JOURNAL OF BACTERIOLOGY, May 1974, p. 640-645 Vol. 118, No. 2Copyright 0 1974 American Society for Microbiology Printed in U.S.A.

Role of Methionine in Bacterial ChemotaxisDANA ASWAD AND D. E. KOSHLAND, JR.

Department of Biochemistry, University of California, Berkeley, California 94720

Received for publication 28 January 1974

The effect of methionine starvation on the motility and behavior of normal andnonchemotactic mutants of Salmonella typhimurium was investigated. Methio-nine starvation eliminates tumbling in the wild type, but fails to do so in anuncoordinated (frequent tumbling) mutant. In the mutant, methionine starva-tion significantly extends the period of smooth motility that follows a sharptemporal increase of attractant. These results suggest that methionine metabo-lism is not tightly coupled to the generation of tumbles, but rather is necessaryfor the return of some tumble-regulating parameter to a steady-state level.

Bacterial chemotaxis provides a convenientsystem for studying the processes of sensoryreception and simple behavior. The migrationalbehavior of bacteria can be measured by thecapillary assay of Adler (1), the populationmigration apparatus of Dahlquist et al. (7), andthe tracking apparatus of Berg (5). Macnab andKoshland (9) have shown that chemical gradi-ents are sensed by a temporal comparison ofconcentrations. Hazelbauer and Adler (8) dem-onstrated that periplasmic proteins are appar-ently the receptors that bind specific attract-ants. The biochemical processes that transmitinformation from the receptors to the flagellaare still obscure.A possible clue to these processes has come

from the observation of Adler and Dahl (2) thata methionine auxotroph of Escherichia coli wasnonchemotactic toward all attractants testedwhen starved for methionine. The effect ofmethionine starvation does not result from lackof protein synthesis because starvation for otheramino acids such as threonine or leucine had noeffect on chemotaxis. Moreover, methioninestarvation does not lead to loss of motility butrather to an alteration in the type of motilitydisplayed.

In this paper we describe studies on the effectof methionine starvation on normal and chemo-tactically defective strains of Salmonellatyphimurium in an attempt to gain furtherinsight into the nature of this phenomenon.A preliminary account of this work was pre-

sented at the 57th Annual Meeting of theFederation of American Societies for Experi-mental Biology, April 1973.

MATERIALS AND METHODSBacteria. All strains used in this study are deriva-

tives of S. typhimurium, strain LT2, and are de-

scribed in Table 1.Media. Minimal citrate (VBC) medium as de-

scribed by Vogel and Bonner (12) was supplementedwith glycerol (1% wt/vol) and any required aminoacids (20 ug/ml). Liquid nutrient broth consisted ofnutrient broth (0.8% wt/vol; Difco) and sodium chlo-ride (0.5% wt/vol). Nutrient agar plates were agar(1.5%; Difco) in liquid nutrient broth. Tryptonesemisolid plates were agar (0.25% wt/vol; Difco),tryptone (1% wt/vol; Difco), and sodium chloride(0.5% wt/vol).

Growth, harvesting, and washing of bacterialcultures. All experiments described in this paperwere done on cultures grown aerobically in minimalmedium at 30 C to mid-log phase (a density of about 5x 108 bacteria/ml). They were then centrifuged at3,000 x g for 15 min at 4 C and resuspended in theappropriate medium. The bacteria were washed oneadditional time before use in an experiment.

Microscopy and photography. Bacteria were ob-served in dark field. One-half drop of culture, 5 x 106cells/ml, was placed in a "chamber" constructed bybridging one cover slip over two others on a glass slide.

Motility tracks were photographed using a strobo-scopic light source as has been described previously(9).

RESULTSEffect of methionine starvation on the

chemotactic response of Salmonella. The ef-fect of methionine starvation is shown in Fig. 1,which demonstrates that Salmonella, like E.coli, require methionine for chemotaxis. In amethionine auxotroph, the chemotactic re-sponse is constant when the methionine concen-tration in the medium is varied over the range10' to 106 M. Below 106 M the responsedrops sharply, approaching zero at 108 M. Thecurve is qualitatively very similar to that ob-tained by Armstrong (3) for E. coli.

