functional homology of chemotaxis genes in...

JouRNAL OF BACTERIOLOGY, July 1979, p. 107-1140021-9193/79/07-0107/08$02.00/0

Vol. 139, No. 1

Functional Homology of Chemotaxis Genes in Escherichiacoli and Salmonella typhimurium

ANTHONY L. DEFRANCO,' JOHN S. PARKINSON,2 AND D. E. KOSHLAND, JR.`*Department ofBiochemistry, University of California, Berkeley, California 94720' and Department of

Biology, University of Utah, Salt Lake City, Utah 841122

Received for publication 23 April 1979

Generally nonchemotactic mutants of Escherichia coli and Salmonella typhi-murium were analyzed by interspecies complementation tests to determine thefunctional correspondence between the che genes of these two organisms. The E.coli che region was introduced into Salnonella recipients by means of a series ofF-prime elements. Wild-type che genes of E. coli F'420 complemented all chemutants of Sabnonella except cheS, cheV, and a subclass of cheU. A series oftester episomes carrying E. coli che mutations were then used to determine whichE. coli che function was responsible for the complementation of each Salmonellache defect. By this method, the following correspondences were determined:cheAE and chePs, cheWE and cheWs, cheXE and cheRs, cheeYE and cheQs, cheCEand cheUs, cheBE and cheXs, and cheZE and cheTs. (The subscripts E and Srefer to E. coli and S. typhimurium genes, respectively.) In some tests, especiallythose involving the last two pairs of genes, poor complementation was observedbetween noncorresponding genes. A model explaining these observations in termsof subunit interactions is proposed.

Bacterial chemotaxis has attracted interest asa simple behavioral system which exhibits manyof the properties of neuronal and hormonal sys-tems in eucaryotes (1, 9, 10, 14, 18). For example,Escherichia coli and Salmonella typhimurium,the best-studied organisms, detect chemicalstimuli by means of specific receptors that inturn transmit sensory information to the flagellato elicit motor responses. The information-proc-essing machinery in these bacteria is capable ofintegrating receptor signals and of undergoingboth adaptation and potentiation (16).One of the major advantages of bacterial

chemotaxis as a model sensory system is itsamenability to genetic analysis. Mutants defec-tive in chemoreception, signalling, and flagellarresponses have served to dissect the path ofinformation flow during chemotaxis, as summa-rized in Fig. 1. Receptor signals are funnelledthrough a series of signalling elements to a cen-tral processing machinery which integrates andanalyzes all inputs from the sensory networkand then initiates an appropriate flagellar re-sponse. Mutants defective in the central machin-ery are motile, but generally nonchemotactic,and have been the subject of considerable inves-tigation in both E. coli and S. typhimurium.

In E. coli eight genes and in Salmonella ninegenes that can mutate to a generally nonchem-otactic phenotype have been identified (Fig. 1).

In E. coli they are as follows: cheA, cheB, andcheC, first defined by Armstrong and Adler (2);cheD, first defined by Parkinson (12); and cheW,cheX, cheY, and cheeZ, which were recently de-scribed by Silverman and Simon (18), whoshowed that the original cheA and cheB "genes"were actually complex loci. Work by Aswad andKoshland (3), Collins and Stocker (5), and War-rick et al. (22) with S. typhimurium establishedthat there are at least nine che genes in thatorganism. The genes were designated cheP,cheQ, cheR, cheS, cheT, cheU, cheV, cheW, andcheX (Fig. 1).Because S. typhimurium and E. coli each offer

certain advantages in chemotaxis studies, andbecause their genetic similarity is of generalinterest, it seemed worthwhile to investigate therelationship between che functions in the twoorganisms. We therefore carried out complemen-tation tests to establish the correspondences be-tween che genes and compared these geneticdata with the known biochemical and physiolog-ical properties of che mutants in the two orga-nisns. In general, che functions in E. coli and S.typhimurium appeared to be interchangeable.However, several interesting anomalies werenoted, and these are also discussed.

