JOURNAL OF BACTERIOLOGY, Nov. 1967, p. 1431-1436 Vol. 94, No. 5Copyright © 1967 American Society for Microbiology Printed in U.S.A.

Inhibitio of Flagellar Coordination inSpirillum volutans

NOEL R. KRIEG, JOSEPH P. TOMELTY, AND J. SCOTT WELLS, JR.Department of Biology, Virginia Polytechnic Institute, Blacksburg, Virginia 24061

Received for publication 24 August 1967

The motility of Spirillum volutans is caused by the rotation of each polar flagellarfascicle in a direction opposite to that of the more slowly rotating cell. Both flagellaform cones of revolution oriented in the same direction. When the cell reverses itsmotion, both fascicles simultaneously reverse their rotation and also the orientationof their cones of revolution, with the tail fascicle becoming the head and vice versa.Chloral hydrate and phenol were found to cause uncoordination, with both fasciclesbecoming the head type; MgSO4, Mg(NO3)2, NiSO4, NiC12, CuSO4, and CuC12also caused uncoordination, with both fascicles becoming the tail type. In all cases,the flagellar fascicles remained highly active but the cells were motionless becauseof the opposing propulsion; the rotation of the fascicles was in a constant directionwithout reversal. Uncoordinated states could be maintained for 30 to 60 min.Neutralization of the dual-tail flagellation caused by NiSO4 could be accomplishedwith chloral hydrate. At the null point, the flagellar orientation was intermediatebetween head and tail; the fascicles continually reversed direction of rotation, and,now coordinated, caused the cells to move back and forth. Higher concentrations ofchloral hydrate completely overcame the effect of NiSO4 and caused dual-headflagellation. Optimal concentrations of test compounds were determinied with theuse of pure cultures and a reproducible growth medium.

The visibility of the flagellar fascicles ofSpirillum volutans with the light microscopemakes this species a unique research tool. In1919, Metzner (3) was the first to take advantageof this in an investigation of the dynamics offlagellar propulsion. S. volutans appeared topossess a large flagellar fascicle at each pole;during motion, each fascicle rotated at about 40rev/sec, while the cell body rotated in an oppositedirection at one-third this speed, obeying thelaw of action and reaction. The helical cell bodywas propelled through the medium as a result ofits rotation. Reversal of direction was caused by asimultaneous reversal of the rotation and also ofthe orientation of the cone of revolution of bothfascicles (Fig. 1).

Metzner noted two responses of especialimportance to flagellar coordination. By placingcompounds at the edges of wet-mount prepa-rations, it was noted that, as the spirilla ap-proached chloroform, ether, or acetone, theorientation of their flagellar fascicles was alteredso that both became the head type, folded intoward the cell body. Such cells were motionlessbut had highly active flagella, each fascicleopposing the action of the other. Halogen salts(NaCl, KCI, KBr, and KI) were found to cause

both fascicles to become the tail type, flaredaway from the cell body; again, cells were motion-less but the flagella were highly active.

In 1965, pure culture isolates of S. volutansbecame available for the first time (4). Previousfailures in isolation were attributed to the un-suspected microaerophilic nature of the organism.In wet-mount preparations, a characteristic bandof spirilla formed near small air bubbles or theedges of the cover slip in zones of optimal oxygenconcentration, and in such a band the spirillawere highly motile, reversing back and forthwithout leaving the band. Such behavior sug-gested two hypotheses: (i) the presence of twokinds of sensors in a cell-one which sensed toomuch oxygen, and the other, too little oxygen; or(ii) the presence of a single sensor, which simplydetected an unfavorable oxygen concentration.In any case, the flagellar motors appeared toreceive a signal to reverse their rotation andorientaticn, resulting in a reversal of cell motionand a consequent withdrawal from the unfavorableregion. The most remarkable aspect, however,was that in these spirilla both flagellar fascicleswere reoriented simultaneously, and it seemedalmost inescapable that some kind of coordina-tion mechanism existed between the cell poles,

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FIG. 1. Reversal of motion andflagellar orientationin normal cells of Spirillum volutans.

despite the large distance (ca. 50 IA) that inter-vened. In view of Metzner's report, it seemedlikely that certain compounds could interferewith such a coordination.The present report is concerned with (i) the

discovery of uncoordinating compounds moreeffective than those employed by Metzner, withdetermination of their optimal concentrations,effect on cell growth, and interactions, and (ii)the utilization for these purposes of a reproduciblegrowth medium and pure cultures of the or-ganism for the first time.