This effect is specific for methionine starva-tion because chemotaxis was not inhibited by

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TABLE 1. Genotypes and sources of bacterial strains

Strain no. Description Source/reference

ST2 metE Diethylsulfate mu-tagenisis of ST1a

SL4041 trpA8 P. Vary and B. A. D.hisC527(amber) Stocker (11)che(amber)

ST4 trpA8 Diethylsulfate muta-hisC527(amber) genesis of strainche(amber) SL4041met

ST41 trpA8 met His' Selected as a sponta-Che+ b neous Che" revertant

of strains ST4`ST42 trpA8 Selected as a sponta-

hisC527(amber) neous Che+ revertantmet of strains ST4'

a ST1 is a wild-type S. typhimurium LT2 havinggood motility and, chemotaxis. It was formerly re-ferred to as LT2-S2 (9).

'This strain is probably hisC527(amber) andche(amber) containing an amber suppressor. Theevidence for this is that the histidine requirement waslost concomitant with reversion to the Che+ pheno-type. Also, the motility of this mutant, i.e., tumblingfrequency, is intermediate between that of the wildtype and strain SL4041, indicating a leakiness charac-teristic of suppressed amber mutations.

' Che+ revertants of ST4 were isolated as follows. Adrop from a full grown nutrient broth culture of ST4was placed on a tryptone semisolid plate and incu-bated at 30 C in a wet incubator. After 24 to 48 h,asymmetric rings indicative of chemotactic swarmingbacteria formed. Samples were taken from the leadingedge of the swarms, and single colonies were isolatedfrom them. These were checked for amino acid growthrequirements and the ability to form symmetricalrings in tryptone semisolid medium.

starvation for histidine of tryptophan in a his-,trp-, met- triple auxotroph. Adler and Dahl (2)found no effect during starvation of E. coli forthreonine or leucine, and Armstrong (3) re-ported no effect during arginine, histidine, pro-line, shikimic acid, or tryptophan starvation.

Chemotaxis is blocked regardless of how me-thionine starvation is imposed. Starvation in ametE mutant (blocked in the conversion ofhomocysteine to methionine) and a metA mu-tant (blocked in the conversion of homoserine toO-succinylhomoserine) blocked chemotaxisequally well. Growth of wild-type cells in thepresence of methionine will repress all of theenzymes in the methionine biosynthetic path-way (10). Chemotaxis is blocked in repressedcells if methionine is absent from the medium.These results show that the methionine effectdoes not result from accumulation of a methio-nine precursor.

Effect of methionine starvation on motility.

Methionine-starved cultures of S. typhimuriumare motile, but there is a striking differencebetween their motility and that of nonstarvedcultures. Normal motility consists of "runs" inwhich bacteria swim in a generally straight linefor a few seconds. This period of coordinatedmotility, the run, is then interrupted by a briefperiod of apparent rapid turning, which iscalled a "tumble." The tumble usually lasts lessthan 1 s. When coordination resumes, the bacte-ria swim off again in a new direction that israndom with respect to the former direction.Figure 2A shows this normal motility pattern inthe presence of methionine.The absence of methionine shown in Fig. 2B

causes the trajectories of the bacteria to besmoother, showing much less frequent tum-bling. Although the bacteria do not swim inprecisely straight lines, vigorously motile indi-vidual bacteria were not observed to display abona fide tumble for up to 1 min of continuousobservation. The same impression is gainedfrom looking at a field of bacteria (20 to 50 inview at any one time). Any bacterium that isseen not to be swimming in a smooth coor-dinated manner is invariably either nonmotileor poorly motile. Thus, in normally motile cells,tumbling is completely suppressed by methio-nine starvation.

Effect of methionine starvation on the re-

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0 l-8 10-7 10-6 10-5 10-4 10-3L-Methionine Molarity

FIG. 1. Methionine dependence of chemotaxis in amethionine auxotroph. A culture of strain ST2 waswashed in methionine-free VBC-glycerol, and samples(0.03 ml) of the washed suspension were then dilutedinto tubes containing 3.0 ml of VBC-glycerol and L-methionine at the concentration indicated on the ab-scissa. The tubes were incubated at 30 C with shak-ing for 15 min before assaying for chemotaxis. Chemo-taxis was measured at 30 C for 30 min by the Pfeffercapillary assay described by Adler (1) using 1 mM L-serine as an attractant.