MATERIALS AND METHODSBacterial ains. S. typhimurium and E. coli

107

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108 DeFRANCO, PARKINSON, AND KOSHLAND

E. coli(A,B,C,D,W,X,Y,Z)

CentralChemical Sigl machinery Motor._Receptors-. _ mcnr _Paestimiuli path-ays (che functions)

reapona

S. typhimurium(P,Q,R,S,T, U,V, W,X

FIG. 1. General scheme for bacterial chemotaxisin E. coli and S. typhimurium. This paper is con-cerned with those genes required for chemotaxis toall chemical stimuli, referred to here as the centralmachinery of chemotaxis. The che genes that havebeen identified to date are listed.

strains and F-prime elements used in this work arelisted in Table 1.Media. Tryptone swarm plates contained 1.0%

tryptone, 0.5% NaCl, and 0.3% agar (Difco). Vogel-Bonner citrate medium (21) was supplemented with1% glycerol (VBC-glycerol) or 0.4% glucose (VBC-glu-cose). M9 glucose minimal medium (11) was used forgrowth of E. coli F' che strains. Minimal media weresupplemented with required amino acids (0.1 mM) andrequired vitamins (1 ,Lg/ml).Complementation tests. Complementation be-

tween E. coli and S. typhimurium chemotaxis mutantswas determined by transferring F' che episomes con-taining the region of the E. coli chromosome around42 min (Fig. 2) into S. typhimurium recipients. Bothdonors and recipients were grown to early stationaryphase at 37° C in minimal medium and spotted to-gether on appropriately supplemented VBC-glucoseplates to select for his' recombinants which had re-ceived F-prime elements from E. coli. Partial diploidsformed in this way displayed a mucoid morphology, aspreviously reported forE. coli (13). After single-colonypurification, these F-ductants were tested for chemo-taxis on tryptone swarm plates. When negative orweak complementation was observed, the partial dip-loids were further analyzed by observation in a dark-field microscope after growth in VBC-glycerol at 370C.The F' che episomes were checked after transfer

into Salmonella, in some cases, by transferring theepisomes to various E. coli che mutants. In all suchcases the complementation behavior was exactly asexpected, indicating that the F' element had not beenaltered during transfer to S. typhimurium.Construction of ST335. The transfer of F-prime

elements into Sabnonella strains required that therecipients be his. One strain, SL2516 (cheV), was his'.The cheV107 mutation was transduced by P22 intoST23 by cotransduction with a TnlO (tetracyclinetransposon) insertion that maps near cheV. This TntOinsertion, present in strain ST322, was isolated by M.Snyder (M. Snyder and D. E. Koshland, Jr., unpub-lished data). Tetracycline-resistant transductantswere tested for their ability to respond to chemotacticstimuli on tryptone swarm plates. One che transduc-tant, ST335, was used in these experiments.Nomenclature. The nomenclature of the che genes

in Salmonella and E. coli evolved independently, andany correspondence between genes designated by thesame letter in the two species is totally coincidental.For clarity we therefore use a subscript E to indicateE. coli genes and a subscript S for S. typhimuriumgenes. Some further suggestions with regard to no-menclature are made below.

RESULTSCorrespondence of E. coli and S. typhi-

murium che genes. With the exception ofcheD, which maps at 99 min, all of the E. coliche genes are located between 41 and 43 min,near the his locus (Fig. 2). Most of the che genesin S. typhimurium also map near the his locus(22). Various F-prime elements carrying the ma-jor chemotaxis region of E. coli were transferredto recipient che mutants of S. typhimurium toestablish the functional relationship between thegenes for chemotaxis in the two organisms.F'420, which carries wild-type chemotaxis genes

TABLE 1. Bacterial strainsStrain Genotype Reference

S. typhimu-rium

ST23 thyA1981 hisF8786 22ST36 cheRs57 recAl hisF8786 22ST56 cheWs227 recAl 22

hisF8786ST171 cheTs221 thyA1981 22

hisF8786ST172 chePs5l recAl hisF8786 22ST176 cheQs62 recAl hisF8786 22SL4041 cheXslll trpA8 hisC527 5ST108 cheSs58 thyA1981 22

hisF8786ST120 cheUs70 thyA1981 22


hisF8786ST203 cheUs3=3 thyAl981 22


hisF8786SL2516 cheVs1O7 (his+) 5ST335 cheVslO7 thyA198l This pa-

hisF8786 perE. coliRP120 recA aroD his pro 13F-prime ele-

ments(inRP120)

