Aerotaxis in Salmonella typhimurium: Role Electron Transportacchini and Sherris (5) surveyed 24...

JouRNAL OF BACTERIOLOGY, Feb. 1981, p. 990-10010021-9193/81/020990-12$02.00/0

Vol. 145, No. 2

Aerotaxis in Salmonella typhimurium: Role of ElectronTransport

DANIEL J. LASZLO AND BARRY L. TAYLOR*Department ofBiochemistry, School ofMedicine, Loma Linda University, Loma Linda, California 92350

Sensory transduction in aerotaxis required electron transport, in contrast tochemotaxis, which is independent of electron transport. Assays for aerotaxis weredeveloped by employing spatial and temporal oxygen gradients imposed inde-pendently of respiration. By varying the step increase in oxygen concentration inthe temporal assay, the dose-response relationship was obtained for aerotaxis inSabnonella typhimurium. A half-maximal response at 0.4 iLM oxygen and inhi-bition by 5 mM KCN suggested that the "receptor" for aerotaxis is cytochromeo. The response was independent of adenosine triphosphate formation via oxi-dative phosphorylation but did correlate with changes in membrane potentialmonitored with the fluorescent cyanine dye diS-C3-(5). Nitrate and fumarate,which are alternative electron acceptors for the respiratory chain in S. typhimu-rium, inhibited aerotaxis when nitrate reductase and fumarate reductase wereinduced. These results support the hypothesis that taxis to oxygen, nitrate, andfumarate is mediated by the electron transport system and by changes in theproton motive force. Aerotaxis was normal in Escherichia coli mutants that weredefective in the tsr, tar, or trg genes; in S. typhimurium, oxygen did not stimulatemethylation of the products of these genes. A cheC mutant which shows aninverse response to chemoattractants also gave an inverse response to oxygen.Therefore, aerotaxis is transduced by a distinct and unidentified signalling proteinbut is focused into the common chemosensory pathway before the step involvingthe cheC product. When S. typhimurium became anaerobic, the decreased protonmotive force from glycolysis supported slow swimming but not tumbling, indicat-ing that a minimum proton motive force was required for tumbling. The bacteriarapidly adapted to the anaerobic condition and resumed tumbling after about 3min. The adaptation period was much shorter when the bacteria had beenpreviously grown anaerobically.

Bacterial migration in response to oxygen gra-dients was demonstrated as early as 1881. En-gelmann (13) observed microscopic accumula-tion of bacteria about air bubbles, at the edgesof a cover glass, and around photosyntheticallyactive plant cells. On the basis of the macro-scopic bands that forned beneath a cover glass,Beijerinck defined three categories of aerotacticresponse: aerobic bacteria formed a band adja-cent to the air interface, anaerobes moved as faras possible from the interface, and spirillar bac-teria (microaerophiles) congregated a short dis-tance from the interface (6). More recently, Bar-acchini and Sherris (5) surveyed 24 species ofhighly motile bacteria and confirmed that aero-bic and facultative anaerobic species showedpositive aerotaxis, whereas three anaerobic spe-cies of Clostridia showed negative aerotaxis.Evidently, bacteria seek an oxygen concentra-tion that is optimal for their metabolic "lifestyle," and oxygen may function as an attractantor a repellent, depending on the species studied.

The dual properties of oxygen as a chemoeffec-tor, together with evidence for a novel sensorytransduction mechanism, have stimulated an in-vestigation of the mechanism of aerotaxis in ourlaboratory. In the studies described in this paper,aerotaxis was investigated in Salmonella typhi-murium and Escherichia coli, which are at-tracted to oxygen.

Aerotaxis and chemotaxis to amino acids andsugars are functionally equivalent (1, 2). In anisotropic medium, E. coli and S. typhimuriumconsume oxygen and carbon sources in theirenvironment, thereby creating gradients. Thebacteria then move preferentially toward regionsof a more concentrated oxygen or carbon source,depending on which substance is limiting. Con-siderable progress has been made in understand-ing the mechanism of the chemotactic responsein S. typhimurium and E. coli (18, 25, 28, 46; B.L. Taylor and D. J. Laszlo, In D. Norris (ed.),The Perception of Behavioral Chemicals, inpress). Swimming bacteria change direction

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ROLE OF ELECTRON TRANSPORT IN AEROTAXIS 991

(tumble) about once per second and, as a result,move in a three-dimensional, random walk pat-tern (7). When the bacteria detect an increase inattractant concentration or a decrease in repel-lent concentration they temporarily suppresstumbling so that they continue to swim in thefavorable direction (29). The molecular eventsassociated with the regulation of tumbling in-clude formation of a chemoeffector-receptorcomplex and interaction of the complex with amembrane bound signalling protein which ulti-mately affects the probability of tumbling.Methylation of the signalling protein mediatesadaptation to attractant stimuli (22, 43, 46, 47,49). At least 12 gene products are required forsignal transduction and processing (11, 42, 44,55). Neither the electron transport system norchanges in the proton motive force are requiredfor chemotaxis to sugars and amino acids (35,37, 51; M. Snyder, J. Stock, and D. E. Koshland,Jr., personal communication).