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FIG. 2. Effect of methionine starvation on themotility of strain ST41. Bacteria were washed andthen diluted to a cell density of about 2 x 107 cells/mlin VBC-glycerol containing 10-5 M L-histidine, 10'M L-tryptophan, and 10-5 M L-methionine, (A), or

the same without methionine (B). Strain ST41 was

used here instead of ST2 or ST42 because ST41 has a

relatively higher rate of spontaneous tumbling, andthe difference between the presence and absence ofmethionine is more noticeable during the 5-s exposure

period. In the presence of methionine (A), the motilitytracks consist of relatively smooth, gently curvedsegments in which the bacterial soma is orientedalong the direction of the track interrupted by "tum-bles" where the bacterial soma shows random orienta-tion. In the absence of methionine (B), tumbling isvirtually absent and the motility tracks are nearly allsmooth.

sponse to a steep temporal gradient. A more

severe test of tumble suppression by methioninestarvation was performed by subjectingSalmonella to a sharp temporal decrease inattractant concentration. Normally such a

stimulus dramatically increases the frequencyof tumbling for up to 10 to 15 s after thestimulus is applied (9). Under conditions thatnormally elicit a tumbling response for un-

starved bacteria, methionine-starved bacteria

showed no change in behavior. The bacteriacontinued to swim in a smoothly coordinatedmanner. Tumbling is apparently blocked dur-ing methionine starvation so that even a severestimulus cannot reinstitute it.An uncoordinated mutant. Because normal

bacteria exhibit only smooth motility in theabsence of methionine, it seemed of interest tosee whether methionine starvation would alterthe motility pattern of an uncoordinated mu-tant. This mutant, strain SL4041, is non-chemotactic as judged both by the capillaryassay and by the fact that it does not swarm ontryptone semisolid media. The motility patternis one of constant tumbling or uncoordination,with no smooth coordinated swimming. Fivemethionine auxotrophs were independently de-rived from strain SL4041, and their motility wasexamined both in the presence and absence ofmethionine. In all five cases the continuoustumbling was unaffected by methionine starva-tion. Figure 3A and B show this for one of theauxotrophs, ST4.The possibility that the auxotrophs were

leaky and contained enough methionine to sup-port tumbling was considered; however, theprobability that this alternative is correct for allfive mutants is small. In addition, two Che+revertants of strain ST4, ST41 and ST42, wereisolated. When either strain is starved for me-thionine, tumbling is eliminated. Becausestrains ST41 and ST42 contain the same methi-onine biosynthetic mutation as the parent, ST4,it is unlikely that ST4 is a leaky auxotroph.The observation that ST4 continues to tum-

ble in the absence of methionine suggests one oftwo possibilities. The first is that strain SL4041contains a mutation that allows tumbling tooccur in the absence of methionine, and thesecond is that the abnormal motility pattern ofstrain SL4041 is not the result of continuoustumbling, but rather the result of a defectiveflagellar apparatus. In the first alternative, themutation is assumed to effect the regulation oftumbling, i.e., the flagella are capable of coor-dinating properly, but they are constantly re-ceiving signals that prevent smooth swimming.In the second alternative, the flagella are receiv-ing the proper signals, but they are unable tocoordinate their flagella properly, regardless ofwhich signals are being received.To choose between these alternatives, strain

ST4 was subjected to a positive temporal gradi-ent of serine. Normally, chemotactic bacteriasubjected to such a temporal increase in attract-ant concentration respond by suppressing tum-bling for several minutes. We call this a

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FIG. 3. Motility tracks of strain ST4, an uncoordi-nated mutant, under a variety of conditions. (A) Motil-ity in the presence of methionine. The bacteria were

washed and resuspended in VBC-glycerol containingL-methionine, L-histidine, and L-tryptophan, each at10-5 M, prior to photographing them. No smooth mo-

tility tracks are present because the bacteria were

swimming in a highly uncoordinated manner with fre-quent, almost continuous, changes in direction. Manybacteria, out of the plane of focus, appear as lightfuzzy spots in the photograph. (B) Motility in theabsence of methionine. The bacteria were washed andresuspended as in (A) except that methionine was

"smooth response." The result with strain ST4is shown in Fig. 3C. Inhibition of tumbling wasobserved in 90% of the bacteria. These bacteriaswam in a coordinated manner with a linearvelocity typical of normal strains. This period ofsmooth motility died away in a few minutes,giving rise to the tumbling pattern characteris-tic of this mutant strain. These observationsprove that the flagella of strain ST4 (andtherefore strain SL4041) are capable of normalcoordinated motility for a prolonged period oftime, and hence the defect is in the tumbleregulation and not in the flagellar apparatus.