F'410 See Fig. 2 13F'420 See Fig. 2 13

hsfgRQPA(ch*C) motABch-AW cheXBYZhis

F410

F420

FIG. 2. Genetic map of the major chemotaxis andflagellar region ofE. coli (4), showing the location ofche genes and the genetic content of the F-primeelements used in this work. Distances are shownapproximately to scale.

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VOL. 139, 1979

of E. coli (Fig. 2), complemented S. typhimu-rium mutants in seven different genes (chePs,cheQs, cheRs, cheTs, cheUs, cheWs, and cheXs)(Tables 2 and 3). It did not complement cheSsor cheVs (Table 3). Since the E. coli wild-typeche gene functions are able to correct most S.typhimurium che defects, the two systems ap-pear to have considerable functional homology.However, unlike E. coli x E. coli crosses, weobserved no formation of wild-type recombi-nants in these tests (data not shown), indicatinginsufficient base sequence homology for geneticexchanges in the che region. This fact enabledus to examine complementation between E. coliand S. typhimurium che mutants in an unam-biguous manner since wild-type recombinantscould not obscure the results.A series of F'420 derivatives carrying various

che alleles from E. coli were transferred to S.typhimurium che mutants, and the resultingmerodiploids were tested for chemotaxis ontryptone swarm agar (Table 2). In most cases,clear complementation was observed, indicatingthat the functional defect ofthe F-prime elementwas different from that of the recipient. How-ever, some mutant combinations failed to com-plement, which implies that the donor and re-

cipient were both defective in the same chemo-taxis function. Each of these combinations isdiscussed below.cheAE x chePs and CheWE x cheWs. The

chePs recipient was complemented by all F'testers except those bearing cheAE mutations;conversely, the F' cheAE testers complementedall but the chePs recipient (Table 2). Theseresults indicate that cheAE and chePs mutantshave the same functional defect. Several otherobservations are also consistent with this conclu-sion. First, both cheAE and chePs strains havesimilar mutant phenotypes: they are unable totumble and cannot respond to any type of chem-otactic stimulus. Second, the cheAE locus hasthe largest mutational target size ofany che genein E. coli (15), and similarly, chePs mutants areby far the most frequent class of che isolate in S.

HOMOLOGY OF CHEMOTAXIS GENES 109

typhimurium (22). (Although the factors influ-encing mutational target size are not well under-stood, it is interesting that this property of thesetwo homologous genes has been conserved.)The data in Table 2 also indicate that the

cheW functions of E. coli and S. typhimuriumare homologous. Strains with these mutations,like those with cheAE and chePs, cannot tumbleor respond to chemotactic stimuli.CheXE and CheYE x cheRs and cheQs. The

data in Table 2 indicate that cheXE may corre-spond to cheRs and that cheYE may correspondto cheQs since these mutant combinations neverexhibited any complementation for chemotaxis.In E. coli certain pairs of cheXE and cheYEmutants complement rather poorly, even thoughthe individual mutations are clearly in differentgenes and are recessive to wild type (14). Similareffects were observed in the interspecies tests(Table 2): cheXE and cheQs complementedrather poorly, as did cheYE and cheRs. Thisbehavior may be due to interaction of the cheXE(cheRs) and cheYE (cheQs) gene products, apossibility which is considered in more detailbelow. In any event, gene correspondences basedon the complementation data alone must be

TABLE 3. Complementation of cheUs, cheVs, andcheSs mutants with E. coli F' episomes

Complementation with:aRecipient F'410 F'410

F'420 F'410 cheC497 flaA371cheUs (tumbly)ST120 + + + -ST134 ND + ± ±

cheUs (smoothswimming)

ST203 ND 0 0 0ST213 ND 0 0 0

cheVsST335 0 0 0 0

cheSsST108 0 0 0 0a For explanation of symbols, see Table 2, footnote

a. ND, Not done.