In contrast to the extensive studies of chem-otaxis, the mechanism of aerotaxis is relativelyunexplored; even the identity of the oxygen re-ceptor has not been clearly established. Clayton(9) observed that cyanide inhibits aerotaxis inRhodospirillum rubrum and suggested thatboth electron transport and the rate of oxidationof the carbon source are important in aerotaxis.Cytochrome c oxidase has been proposed as thereceptor for aerotaxis in Euglena sp. (38). Re-cently, Taylor et al. (51) confirmed that cyanidealso inhibits aerotaxis in S. typhirnurium andproposed that aerotaxis is a specific example ofa more general phenomenon that they namedelectron acceptor taxis. S. typhimurium is at-tracted by a variety of alternative electron ac-ceptors for the respiratory chain, including ni-trate, fumarate and trimethylamine oxide. Elec-tron acceptor taxis to nitrate and fumarate re-quires the appropriate electron transport path-way and is abolished by specific mutations inthe pathway. As a result of these studies, it washypothesized that electron transport mediatedchanges in the proton motive force are a com-mon factor in electron acceptor taxis (51). Thispaper presents direct evidence for the role ofelectron transport and the proton motive forcein aerotaxis in S. typhimurium.

(Preliminary reports of this work have beenpresented elsewhere [B. L. Taylor and D. J.Laszlo, Fed. Proc. 39:2103, 1980; D. J. Laszloand B. L. Taylor, Clin Res. 28:9A, 1980.].)

MATERIALS AND METHODSBacterial strains and growth conditions. The

strains of S. typhimurium and E. coli used in thisstudy are described in Table 1. Cells were grown at30°C in Vogel-Bonner citrate medium (53), pH 7.0,

supplemented with glycerol (1.0% [vol/vol]) or glucose(0.6% [wt/vol]) and the auxotrophic requirements ofthe strain. Early log-phase cells (optical density at 600nm - 0.2 to 0.25) were washed and suspended inVogel-Bonner medium with either glucose or glyceroland with appropriate supplements.Observation of motility and measurement of

speed. Bacteria were observed at a magnification ofx500 or x800 by using a Leitz Dialux trinocular mi-croscope with dark-field optics and an objective lenswith a long working distance (UMK50 or L32). Themicroscope was fitted with interchangeable photo-graphic (Nikkormat ELW) and video equipment (cam-era, Sanyo model VC3300X; recorder, Sanyo modelVTC7100; monitor, Hitachi model VM-172).The photographic analysis of motility was similar

to the procedure of Spudich and Koshland (48). A 2-sexposure was made of bacteria with 5-Hz stroboscopicillumination. The fraction of smoothly swimming bac-teria was determined as the ratio of tracks withoutabrupt change in direction to the total number oftracks.The video analysis of motility was made during

replay in slow motion. The path of a bacterium duringa timed interval was marked on the monitor screen,and the speed was calculated. Tumbling frequencywas determined as the reciprocal of the mean timeinterval between consecutive tumbles by a bacterium.

For observation of the light response, bacteria wereilluminated with a high-intensity mercury arc lamp(Osram HBO100w/2) as described previously (50, 51).For normal observation, a long-pass orange ifiter (Oriel5150, 50% tnsmission, 530 nm) was inserted in thelight path to protect bacteria from intense blue light(30). The effect of a pulse of blue light on cell motilitywas observed when the filter was briefly removed fromthe light path.

Aerotaxis assays. Both the spatial and temporalaerotaxis assays utilized a Lucite microchamber (3.8by 8.0 by 0.4 cm) with a cover glas top (2.4 by 6.0 cm,no. 1 thickness) and glass bottom (3.8 by 7.5 by 0.1cm); one end of the chamber was removable. Gas wasintroduced via Teflon tubing (type FEP) which ex-tended into the closed end of the microchamber. Thegas stream from the perforated end of the tubing wasdiffused by a baffle of polyurethane foam. The micro-chamber was placed on the stage of a Leitz Dialuxmicroscope equipped with a Leitz 606 dark-field con-denser. A drop of culture was placed near one end ofa microscope slide which was then inserted into thechamber. Observation of the bacteria through the topof the chamber was possible with a long-working-dis-tance objective.

For the spatial assay, bacteria grown in Vogel-Bon-ner medium with glycerol were washed and suspendedin Vogel-Bonner medium with glucose (optical densityat 600 nm = 0.10). A 3-ml amount of cell suspensionwas placed in a test tube (15 by 1.8 cm) and sealedwith a stopper perforated by a gas inlet, an outlet, anda length of needle stock (24 gauge) that reached thebottom of the tube and was bent at a right angle atthe upper surface of the stopper. The cells were gassedwith nitrogen for 30 min. By briefly closing the gasoutlet, a drop of the culture was injected between aslide and cover glass spaced by capillary fragments

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992 LASZLO AND TAYLOR

TABLE 1. Bacterial strain8Strain Relevant genotypea Source or reference

S. typhimuriumST1 Wild type (selected for serine 3

chemotaxi from LT2)ST23 ST1 hisF8786thyAl981 3ST171 ST23 cheZ22l 3ST832 trg D. E. Koshland, Jr.ST352 unc T. D. Ingolia and D. E.

