-shaped morphology,lacks any orthologs of these proteins. It is possible that thereare different effectors of gliding motility even in these twoclosely related organisms. Unfortunately, genetic manipulationshave not yet been possible in M. mobile despite much successwith mutagenesis techniques in other mycoplasmas (9, 10, 17,42). Perhaps the upcoming report of the genome sequence of M.mobile combined with the power of comparative genomics willhelp to shed light on the mechanism(s) of gliding in the mycoplasmas(J. Jaffe et al., submitted for publication). Ultimately, itwill be important to understand this new class of motor andits mechanism at the molecular level.

ACKNOWLEDGMENTS

We express our gratitude to Woody Hastings for the use of hisluminometer and fluorimeter.

This work was supported by NIH grant number AI16478 to H.C.B.

There are no known competing financial interests involved inthis work.

FOOTNOTES

* Corresponding author. Mailing address: 16 Divinity Ave., Biological Laboratories 3063, Cambridge, MA 02138. Phone: (617) 495-0924. Fax: (617) 496-1114. E-mail: hberg{at}mcb.harvard.edu.

REFERENCES

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