TABLE 2. Complementation of Salmonella che mutants with E. coli F-prime elements

S. typhimurium re- Complementation with che allele of E. coli F' tester:acipient

Strain |ce allele che| AE114 AE115 WE1I3 XE202 XE203 Y2ON YF,219 YE220 BE274 BE;275 ZE280 ZF281ST172 PS51 + 0 0 + + + + + + + + + +ST56 Ws227 + + + 0 + + + + + + + + +ST36 RS57 + + + + 0 0 ±_ o0 + + + +ST176 Qs62 + + + + T ± 0 0 0 + + + +SL4041 Xslll + + + + + + + + + + 0 0 ±ST171 ITs221 + + + + + + + + + 0 :F T 0

a Symbols: +, complementation; ±, weak complementation; :F, very weak complementation; 0, no detectablecomplementation.


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considered as tentative in this case, since mu-tants believed to be defective in different genesshowed only partial complementation.A number of the crosses were checked for

complementation in another way. Since the abil-ity to tumble and the ability to modulate tum-bling frequency in response to stimuli are basicfeatures of chemotactic behavior, it seemed rea-sonable to examine the swimming pattern ofcheR8/F' cheXE and cheRs/F' cheYE merodip-loids. No tumbling was seen in the formerstrains, whereas two of four F' cheYylcheRsstrains did exhibit spontaneous tumbling behav-ior while swimming, indicating that cheYE andcheRs mutants are not defective in the samechemotaxis function (data not shown).Other lines of evidence indicate that these

assignments are correct. For example, cheYEmutants are the second most frequent type ofche isolate in E. coli (15), which is also true forcheQs mutants of S. typhimurium (22). Thus,cheYE and cheQs have similar mutational targetsizes relative to other che genes in the twoorganisms. Moreover, both mutant types areunable to tumble or to respond to chemotacticstimuli. Mutants defective in cheXE or cheRsfunction also lack spontaneous tumbling behav-ior, but, unlike cheYE and cheQs, they can beinduced to tumble by certain types of stimuli(22). Both cheRs and cheXE mutants have beenfound to be defective in a methyltransferaseactivity that somehow controls tumbling fre-quency (19).

cheBie and cheZz x cheXs and cheTs. Thesefour classes of mutants tumble constantly andgenerally complement well with all of the non-tumbling mutants discussed above (Table 2).Although none of the tumbly mutants we stud-ied were dominant, cheBE and cheZE comple-mented cheXs and cheTs poorly in all combina-tions (Table 2). This result was also confirmedby examining the swimming patterns of the var-ious merodiploids (Table 4). These complemen-tation tests were also performed with threenewly isolated cheTs mutants (obtained from B.Taylor, Loma Linda University) as recipients,with similarly equivocal results.

Since cheXs and cheTs mutants ofSalmonellaare complemented by the wild-type F-prime ele-ment from E. coli and by other F-prime ele-ments that furnish wild-type alleles of both thecheBE and cheZE genes, it seemed possible thatthe cheBE and cheZE gene products were func-tioning as a complex and that this complex wascompatible with the rest of the Salmonellachemotaxis machinery, whereas the individualsubunits were interacting in a species-specificmanner and were not readily interchangeable.

This model suggests that formation of a func-tional hybrid complex containing subunits fromboth species might be facilitated by using amutation that leads to complete loss of the poly-peptide product as opposed to one that leads toproduction of an altered, nonfunctional subunit.To accomplish this, complementation tests werecarried out with an E. coli F-prime element thatcarried a polar mutation in cheYE. This muta-tion should prevent synthesis of any product ofthe CMeZE gene and should leave cheBE expres-sion unaffected. We reasoned that in the com-plete absence of CheZE product, the cheBE sub-unit might be able to interact with the appro-priate SalmoneUa subunit and restore a meas-urable degree of chemotaxis. The results of thistest are summarized in Table 5 and demonstratethat complementation occurred with cheXs re-cipients but not with cheTs recipients, suggest-ing that cheBE corresponds to cheXs and cheZEcorresponds to cheTs.These conclusions are consistent with the phe-

notypic properties of the mutants (Table 6). Inthese mutants, as in the wild type, tumbling canbe transiently suppressed by large temporal in-creases in attractant concentration. The wild-type responses are longer, indicating that thetumbly strains are partially defective in theirresponse to attractant stimuli. In general, cheBEmutants respond better to serine than to aspar-tate, whereas cheZE mutants respond better toaspartate than to serine (Table 6). These re-