Koshland, Jr.ST134 ST23 cheC84 43ST55 ST23 cheWM26 55ST162 ST23 cheR212 55ST108 ST23 cheS58 47ST176 ST23 cheY62 55SL2516 SL4213 cheV107 10SL3730 galK galU463 str M. SnyderBT10 ST23 cheZ22l metE862::TnlO 14BT11 ST23 cheBlll::TnlO 14

E. coliRP477 F' thi thr(Am) leu his A(gal-att, 39

eda, rpsL)RP437 (tar) RP437 eda+ tar-539 J. S. ParkinsonRP4790 RP477 thr+ tsr-14 40AW702 trg thr leu his thi 24

a The gene designations shown are the nomenclature adopted recently by workers in chemotaxis. For S.typhimurium che genes, the original P, Q, T, U, and Y have been replaced by A, Y, Z, C, and B, respectively(14).

within the nitrogen-filled microchamber. A capillarytube (1-!d Drummond Microcap) was filled with eitheroxygen or nitrogen and sealed at one end. The open N2end was insrted into the drop, and the behavior of 02 14the cells around the end was observed. KCN, when cvused, was added anaerobically to the test tube to afinal concentration of 5 mM.

For the temporal asay the microchamber was con- cv

nected to a gas proportioner (Fig. 1). A flow meter(Matheson 1472) with a mixing chamber was fitted.with stainless steel inlet tubes connected to cylinders Flow oof prepurified nitrogen and oxygen or 1% oxygen in MMfosnitrogen (Matheson). In some of the early experi-ments, argon was used in place of nitrogen. A three- H20. m.olbway valve permitted selection between two ranges ofoxygen flow rates. An additional system provided hu-midified, oxgen-free nitrogen; gas from a low-presureregulator was diected through a pair of gas washingbottles (Kimax) connected with butyl rubber tubing(Fisher Scientific). One of the bottles contained a Mixing, Tub.solution of 0.15 M pyrogallol in 10 M KOH to removeresidual oxygen, and the other bottle held distilled Chbwater. A four-way valve permitted selection betweenscrubbed nitrogen and the oxygen mixture. A Beck- FIG. 1. Gasflowdagramforoxygentemporalgra-man E2 oxygen analyzer was used to calibrate the dient apparatus. The cylinder designated 02 con-proportioner. Direct measurements were obtained tained either oxygen or 1% oxygen in nitrogen. Inover the range of 24 to 0.1% oxygen by mixing pure some earlier experiments, argon was substituted foroxygen and nitrogen. More dilute oxygen mixtures nitrogen. Thefour-way valve betueen the mixing tube(0.002 to 0.24%) were formed by mixing 1% oxygen in and chamber was used to switch the ventilating gasnitrogen with nitrogen. from nitrogen to an oxygen plus nitrogen mixture.The response to a temporal gradient of oxygen was Gas regulators are shown as small circles, with the

determined by placing 2 to 10 id of culture on a selectedpresue(in kiogramsper square centimeter)microscope slide in the microchamber and ventilating indicated within the circle. cv, One-way check valve.

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the chamber with nitrogen for 4 min. Oxygen wasreadmitted to the chamber as a mixture with nitrogenor as air that rapidly diffused into the chamber whenthe nitrogen flow was stopped. The behavior of thebacteria was observed and videotaped for analysis. Forthe dose-response studies, 2 pl of culture was spreadon the slide to a diameter of 6 mm. The culture wasequilibrated with nitrogen for only 1 min (to minimizeevaporative loss) before exposure to the desired oxy-gen concentration.

Fluorometric measurement of membrane po-tentiaL A 1-dl amount of an ethanol solution (1 mM)of the cyanine dye diS-C3-(5) (45) or 2 !d of 8-anilino-1-naphthalene sulfonate (5 mM) was added to 3 ml ofcell suspension (optical density at 600 nm - 0.3) in afluorometer cuvette. The cells were stirred continu-ously by a magnetic bar, and a stream of air or nitrogenwas directed into the capped cuvette. Fluorescencewas measured in a Spex Fluorolog spectrofluorometerwith mulilkali photodetector, with excitation andemission wavelengths of 622 and 670 nm, respectively,for diS-C3-(5) and 375 and 472 nm, respectively, for 8-anilino-l-naphthalene sulfonate. The band-pass was10 nm. Simultaneous measurement of oxygen concen-tration was made with a chemical microsensor (Tran-sidyne General) connected to a needle oxygen elec-trode (Transidyne General no. 760) and a silver-silverchloride reference electrode positioned in oppositecorners of the fluorometer cuvettes so that they wereout of the light path. Additions were made with aHamilton syringe through the gas outlet pore in thecuvette cap. At the end of the experiment, 1 drop ofculture from the cuvette was examined under themicroscope to confirm that the bacteria were motileand had not been killed by the photodynamic actionof the dye (36, 50).

RESULTSSpatial assay for aerotaxis. The aerotaxis

assays used by most previous investigators de-pended upon bacterial oxygen consumption togenerate the gradients to which the bacteriaresponded (2, 5, 6). This type of assay is clearlyunsatisfactory for investigating the role of elec-tron transport in aerotaxis. For example, cyanidewould inhibit respiration-mediated formation ofthe gradient whether or not respiration is re-quired for aerotaxis. We have developed alter-native assays which can be used in the presenceof respiratory inhibitors. An artificially gener-ated spatial gradient of oxygen was used forqualitative measurements, and a temporal assaywas used to quantitate the aerotactic responseto precisely determined oxygen concentrations.For the spatial assay, a dilute culture of S.