MATERIALS AND METHODSThe organism used was S. volutans ATCC 19554.

Cultures (24-hr) in peptone-succinate-salts mediumwere prepared as described previously (2). Test com-pounds were added to 10-ml portions of growing cul-ture; wet mounts were prepared at 0, 10, 30, 45, 60,90, and 120 min for dark-field microscopy. In the caseof normal medium, medium plus chloral hydrate, andmedium plus MgSO4, high-speed cinephotomicrog-raphy was used to record results. For this purpose, aBolex H 16 M camera, mounted on a Leitz Ortholuxmicroscope, equipped with a 10 X eyepiece, 25 Xobjective, dark-field condenser (numerical aperture,1.20), and XBO 150 Osram Xenon lamp, was used.Pictures were taken on Ansco Super Hypan film, withan exposure time of 1/120 sec and a frame speed of48 per sec.

In wet-mount preparations of peptone-succinate-salts medium cultures, the spirilla were found to re-main homogeneously dispersed for 10 to 15 min, re-versing their direction of motion with high frequency,until oxygen became limiting in the central portion ofthe mount. At this point, a microaerophilic bandbegan to form near the edge of the cover slip. All ob-servations of flagellar uncoordination were made im-mediately after preparation of wet mounts.

For those test compounds which preliminary studiesshowed to produce a significant and prolonged effect,it was desirable to estimate the length oftime that sucheffect could be maintained. Since some of the effec-tive compounds caused an alteration of the pH of the

culture from 6.8, the optimal pH for the growth ofS. volutans, this was readjusted immediately afteraddition of these compounds. Microscopic examina-tion of fresh wet mounts was undertaken at varioustimes during subsequent incubation, and the time re-quired for approximately half of the cells, by inspec-tion, to lose all flagellar activity was recorded.The growth tolerance of S. volutans to optimal un-

coordinating concentrations of test compounds wasdetermined by the use of 20-ml portions of sterilemedium, each containing one of the compounds andbeing adjusted to pH 6.8. These were placed in 50-mlcotton-stoppered flasks, inoculated with 0.25 ml of a24-hr culture of S. volutans, and incubated at 30 C inan atmosphere of 94% nitrogen and 6% oxygen. Theflasks were examined for turbidity at 2- and 7-dayintervals.

Satisfactory plate-count methods for enumeration ofS. volutans cells have not yet been developed; conse-quently, viability studies of the organism in the pres-ence of the test compound could not be carried out.

RESULTS

Figure 2 presents a short cinematographicsequence of normal cells of S. volutans in peptone-succinate-salts medium. Such cells were highlymotile and reversed their direction frequently,despite the fact that a microaerophilic band hadnot yet formed. It can be seen that the transitionperiod of reversal of flagellar orientation oc-cupied no more than 3X sec, and that bothfascicles reversed their orientation simulta-neously.

Figure 3 illustrates the uncoordinating effectof chloral hydrate (0.038 to 0.048 M) on S.volutans; in this case, both flagellar fasciclesbecame the head type, folded in toward the cellbody. The fascicles were still highly active androtated in a constant direction with no reversal;yet the cells were almost motionless, apparentlybecause of the rotation of each fascicle in adirection opposite to that of the other, resultingin opposing propulsion. Concentrations doublethose indicated resulted in a significant pro-portion of cells without flagellar activity; con-centrations half those indicated yielded many cellsshowing no uncoordinated effect. During incu-bation, an increasing proportion of cells beganto exhibit more slowly rotating fascicles, witheventual cessation of flagellar activity. After 45min, approximately half the ctlls were inac-tivated; however, a few cells still possessed activeflagella at 75 min. Phenol in a concentration of0.016 to 0.020 M produced an effect similar tothat of chloral hydrate, with half of the cellsbeing inactivated at 30 min.

It was interesting to note that spirilla possessingonly one polar fascicle, which occur in smallnumbers in cultures of S. volutans and presumablyrepresent the products of recent cell division

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FLAGELLAR COORDINATION IN SPJPULLUM VOLUTANS

FIG. 2. Dark-field cinematographic sequence of normal cells of Spirillum volutans in peptone-succinate-saltsmedium. Frames 14 and 15 indicate reorientation of the flagella. The total time for frames I to 28 is only 7/12sec. X 470.

continued to be motile despite the presence of theuncoordinating compounds; however, their direc-tion was unchanging.