TABLE 4. Swimming behavior ofcheTs and cheXsmutants containing F'cheBE and F'cheZE episomesche allele Behavior with the following che allele in recipienton F-

prime ele- c 1eXslII cheTs221mentcheBg274 Similar to wild type Tumbly, with occasional

swimmingcheBs275 Tumbly, with occa- Tumbly, with occasional

sional swimming swuimmngcheZz2&) Similar to wild type Tumbly, with occasional

cheZz281 Tumbly, with occa- Tumbly, with occasionalsional swunimng swunmmg

TABLE 5. Complementation ofE. coli and S.typhiurium che mutants with F'cheYEF200 (polar)

E. coli S. typhimuriumComple- Comple-

Mutant menta- Mutant menta-tiona tion

CheBE + cheXs ±CheYE 0 cheQs 0CheZE 0 cheTs 0

a For explanation of symbols, see Table 2, footnotea.

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TABLE 6. Phenotypes of tumbly mutantsSalmonella E. coli Refer-Property -encercheXs cheTs cheBE cheZE ence

Swimming behavior Tumbly Tumbly Tumbly Tumbly 13, 22Responses to Ser > Asp (1/1)0 Asp > Ser (4/4) Ser > Asp (3/4) Asp > Ser (5/6) 13b,

aspartate andserine (relative towild type)

Response to multiple Potentiation Additivity Potentiation Additivity 16Cstimuli

Level of in vivo 49%d 71% 10% 50% 14, 19methylation

Methylesterase 1%, 3%d 1%, 47% 8% 92% 20activity in vitro'Numbers in parentheses indicate number of mutants that have this phenotype/total number of mutants

examined.b Information also obtained from R. D. Jaecks and B. L. Taylor (personal communication).' Information also obtained from B. Rubik and D. E. Koshland, Jr. (unpublished observations).d Percentage of wild-type level.

sponses have now been examined in the corre-sponding S. typhimurium mutants, with similarfindings: cheXs behaves like cheBE and cheTsbehaves like cheZE. Although there is only onecheXs mutant, four cheTs strains are available,and all four, like cheZE, are more defective intheir response to serine than to aspartate (R. D.Jaecks and B. L. Taylor, personal communica-tion). Rubik and Koshland (16) have also shownthat cheBE and cheXs mutants undergo responsepotentiation. Potentiation is seen when two si-multaneous stimuli produce a significantlylonger response than is expected from the sumof the individual response times. Wild-typestrains and cheTs and cheZE mutants exhibitessentially additive response times to simulta-neous stimuli.CheCE and cheDE x cheS%, cheVs, and

cheUs. Several che loci, cheUs and cheVs inSalmonella (5, 22) and cheCE in E. coli (17),appear to coincide with fla genes. This has beeninterpreted to mean that the products of thesegenes are essential components ofthe basal bodystructure of the flagellum. According to thismodel, loss or gross mutational alteration ofthese gene products prevents formation of flag-ella. However, some mutations in these genesalter the protein such that functional flagellacan be formed, but chemotaxis is prevented.This would occur, for example, if the alteredprotein allowed flagellar rotation in only onedirection or if the flagellar basal bodies in suchmutants were defective in their ability to receiveor respond to the chemotactic signals whichdetermine the direction of rotation of the flagel-lum. The latter explanation appears to be mostconsistent with the properties of cheUs mutants

of Salmonella (7, 14) and cheCE mutants of E.coli (12, 15a).There is reason to believe that cheCE may be