typhinurium was made anaerobic by bubblingwith nitrogen, and 1 drop was then placed in thenitrogen-ventilated microchamber (see above).An oxygen-filled capillary was inserted into thedrop, and the distribution of bacteria was ob-served under the microscope. The results ob-

taimed with the wild-type strain ST1 are shownin Fig. 2. The bacteria formed a distinct clusterat the mouth of the capillary tube within 5 min(Fig. 2A). The cluster persisted for up to 2 h.High magnification (x800) revealed that cellswithin the cluster swam at normal speed (30,um/s), whereas cells at the periphery of the dropmoved at approximately one-half this speed.The presence of aspartate (1 mM) or serine (1mM) did not prevent cluster formation. No clus-ter formed around the mouth of a control capil-lary filled with nitrogen gas (Fig. 2B) or at themouth of an oxygen-filled capillary when themedium contained 5 mM KCN (Fig. 2C). Toensure that the KCN-inhibited cells were anaer-obic, the culture was gassed with nitrogen for 30min before the assay.Temporal assay for aerotaxis. The sensing

mechanism for chemotaxis involves comparisonover time of changes in effector concentrationrather than comparison of simultaneous mea-surements made at different points in space (29).As a result, the bacteria respond to a step in-crease in attractant concentration by suppress-ing tumbling and swimming smoothly for aninterval before relaxing to a normal (random)motility pattern. The time required for 50% ofthe bacteria to resume random motility is a validmeasure of the chemotactic response (48).An analogous temporal assay for aerotaxis was

designed by using the apparatus shown in Fig. 1.A small drop of bacteria was placed in a micro-chamber on the microscope stage. The bacteriarapidly became anaerobic when the chamberwas ventilated with purified nitrogen or argon.After the bacteria adapted to the anaerobic con-ditions they were exposed to oxygen by ventilat-ing the chamber with an oxygen-nitrogen (oroxygen-argon) mixture. The composition of themixture was controlled by a gas proportionerand could be varied to give an equilibrium con-centration of dissolved oxygen in the mediumbetween 0.03 and 300 pM.When anaerobic S. tphimnurium ST1 in glu-

cose medium was exposed to oxygen, the bacte-ria suppressed tumbling and swam smoothly forabout 15 s before resuming random motility (Fig.3). The transition from anaerobic to aerobicconditions was accompanied by changes in ve-locity and tumbling fiequency. As previouslyobserved in the spatial assay, the mean swim-ming speed of anaerobic S. typhinurium in me-dium with glucose was 19 pm/s. Anaerobiosisaffected the population uniformly; the distribu-tion of speeds about the mean was nornal andwas not skewed by a disproportionate numberof sluggish bacteria. When aerated, strain ST1organisms rapidly accelerated with a slight

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m-

FIG. 2. Response ofanaerobic S. typhiurium STIto an oxygen spatial gradient in the presence andabsence ofKCN. Thepreparation wasphotographedwith dark-field iUumination 5 min after insertion

(described in the text) of a gas-filled capillary intoanaerobic S. typhimurium STI in Vogel-Bonner me-

dium with 0.6% (wt/vol) glucose. (A) CapiUary con-

tained oxygen. (B) Capillary contained nitrogen. (C)Capilary contained oxygen, and the meduam con-tained 5mM KCN.

50 A

40.

002

20O

I0.

35 B

30-

-.325

020 2

Tirn.(s)FIG. 3. Effect of a temporal gradient of 0 to 270

,uM oxygen on the motility pattern of S. typhnriuwnSTI. Stain STI in Vogel-Bonner medium with glu-cose wasplaced in a microchamber and made anaer-obic by ventilaion with argon gas. After 4 min ofpreincubation, 21% oxygen in argon was introducedas the ventilating gas at zero time. The motilty ofstrain STI was photographed with a stroboscopiclamp; the tumbling frequency (A) and speed (B) ofthebacteria were measured as described in the text.

overshoot before steady-state swimming speedswere approached. The smooth response to oxy-gen was also followed by an overshoot in whichthe tumbling frequency was increased over thenQrmal aerobic tumbling frequency of this or-ganism. The speed overshoot and the tumblingovershoot were not simultaneous.The random motility of anaerobic S. typhi-

murium ST1 at zero time in Fig. 3 was observedonly after the bacteria had been anaerobic for 2to 3 min. The time course of change in motilityduring the temporal aerotaxis assay is seen moreclearly in Fig. 4, which shows a longer timeinterval. At the commencement of anaerobiosis,the celLs began to swim smoothly. This was notthe result of a favorable stimulw caused by theloss of oxygen. On the contrary, when an aerobicculture of S. typhiunwium ST1 was flushed withnitrogen, the cells first entered a tumbling phase,then slowed down and swam smoothly. Al-

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0.2.