Effect of methionine starvation on durationof the smooth response of the uncoordinatedmutant. The influence of methionine starva-tion on the smooth response of strain ST4 wasinvestigated, and the results are shown in Table2. For an aspartate gradient in the absence ofmethionine, the response lasted up to 10 min,whereas in the presence of methionine theresponse lasted only 1.5 min. A similar effect

TABLE 2. Effect of amino acid starvation on durationof the smooth response

Starvation ResponseStrain condition durationaconditions (min)

ST42 (che+, control) - Histidine 2.3 ± 0.3- Tryptophan 2.5 ± 0.3- Methionine b

ST4 (uncoordinated) - Histidine 1.2 + 0.3- Tryptophan 1.5 -i± 0.3- Methionine 10 i 2.0

a Stimulus is a temporal gradient of 0 1 mML-aspartate generated in the apparatus described byMacnab and Koshland (9).bNo response listed because methionine-starved

che+ strains always swim in a smooth coordinatedmanner.

omitted. The motility tracks of ST4 under theseconditions is similar to photograph (A), indicatingthat methionine starvation does not eliminate tum-bling in this mutant. (C) The effect of a positivetemporal gradient of attractant on the motility tracksof ST4. Bacteria were washed and resuspended inVBC-glycerol containing all three amino acids as in(A). To create a temporal gradient, 0.5 ml of bacteriasuspension was rapidly pipetted into 0.5 ml of resus-pension medium containing 2 mM L-serine in a smalltest tube. A vortex mixer was used to assure rapidmixing. Immediately after mixing, a drop of thesolution was taken for photomicrography. The photo-graph seen here was taken approximately 30 s afterthe mixing. The motility tracks indicate smoothcoordinated motility.

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was observed with a serine gradient. This resultsuggests that methionine starvation might infact improve the chemotactic response of themutant when swimming in a spatial gradient,i.e., in the capillary assay. However, strain ST4failed to respond in the capillary assay in eitherthe presence or absence of methionine. If im-provement occurred, the effect on the uncoor-dinated bacteria was not sufficient to allowchemotaxis. The positive results in the tempo-ral gradient apparatus therefore were undoubt-edly caused by the much steeper gradient perunit time in that apparatus.

DISCUSSIONThe methionine effect, originally discovered

by Adler and Dahl (2) in E. coli, has now beenshown to occur also in Salmonella. Our studiesof this effect indicate that tumbling is notmerely reduced but completely eliminated in amethionine-starved culture. Moreover, methio-nine-starved Salmonella fail to tumble whenstimulated by a steep temporal decrease inattractant concentration, an experiment thatnormally causes violent tumbling in unstarvedSalmonella. The loss of tumbling explains atthe behavioral level why methionine starvationcauses loss of chemotaxis. As demonstrated byBerg and Brown (6) and Macnab and Koshland(9), when bacteria experience an increase inattractant concentration, e.g., by swimming upa gradient of attractant, tumbling is suppressedand the bacteria swim longer than usual in theup-gradient direction. If the bacteria find them-selves swimming down the gradient, the fre-quency of tumbling is increased only slightlyrelative to normal. As a result, chemotacticmigration occurs essentially from a selectivesuppression of spontaneous tumbling. Becausemethionine starvation removes this capacity totumble, selectivity of direction is also elimi-nated.

Although our results show that a supply ofmethionine is necessary for tumbling in normalstrains, the experiments with ST4 indicate thatmutants can be isolated that continue to tum-ble in the absence of methionine. This putssevere restrictions on the possible role of methi-onine in relation to tumble regulation. Anymechanism in which methionine or a methio-nine derived metabolite is a substrate for atumble generating reaction seems eliminated. Ahypothetical example of such a mechanismwould involve S-adenosyl-methionine donatinga methyl group to a protein in the basal regionof the flagella that would perturb the orienta-tion of the flagella and result in a tumble.