the E. coli gene that corresponds to cheUs. Themap positions of the cheUs (flaQ) gene (5, 22)and the cheCE (tlaA) gene (17) are very similar(6). Furthermore, both can yield two sorts ofnonchemotactic mutants characterized by verylow or very high tumbling rates, respectively.To test whether cheUs and cheCE are corre-

sponding genes, various F' episomes from E. coliwere transferred into one cheVs, one cheSs, andfour different cheUs mutants (Table 3). ThecheUs mutants that have a tumbly phenotypewere complemented by F'420 and F'410. TheF'410cfieC tester complemented these two mu-tants only partially, and the F'410faA tester didnot complement them at all (Table 3), indicatingthat cheCE (flaA) and cheUs mutants are mostlikely defective in homologous chemotaxis func-tions.

cheVs, cheSs, and cheUs mutants with lowtumbling frequencies were not complemented byeither F'420 or F'410 (Table 3). F'410 carriesmany of the same genes that F'420 carries, in-cluding cheCE. It does not, however, carry themain cluster of che genes (near motB; Fig. 2).The lack of complementation may be due todominance by the mutant alleles of cheUs,cheSs, and cheVs, as seen previously in abortivetransduction complementation (22; DeFrancoand Koshland, unpublished data). Alternatively,the F-prime elements may not carry the corre-sponding E. coli gene(s), or these particularfunctions may not be interchangeable in the twospecies. Since the cheSs gene has not beenmapped and could lie elsewhere in the genome,

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this may explain the failure of F410 and F420to complement cheSs. The gene correspondingto che Vs, on the other hand, is probably carriedby 7410. cheVs maps in the flaAII gene (5), andit is known that this gene corresponds to flaC inE. coli (6), which maps near cheCE. Thus, lackof complementation of cheVs by F7410 or F420is probably due to dominance by cheV8.The cheDf3 mutants appear to have defects in

the tsr gene in E. coli (12), a locus involved inchemotactic responses to serine, alanine, andsome repeIlents. These mutants are dominent incomplementation tests and often revert to a Tsr-phenotype (Parkinson, manuscript in prepara-tion). A number of revertants of cheSs wereemined for the Tsr- phenotype but all wereTsr+ (data not shown), suggesting that cheDz)and cheSs mutants are different. Thus, it seemslikely that neither mutants in Salmonella simi-lar to ckeD5 nor mutants in E. coli similar tocheV8 or cheSs have been isolated yet.

DISCUSSIONThe correspondences between che functions

in E. coli and S. tykhiurwm have been estab-lished through interWecies complementationtests and comparisons of mutant phenotypes.Our findings are ized in Fig. 3, whichdepicts theE. coli che genes i their correct maporder with each che gene of S. tyhiunurumndirectly below the corresponding E. coli gene. Insome case, complementation tests providedstrong evidence in favor of the conclusions pre-sented here (for example, the correspondence ofchePs and eheAM). In other cases, two differentcorrespondences could-not be ruled out entirelyby the complementation data. For example,cheRs was clearly different from cheAs, cheWs,cheBg, and chez5 because it was complementedwell by F-prime elements carrying mutations inthose genes. But it was not complemented wellby some allels of both cheXz and cheYs. Com-plementation studies with a number of dierentalleles showed that, although cheRs and cheX5never complemented each other, and neither didcheQs and cheYE, some combinations of cheRsand cheYz alleles did result in detectable com-plementation, as did some combinations of

S. c ij | Z RY |B |X |W | A | |C | | D |

Fm. 3. Correspondence of the genes for chemo-taxis in S. tphimurium and E. col& The che genesare listed according to the mqp order in E. coli witheach Salmonela gene directly below the correspond-ingE. coli gene.