0

E

:20 d

10-

0 3 ; 5

rm. (mn)FIG. 4. Effect of sequential temporal gradients of

oxygen on the motilitypattern ofS. typhimurium STI.The experimental procedure was similar to that de-scribed in the legend to Fig. 3 except that the venti-lating gas was N2 with two step increases in oxygenconcentration to give the dissolved oxygen concentra-tions indicated. The response of the bacteria wasvideotaped, and the tumblingfrequency (A) andspeed(B) were determined as described in the text. Thedata points shown are the means of measurementsmade of5 to 10 bacteria.

though the tumbling phase was consistently ob-served in the microscope, it was not evident inFig. 4 because the response was brief (approxi-mately 3 s) compared with the time scale in thefigure. This led us to consider whether the tum-bling response might be interrupted by a loss ofenergy required for tumbling.Proton motive force in anaerobic and

aerobic cells. The energy for the flageilar mo-tors in bacteria is supplied by the proton motiveforce across the plasma membrane (20, 26, 32-34, 52). At energy levels that are less than satu-rating for the motors, the swimming speed is

proportional to the proton motive force.Changes in swimming speed are therefore anindication of change in the proton motive force.Anaerobic bacteria were immotile in glycerolmedium but swam slowly in glucose medium,indicating that the proton motive force of S.typhimurium in glucose medium was interme-diate between the normal aerobic value and thevalue near zero expected in glycerol medium.This was confirmed by using fluorescence of 8-anilino-1-naphthalene sulfonate, which is in-

versely related to membrane potential (4, 54). S.typhimurium SL3730 was used, since it is amotile strain that is permeable to the fluorescentdyes 8-anilino-1-naphthalene sulfonate and diS-C3-(5) (M. Snyder, J. Stock, and D. E. Koshland,Jr., personal communication). When this strainbecame anaerobic in glucose medium, there wasan increase in fluorescence corresponding to adecrease in potential (Fig. 5). The addition ofthe uncoupler carbonyl cyanide-m-chlorophenylhydrazone further decreased membrane poten-tial and abolished motility. Thus, the membranepotential in anaerobic S. typhimurium is inter-mediate between the values for aerobic and de-polarized celLs.The observation that the slowly swimming S.

typhimurium organisms in anaerobic glucosemedium do not tumble suggested that there wasa mininum proton motive force required fortumbling. Tumbling is resumed after 3 to 5 min

A

2.5

J2.0-

1.5

1.0-

10 B

o r

0 8 12 16

Time (min)FIG. 5. Effect of carbonyl cyanide-m-chlorophenyl

hydrazone (CCCP) addition on fluorescence of 8-an-ilino-l-naphthalene sulfonate in the presence of an-aerobic S. typhiunurum SL3730. The bacteria wereprepared in Vogel-Bonner medium with glucose asdescribed in the text except that EDTA (0.1 mM) wasadded to the medium in which the cells were sus-pended. The bacteria became anaerobic after the airspace in the cuvette was flushed with nitrogen; fluo-rescence and oxygen concentration were measuredsimultaneously. CCCP (1 M) was added through thegas outlet with a Hamilton syringe. Fluorescenceintensity here and in Fig. 8 isgiven in arbitrary units:the base-line fluorescence in Vogel-Bonner mediumwas given the value 1.

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but there is no reproducible change in fluores-cence during this time. Either the ApH compo-nent of the proton motive force increases or thebacteria adapt to the lower proton motive force.The aerotactic response of smooth swimming

did not result from a partial depolarization ofthe membrane. The bacteria experienced twosequential step increases in oxygen concentra-tion (Fig. 4). The first oxygen level (0.5 pAM) wassufficient to support normal random motility yetthe bacteria responded to a further increase inoxygen concentration by suppressing tumblingand swimming smoothly for a brief intervaLDose-response relationship. In a series of

temporal assays S. typhimurium ST1 in glucosemedium was kept anaerobic for 1 min and thenexposed to a predetermined concentration ofoxygen, and the response (smooth swimmig) ofthe bacteria was videotaped and timed (Fig. 6).The half-maximal aerotactic response was elic-ited by 0.4 uM oxygen, which is similar to theKm (0.2 uM) for cytochrome o in E. coli (41).The accuracy of the dose-response plot de-pended in part on a rapid equilibration ofoxygengas with the medium in which the bacteria weresuspended. We have calculated that by using a2-pl drop of culture spread to 6 mm in diameter,the dissolved oxygen concentration would reach95% of the equilibrium value within 1 s. It isassumed that the profile ofan increase in oxygenconcentration is not important and that bacteriarespond to the change in oxygen bound ratherthan the time rate of change in oxygen bound.This assumption is supported by the character-istics of the chemotactic response to ribose by S.typhimurium (48).No evidence was found for oxygen receptors

with a dissociation constant of greater than 10-6M. The response time for 300 pM oxygen wassimilar to that for 2 pM oxygen. When the spatialassay for aerotaxis was performed in the pres-ence of 2.5 pM oxygen in the bacterial medium,there was no accumulation at the mouth of theoxygen-filled capillary. No evidence was foundin the temporal or spatial assay that S. typhi-murium is repelled by high concentrations ofoxygen.Role ofelectron transport. Oxidative phos-

phorylation is not essential for aerotaxis. S. ty-phimurium ST352, which is deficient in the en-ergy transducing ATPase (T. Ingolia and D. E.Kohland, Jr., personal communication), had a.normal aerotactic response. Taylor et aL (51)proposed that electron transport-inducedchanges in the proton motive force were thesignal that regulated tumbling frequency in aero-taxis. The correlation of changes in oxygen con-centration with membrane potential and swim-ming pattem provide direct support for this