Methionine starvation would deplete the supplyof S-adenosyl-methionine and turn off the tum-ble-generating reaction. If bacteria operated bythis mechanism, a mutant that tumbled in theabsence of methionine could not occur. StrainST4, an uncoordinated mutant, does just that,eliminating this hypothesis.An alternative is that methionine, or its

metabolite, has some less direct, perhaps regu-latory role in the control of tumbling. The dataof Table 2 suggest that this regulatory role is tocreate a biochemical environment that turns offthe smooth response. In the presence of methio-nine, strain ST4 showed a smooth response ofqnly 1.5 min, whereas in the absence of methio-nine, the response duration is increased sixfoldto a value of roughly 10 min. Thus, the presenceof methionine in the cell is necessary for rapidreturn to an environment that allows tumblingfrom an environment that causes smooth swim-ming.At present little is known about how tumbles

are regulated at the molecular level. Presum-ably there exists within the cell some parame-ter, e.g., metabolite concentration, ion concen-tration, or membrane potential, that serves tointegrate information from chemoreceptors andmodify motility. When a stimulus is encoun-tered, the value of this parameter changes butmust return to its steady-state level soon afterthe stimulus has ended. Methionine may serveto assume that the tumble-regulatory parame-ter returns to its steady-state value.The observation on the che+ and mutant

strains agree with this hypothesis. In che+bacteria, this regulatory parameter is appar-ently delicately balanced so that only sporadictumbling occurs. A slight perturbation in itssteady-state level caused by methionine starva-tion would thus effectively eliminate tumbling.Strain ST4, however, has a much higher fre-quency of spontaneous tumbling. In this casemethionine starvation could decrease the prob-ability of tumbling without completely elimi-nating it.The validity of our interpretation rests ulti-

mately on the nature of the mutation in theuncoordinated mutant. We have implicitly as-sumed that strain ST4 is qualitatively similar tothe wild type, differing only in the quantitativeaspects of gradient sensing or tumble regula-tion. The temporal gradient results support thisview (Table 2).

In recent publications, Armstrong (3, 4) pre-sented evidence that in E. coli the methionineeffect reflects a need for S-adenosyl-methioninein chemotaxis. Experiments in our laboratorywith Salmonella concur with this conclusion.

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Thus, the methionine requirement may reflect a

need for S-adenosyl-methionine in turning offthe smooth response.

ACKNOWLEDGMENTS

We would particularly like to acknowledge the valuablehelp and suggestions of Robert Macnab during the course ofthis work.

This work was supported by U.S. Public Health Servicegrants 5 TO1 GM 31-15 and AM-GM-09765 from the NationalInstitute of General Medical Sciences and the National Insti-tute of Arthritis, Metabolism, and Digestive Diseases.

LITERATURE CITED

1. Adler, J. 1973. A method for measuring chemotaxis anduse of the method to determine optimal conditions forchemotaxis by Escherichia coli. J. Gen. Microbiol.74 :77-91.

2. Adler, J., and M. M. Dahl. 1967. A method for measuringthe motility of bacteria and for comparing random andnon-random motility. J. Gen. Microbiol. 46:161-173.

3. Armstrong, J. B. 1972. Chemotaxis and methioninemetabolism in Escherichia coli. Can. J. Microbiol.18:591-596.

4. Ar,mstrong, J. B. 1972. An S-adenosylmethionine require-ment for chemotaxis in Escherichia coli. Can. J.Microbiol. 18:1695-1701.

5. Berg, H. C. 1971. How to track bacteria. Rev. Sci.Instrum. 42:868-871.

6. Berg, H. C., and D. A. Brown. 1972. Chemotaxis inEscherichia coli analysed by three-dimensional track-ing. Nature (London) 239:500-504.

7. Dahlquist, F. W., P. Lovely, and D. E. Koshland, Jr.1972. Quantitative analysis of bacterial migration inchemotaxis. Nature N. Biol. 236:120-123.

8. Hazelbauer, G. L., and J. Adler. 1971. Role of thegalactose binding protein in chemotaxis of Escherichiacoli toward galactose. Nature N. Biol. 230:101-104.

9. Macnab, R. M., and D. E. Koshland, Jr. 1972. Thegradient-sensing mechanism in bacterial chemotaxis.Proc. Nat. Acad. Sci. U.S.A. 69:2509-2512.

10. Smith, D. A. 1971. S-amino acid metabolism and itsregulation in Escherichia coli and Salmonellatyphimurium. Advan. Genet. 16:142-165.

11. Vary, P., and B. A. D. Stocker. 1973. Nonsense motilitvmutants in Salmonella typhimurium. Genetics73:229-245.

12. Vogel, H. G., and D. M. Bonner. 1956. Acetvlornithinaseof Escherichia coli: partial purification and some

properties. J. Biol. Chem. 218:97-106.

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