Figure 4 illustrates the uncoordinating effect ofMgSO4*7H20 (0.15 to 0.20 M) on S. volutans;in this case, both flagellar fascicles became thetail type, flared away from the cell body. Again,the fascicles were still highly active and rotated ina constant direction with no reversal; yet the cellswere almost motionless because of opposing

flagellar propulsion. The same considerations asabove concerning increase or decrease in concen-tration and decrease in flagellar rotational speedwith time apply. Ater 60 min, approximatelyhalf the cells had lost flagellar activity, although afew cells still possessed active flagella at 120 min.The following compounds produced an effect

similar to that of MgSO4: Mg(N03)2 .6H20,0.073 to 0.098 M; NiSO4. 6H20, 0.012 to 0.031 M;NiCl2-6H20, 0.026 to 0.034 M; CuS04 5H20,

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FIG. 3. Dark-field cinematographic sequence ofa cell of Spirillum volutans in the presence of 0.048 H chloralhydrate, showing dual-head flagellation. The flagellar fascicles are rotating at high speed (e.g., one completerevolution can be seen easily in frames 10 to 14); yet the cell body is almost motionless because of the opposingpropulsion. X 470.

0.0031 to 0.0040 M; and CuCl2-2H20, 0.0023 to0.0046 M. The time required for half the cells tolose flagellar activity was 30 min in the case of thenickel and copper compounds, and 60 min forMg(NO3) 2.For the compounds cited above causing dual-

tail or dual-head orientation of flagellar fascicles,at least 90% of the cells were affected. Prior todiscovery of the highly effective compoundsindicated above, however, a variety of othercompounds had been screened. These had beenadded to cultures of S. volutans in a series of con-centrations with the use of twofold dilutions. Theresults may be of interest to those who wish touse S. volutans as a tool for study of bacterialmotility, and can be summarized as follows: (i)NaCl, KCJ, KBr, KI, CaCl2, Na2SO4, MnSO4,MnCl2, sucrose, methyl salicylate, and eserinesulfate (an inhibitor of acetylcholinesterase,blocking nerve conduction in higher animals)caused dual-tail flagellation, but affected only a

proportion of the cells; (ii) ethyl ether, acetone,and chlorobutanol caused dual-head flagellationin most of the cells, but the effect lasted no morethan 10 min, followed by complete cessation offlagellar activity. It is possible that all thesecompounds may differ only in degree from theeight finally selected and that a similar mech-anism of action may be involved which is limitedby unrelated side effects.When added to peptone-succinate-salts medium

cultures in optimal concentration, the nickel andcopper salts described previously caused adecrease in pH of the cultures, to as low as 5.3.The pH change was, however, not the cause ofuncoordination, since readjustment ofpH of suchcultures to 6.8 caused no reversal of uncoordina-tion, and also since lowering of pH of normal,untreated cultures to 5.0 with HC1 failed to causeuncoordination.

S. volutans was found to be incapable ofmultiplication in peptone-succinate-salts medium

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FLAGELLAR COORDINATION IN SPIRILLUM VOLUTANS

FIG. 4. Dark-field cinematographic sequence of a cell of Spirillum volutans in the presence of 0.20MMgSO4 7H2 0, showing duai-tai flagellation. Theflagellar fascicles are rotating at high speed (e.g., one completerevolution can be seen easily in frames 14 to 18); yet the cell body is almost motionless because of the opposingpropulsion. X 470.

containing any of the eight effective uncoordinat-ing compounds in their optimal concentration,even when incubated in a suitable oxygen at-mosphere at pH 6.8, in contrast to controlswithout additions.Table 1 presents an interesting illustration of

the neutralization of flagellar uncoordination,achieved by the combination of a compoundinducing dual-tail flagellation and a compoundinducing dual-head flagellation. When NiSO4was used alone in optimal concentration, a dual-tail type of flagellation was produced, as describedpreviously. When NiSO4 was used in combinationwith chloral hydrate, also in optimal concen-tration, the dual-head flagellation effect of thelatter dominated; however, by reducing the con-centration of chloral hydrate to 35% of itsoptimum, a null point could be reached betweenthe two compounds at which reversal of flagellarrotation and also coordination between both