hypothesis (Fig. 7). Fluorescence of the cyaninedye diS-C3-(5) was used to monitor membranepotential (35, 37, 54), and a needle oxygen elec-trode in the fluorometer cuvette permitted si-multaneous measurement of potential and oxy-gen concentrations. At the same time, 1 drop ofthe culture was monitored in the microchamberused for the temporal assay of aerotaxis. Oxygenconcentrations in the microchamber were al-tered in parallel to changes in the fluorometercuvette. Two anaerobic-aerobic cycles are shownin Fig. 7. Membrane potential (measured as neg-ative fluorescence) decreased with anaerobiosisand increased with aerobiosis as predicted bythe hypothesis.The hypothesis further required that the tum-

bling frequency be regulated by changes inmem-brane potential. In Bacillus subtilis, uncoupler-or ionophore-mediated depolarization causes atumbling (repellent) response, and hyperpolari-zation causes smooth swimming (35, 37). Re-sponses to change in the proton motive forcewere less dramatic in S. typhimurium and wereconsequently harder to detect. Nevertheless,there was a brief tumbling response (ca. 2 s) tothe addition of carbonyl cyanide-m-chloro-phenyl hydrazone or KCN and a smooth re-sponse after membrane hyperpolarization byvalinomycin.Competition between oxygen and other

electron acceptors. The electron transportsystems of S. typhinurium and E. coli arebranched pathways that can utilize oxygen oralternative terminal electron acceptors (15). Themost studied alternate electron acceptors arenitrate and fumarate, and the terminal compo-nents of the electron transport systems thatinteract with these acceptors are nitrate reduc-

12-

9- A

6-

0 2 3 4 SCOYGEN (PM)

FIG. 6. Dose-response reltonship for aerotaxis inS. typhiurium STI. The bacteria were prepared inVogel-Bonner medium with glucose, and aerotaxiswas measured by the temporal assay as described inthe text. Timing ofthe response commenced when thebacteria acclerated after exposure to oxygen andended when 50% ofthe bacteria had resumed randomswimming.

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ROLE OF ELECTRON TRANSPORT IN AEROTAXIS

R]P S _L R S R S , R ASI R ,

2-

i~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~A10-0-

0 15

rim.(mmn)FIG. 7. Effect of increasing and decreasing temporal oxygen gradients on the fluorescence ofdis-C3- (5) and

on motility of S. typhimurium SL3730. The bacteria were prepared as described in the text, and fluorescenceand motility were simultaneously examined in the fluorometer and in the microchamber, respectively. Theoxygen concentration in the fluorometer cuvette was altered by flushing the air space at the top ofthe cuvettewith either nitrogen or air. The composition ofthe gas ventilaing the microchamber was regulated manuallyto correspond to the reading of the oxygen electrode in the fluorometer cuvette. Two anaerobic-aerobic cyclesare shown. R, Random swimmingpattern; S, smooth swimming pattern.

tase and fumarate reductase, respectively. Bothnitrate and fumarate are attractants for S. ty-phinurium and E. coli, and in each case thereceptor is the terminal reductase (51). If taxisto electron acceptors is mediated by electrontransport and the proton motive force, aerotaxisshould be competitive with taxis to nitrate andfumarate. This was demonstrated (Table 2). Theaerotactic response was significantly reduced inthe presence of nitrate or fmarate (P < 0.001).Ihibition by nitrate plus fuarate was similarto inhibition by either electron acceptor usedseparately. Induction of the terminal reductasewas required for competition (data not shown).Signalling protein for aerotaxis. The

known snalling proteins for chemotaxis are theproducts of the tsr, tar, and trg genes (23, 24,46). Mutations in these genes in E. coli and intrg in S. typhimurium did not affect aerotaxis(Table 3). This indicated that aerotaxis was notfocused through one of the known signalling

proteins. The response to intense blue light (30,50), which is apparently mediated by the protonmotive force (51), was also found to be normalin tsr, tar, and trg mutants of E. coli. Thenormal aerotactic response in these mutants wasverified by the temporal and spatial assays, and

TABLE 2. Effect of nitrate and fumarate onaerotaxis in S. typhimurium STIaAdditions to medium

Response to oxy-Carbon Electron acceptor gen gradient (a)"source

Glycerol None 16.0 * 1.9 (11)Nitrate 10.9 ± 1.2 (6)Fumarate 10.8 ± 1.1 (5)Nitrate and fumarate 10.3 ± 1.8 (6)

Glucose None 14.7 i 1.5 (7)S. typhmurium STI was grown anaerobically overnight

in Vogel-Bonner medium with glycerol and nitrate (30 mM)or fumarate (30 mM). The cells were washed in aerated Vogel-Bonner medium containing the indicated carbon source andelectron acceptors. Aerotaxis was measured by the temporalassay.

The times shown for the smooth swimming response arethe mean + standard deviation, with the number of determi-nations shown within parentheses.

the RP477 (che+) response was not inhibited byserine (1 mM) or aspartate (1 mM) (31).

Aerotaxis was also examined by introducing aband of bacteria beneath a glycerol gradient inchemotaxis buffer. The bacteria consumed theoxygen in their environment and migrated inresponse to the resultant oxygen gradient (1, 2).

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TABLE 3. Response to a temporal gradient ofoxygen by mutants of S. typhinurium and E. colia

Strain Chemotaxis mu- Unstimulated response Response to oxygen Response to oxygentation decrease increase

S. typhimuriumST1ST832ST134ST55ST162ST108ST176SL2516BT10BT11

E. coliRP477RP437 (tar)RP4790AW702

NonetrgcheCcheWcheRcheScheYcheVcheZcheB

Nonetartsrtrg

RandomRandomRandomSmoothSmoothSmoothSmoothSmoothTumblingTumbling

RandomRandomRandomRandom

TumblingTumblingSmoothNoneNoneNoneNoneNoneNoneNone

TumblingTumblingTumblingTumbling

SmoothSmoothTumblingNoneNoneNoneNoneNoneSmoothbNoneb

SmoothSmoothSmoothSmooth

a Strains were grown in nutrient broth and examined for aerotaxis by the temporal assay procedure.b Observed only after preincubation in anaerobic medium for 1 h or longer.

The rate of migration was similar for E. coliRP477 (che+), RP437 (tar) and RP4790 (tsr),provided that the strains were all highly motile(Fig. 8). Strains that were not highly motile wereslower in moving up the column. RP437 (tar)was the strain most susceptible to poor motilityand was initially thought to be defective in aero-taxis. Subsequent selection of RP437 (tar) forimproved motility on tryptone semisoft agargave a highly motile strain whose migration wasconsistently similar to the results shown in Fig.8. The selected strain was confirmed to be tar.Convergence of pathways for aerotaxis

and chemotaxis. Although electron acceptortaxis is not focused through one of the knownchemotaxis ignalling proteins, it is evident (Ta-ble 3) that the pathways eventually convergeinto a common tumble-regulating system. Thisis most clearly illustrated by S. typhimuriumST134 (cheC). The cheC gene product is a com-ponent of the flagellar basal body and is appar-ently at the interface between the sensing sys-tem and the motor (21, 43). S. typhimuriumST134 has an extreme clockwise bias, resultingin inverse responses to chemoeffectors; that is,repellents cause smooth swimming, and attrac-tants cause tumbling. The response by thisstrain to a temporal gradient to oxygen is alsothe inverse of the response by che+ strains;decreased oxygen causes smooth swimming, andincreased oxygen causes tumbling (Table 3). Inthe spatial assay, S. typhimurium ST134 wasrepelled by oxygen.

Strains with che mutations other than cheCdid not show a response to oxygen in the routinetemporal assay except that S. typhimurium

BT10 responded positively to an increase inoxygen concentration after being incubated an-aerobically for 1 h or longer (Table 3). Theoxygen response of S. typhimurium ST1 waslengthened by aerobic growth in Vogel-Bonnermedium with glucose and by anaerobic incuba-tion or growth in the same medium.

DISCUSSIONThe temporal assay that was developed for

use in these studies has made it possible todetermine the dose-response relationship foraerotaxis in S. typhimurium. This is the firstsuch determination for a microorganism, and themethod should be useful for studying the oxygenresponse by other organisms. For organims witha longer response time, it should be possible toobtain a precise measurement of the half-maxi-mal response from a double-reciprocal plot ofthe data. We estimated the half-maximal re-sponse (approximately 0.4 ,uM) for S. typhimu-rium directly from the dose-response plot.

Certain cytochrome oxidases have Km valuessimilar to the half-mimal response for aero-taxis in S. typhimurium. We conclude from thisand from the inhibition of aerotaxis by KCN(Fig. 2) that the oxygen receptor for aerotaxis isa terminal oxidase. The known cytochrome oxi-dases (d and a,) in S. typhimurium (12) are notgood candidates for the aerotaxis receptor. In E.coli, cytochrome d (Km = 0.02 AM) and cyto-chrome a, are found in stationary-phase cells(8), whereas our studies utilized early exponen-tial-phase cells. Furthermore, during the dose-response investigations, it was evident from therelationship between oxygen concentration and

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12-

ES

2 4 6 8Ti,m (h)

FIG. 8. Rate of migration of E. coli mutants inresponse to a self-generated oxygen gradient. A 1 to3% glycerol gradient in 10mMpotassium phosphate,pH 7.0, 0.1 mM EDTA, and 0.3 mM (NH42sSO4 waspreformed in glass tubes (7.5 by 1.0 cm). E. coli RP477(che+) (0), RP437 (tar) (C]), and RP4 790 (tsr) (A) weregrown at 30°C in Vogel-Bonnerglycerol medium withrequired supplements until the optical density at 600nm reached 0.45 to 0.50. The cells were washed twiceand suspended in phosphate-EDTA-(NHU2SO4 with5% glycerol. A 0.1-ml portion of the washed bacteriawas introduced beneath the glycerol gradient; thesurface ofthe gradient wasgassed with nitrogen, andthe tubes were stoppered and sealed with Lubriseal.The bacteria consumed the oxygen in their environ-ment and migrated as a band in response to theresulting oxygen gradient. The distance that eachmutant had migrated from the bottom ofthe tube wasmeasured at the times indicated.