flagellar fascicles was now permitted. As a result,the cells became motile and moved back andforth with high frequency. Although a consider-able degree of normalcy had been regained bythe cells, they were still not entirely normal,since the orientation of both fascicles appearedto be neither head nor tail, but intermediate inconfiguration. At the null point, the cells re-mained vigorous for about 15 min, but eventuallybecame motionless with increasing loss of flagel-lar activity. It was important to add both reagentssimultaneously in order to insure a maximal nullperiod for the cells. A similar null point could bedemonstrated with CuS04 and chloral hydrate;in this case, 40% of the optimal concentration ofthe chloral hydrate proved to be suitable. Itwas not possible to demonstrate a null pointwith MgSO4 and chloral hydrate, however, sincethe addition of as little as 25% of the optimal

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TABLE 1. Neutralization offlagellar uncoordination

Experimental system Appearance and behaviorof cells

NiSO4-6H20 (0.026 M) Dual-tail flagellation;flagellar rotation di-rection constant;cells motionless be-cause of opposingpropulsion

NiSO4-6H20 (0.026 M) Flagellation interme-+ chloral hy- diate between headdrate (0.017 M) and tail orientation;

fascicles continuallyreversing directionof rotation; fasciclesnow coordinated andcell motile; cellsmove back and forthwith high frequency

NiSO4-6H20 (0.026 M) Dual - head flagella-+ chloral hy- tion; flagellar rota-drate (0.048 M) tion direction con-

stant; cells motion-less because of op-posing propulsion.

concentration of chloral hydrate resulted incessation of all flagellar activity.

DISCUSSION

Two considerations suggest that the effect ofthe uncoordinating compounds is indirect andprobably the result of cellular changes, which,if unchecked, lead to cell death: (i) the failure ofS. volutans to multiply in the presence of any ofthe compounds in their optimal uncoordinatingconcentration, and (ii) the gradual cessation offlagellar activity with increasing time of exposureof the cells to the compounds. In the absence ofsatisfactory viable-count methods, however, thepossibility that loss of flagellar activity may notnecessarily reflect loss of cell viability must beconsidered, and the action of the uncoordinatingcompounds may be specifically directed towardthe flagellar coordination mechanism. By anal-ogy to animal nerve cells, the spirillar cell mem-brane is a likely candidate for the location of thiscoordination mechanism, and the generation ofan action potential in this membrane by a waveof depolarization, with resulting transmission

of an electrical impulse, is not inconsistent withthe rapidity with which simultaneous reori-entation of flagellar fascicles occurs in normalcells of S. volutans. If such a mechanism wereoperative, the action of uncoordinating com-pounds might be directed at this mechanism. Inthe case of magnesium, nickel, and copper salts,the cation appears to be relevant moiety, and it isconceivable that such cations could interferewith the ion imbalance necessary to produce apolarized membrane; in the case of phenol, thewell-known effects of this compound on bacterialpermeability might also interfere with the main-tenance of a polarized membrane. In this con-nection, the possibility that the flagellar fasciclesthemselves, rather than the cell membrane, mayrepresent the sites of action for the uncoor-dinating compounds must be borne in mind.Electron microscopy of the fascicles of affectedcells may prove to be useful in clarifying this.One of the major functions of the present

report is to stimulate further investigation of theflagellar coordination mechanism of S. volutans.For example, the discovery of inhibitors whichare less toxic than those now available would beof value. Also, an investigation of the ability ofS. volutans to discriminate between Na and Kwith regard to permeability may bear directly onthe problem of the existence of a polarized cellmembrane. Furthermore, the approach of Adler(1), who has demonstrated the usefulness ofmutants in a study of bacterial chemotaxis, maybe applicable to S. volutans. Certainly a varietyof information will be required to illuminatefurther the fascinating frontier of bacterial"nervous systems."

LITERATURE CITED

1. ADLER, J. 1966. Chemotaxis in bacteria. Science153:708-716.

2. MCELROY, L. J., J. S. WELLS, JR., AND N. R. KRIEG.1967. Mode of extension of cell surface duringgrowth of Spirillum volutans. J. Bacteriol. 93:499-501.

3. METZNER, P. 1919. Die Bewegung und Reizbeant-wortung der bipolar gegeisselten Spirillen.Jahrb. Wiss. Botan. 59:325-412.

4. WELLS, J. S., JR., AND N. R. KRIEG. 1965. Culti-vation of Spirillum volutans in a bacteria-freeenvironment. J. Bacteriol. 90:817-818.

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