swimming speed that the principal cytochromeoxidase determining the energy production ratein early exponential-phase S. typhimurium hada Km similar to the half-maximal response foraerotaxis. We have determined that the oxidasein--S. typhimurium used in these studies wascytochrome o (B. Fandrich and B. L. Taylor,unpublished data) which is the principal cyto-chrome oxidase in E. coli and has a Km of 0.2,uM (41). The dose-response relationship sug-gests that cytochrome o is also the receptor foraerotaxis. The terminal oxidase appears to bethe receptor for aerotaxis in Euglena sp. (38).The evidence that the aerotaxis receptor is cy-tochrome o is important in view of the findingby MacGregor and Bishop (27) that cyto-chromes do not function as oxygen sensors inthe regulation of nitrate reductase biosynthesis.It is possible that cytochrome d is also a receptorfor aerotaxis but this would not have been de-tected in our studies.Competition among nitrate, fumarate, and ox-

ygen (Table 2) suggests that there is a commoncomponent in the signalling pathways for taxisto electron acceptors. The receptors (nitrate re-ductase, fumarate reductase, and cytochrome o)could interact directly with a common signalling

protein; however, the evidence suggests that thecommon component is a product of the electrontransport system. 2-Heptyl-4-hydroxyquinoline-N-oxide, which blocks electron transport at asite other than the terminal oxidase, inhibitsaerotaxis in B. cereus (W. W. Goral and B. L.Taylor, Clin. Res. 27:5A, 1979). If electron ac-ceptor taxis is mediated by the electron trans-port system, the receptors for this type of taxisare not receptors in the same sense used forother chemoattractants because the classicalchemoreceptor interacts directly with the chem-otaxis sensing system.The role of the electron transport system in

aerotaxis was previously assumed to be in ATPsynthesis (9). However, this possibility is ex-vluded by the observed normal aerotaxis in S.typhimurium ST352 which is deficient in theenergy-transducing ATPase (unpublished data).The following evidence is consistent with elec-tron transport-mediated changes in the protonmotive force as the parameter that regulates thebehavioral response in aerotaxis: (i) the corre-latiob between membrane potential and oxygenconcentration (Fig. 8); (ii) the response (smoothswimming) to valinomycin-induced hyperpolar-ization of the proton motive force; and (iii) inthe competition studies (Table 2) the magnitudeof the increase in swinmming speed after thebacteria became aerobic appeared to correlatewith the response time. The increase in swim-ming speed reflects a change in the proton mo-tive force (19). If the proton motive force is thefirst common component for electron acceptortaxis, we assume that there is a signaling proteinresponsive to changes in the proton motive force.One of the surprising findings was the shortresponse of S. typhimurium to a temporal oxy-gen gradient. This made it difficult to determinethe quantitative relationship between responsetime and change in the proton motive force.Smooth swimming observed after S. typhi-

murium became anaerobic is the result of adecrease in the proton motive force, which, inturn, decreases the swimnmg speed. Tumblingwas prevented in aerated S. typhimurium whenthe membrane potential was lowered by additionof valinomycin or arsenate (unpublished data).This relationship has been quantitated by Khanand Macnab (19), who found that the clockwise-counterclockwise bias of the flagella motor is anexponential function of the speed of rotation.Whenever the proton motive force is decreased,the motor becomes more counterclockwisebiased, thereby reducing the tumbling fre-quency. Smooth swimming in response to local-ized conditions that decrease the proton motiveforce might disperse the bacteria and enhance

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their survival (19). However, such a responsewould be disadvantageous to facultative anaer-obes in generalized anaerobiosis because itwould preclude chemotaxis to fermentable en-ergy sources. It is therefore ignificant that S.typhivnwium adapted to anaerobiosis within 2to 3 min and resumed random swimming. Thisadaptation, which is independent of protein syn-thesis, might result from a metabolic shift thatincreases the anaerobic proton motive force, al-though the possibility that the tumble regulatoradapts to a decreased proton motive force can-not be excluded at this time.

It is clear from both spatial and temporalassays that aerotaxis is normal in E. coli mutantsthat are defective in the tsr, tar, or trg gene(Table 3; Fig. 8). Thus, sensory transduction inaerotaxis does not involve the known signallingproteins, which are the products of these genes(23, 46). The observation that oxygen did notstimulate methylation of these gene products inS tyhiurium supports a similar conclusionfor that species. The response to blue light isalso independent of these signalling proteins.Sugars that are attractants and that are trans-

STIMULI RECE"ORS SIGNALING PROTEINS

FIG. 9. Diaram showing a minimum of five sig-nalling proteiss that are involved in focusing chem-otaxis signals into the common response system Ev-idence for the membrane potential signallingproteinis found in the text, and evidence for the trg, tar, tsr,andphosphotransferase system (PTS) sinallingpro-teins has been reported previously (17, 23, 24, 46).MCP, Methyl-accepting chemotaxis protein.

ported by the phosphotransferase system arefocused through a distinct ignalling protein (17,23). In view of this, it seems that there are atleast five signalling proteins (Fig. 9). We wouldassume that effectors such as light (16, 51),which also modulate the proton motive force,would be focused through the ignalling proteinfor aerotaxis. The results obtained with the cheCmutant of S. typhimunum ST134 indicate thatthe point of convergence for the chemotaxis andaerotaxis pathways is either before or at theswitch in the flagellar basal body. It will be ofconsiderable interest to discover how changes inthe proton motive force are tamnduced into achemotactic signal that impinges on the com-mon response system.

ACKNOWLEDGMNSWe thank James Engelhardt for excellent tecnical assist-

ance, Alan S. Waggoner for the generoua gift ofdiS-Cr(5), andRobert M. Macnab and Daniel K hland, Jr., for providingcopies of unpublished manus t Straim were kindly pro-vided by the sources listed in Table 1. We are grateful to MarkSnyder for suggesting the use of SL3730 for the fluorescencestudies.

This work was supported by a grant-in-aid from the Amer-ican Heart Asociation and with funds contnbuted in part bythe California Heart Association.

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