cheQs and cheXz. The most likely conclusionfrom these results is that ckeRs is homologousto cheXz and cheQs is homologous to cheYE.One explanation for the lack ofcomplementationbetween strains with mutations in nonhomolo-gous genes is given below.The correspondence between the four genes

that result in tumbly behavior was more difficultto determine. The main piece ofgenetic evidencein favor of the asiments indicated in Fig. 3 isthat a polar mutation in cheYE complementscheXs and not ckeTs, a result that would beexpected by the homologies indicated. A secondindication that the poslated homolgies arecorrect is obtained from analysis ofthe behaviorof these mutants. Particularly iing are thedifferences in their responses to multiple imuli;cheBz and cheXs mutants display rewonse po-tentiation (extremely long responises to multiplestimuli), whereas cheZE and ckeTs mutants haveessentially additive responses to two stimuliadded at the same time. Thus, although theevidence clearly favors che.B and cheXs as wellas cheZE and ckeTs as being homologous, thealterative possibility for correspondence ofthese four genes is not entirely excluded.Nomenlature sugestion. Studies of the

genetic and biochemical properties of the chegenes are proceeding in both E. coi and S.typhim . The functional homology of theche genes of these two organsms, as demon-strated in this paper, means that reults obtainedin one organism can be compared with reults inthe other. At present, the ability to compareresults is hindered by the confuing nomencla-ture.

Because the true function of all of the chegenes has not yet been elucidated, it seems un-nec_arily complicated to rename all the geneswith a common system at this point. It seemsthat a gradual approach to a more rationalo-menclature is needed, and we suggest the follow-iDg.

(i) Wherever ambuity is to be avoided oremphasis is needed, the subscript E or S shouldbe used to designathe iorin of the gene.

(ii) For new genes in one spies which areshown to correspond to existing genes in theother species, the letter already utilized shouldbe kept. Thus, a gene in E. coli which corre-sponds to cheS in Salmonella should (whendiscovered) be called cheS.Effets of gene product interacdons on

complementation patterns. In E. coli, poorcomplementation has been observed betweencertain combinations of cheXc and cheYE mu-tations and between some cheB5 and cheZz mu-

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tations (14). Although these four genes are co-transcribed, polarity effects cannot be responsi-ble for these phenomena because only specificcombinations of mutant alleles fail to comple-ment and, moreover, cheXE and cheYE alwayscomplement cheBE and cheZE. There are severalways in which apparently nonpolar, recessivemutations in different genes could fail to com-plement, providing that the products of the twogenes interact in some fashion, presumablythrough direct protein-protein contacts. Thedata on cheXE x cheYE and cheBE X cheZE aremost consistent with a dominant complex modelin which altered subunits synthesized by the twomutant genes associate to form an aberrant com-plex that inhibits or masks the activity of thewild-type product complexes, perhaps by com-peting for substrate or by blocking binding sites(Fig. 4a). This model correctly predicts that poorcomplementation should be allele specific sincemany combinations of mutant subunits wouldnot be expected to interact in this way. More-over, null defects in one of the mutant genesshould not lead to poor complementation be-cause no aberrant product would be made. Thisprediction has been confirmed by examining thecomplementation properties of polar mutations.For example, polar defects in cheYE, which pre-vent cheZE expression, complement fully withall cheBE mutants. Similarly, polar mutations ineither cheYE or cheBE, which abolish cheYE

expression, fully complement all cheXE mutants(Parkinson, unpublished data).

It seems reasonable to suppose that similarinteractions take place among the correspondingche gene products of S. typhimurium, and, infact, Stock and Koshland (20) have obtainedbiochemical data in support of one such inter-action. They were able to assay an enzyme thatremoves the methyl group from the "methyl-accepting chemotaxis protein" (8). This proteinmethylesterase activity was absent in cheBEstrains but present in CheZE strains (Table 5).This activity was also lacking in cheXs strainsand in a cheTs point mutant. A cheQs-cheTsmutant (probably a polar mutant), however, hadabout one-half the wild-type level of this en-zyme. From these data, the authors concludedthat cheBE in E. coli and cheXs in Salmonellaare probably the genes coding for the methyles-terase and that the cheZE and cheTs gene prod-ucts may interact with the enzyme in somemanner to regulate its activity.Formation of product complexes could ac-

count for some of the anomalies we encounteredin using E. coli F-prime elements to correctchemotaxis defects in Salmonella. For example,E. coli mutants defective in either cheBE orCheZE function failed to complement both cheXsand cheTs mutants of Salmonella. Similarly,cheXE and cheYE mutants of E. coli did notcomplement cheRs and cheQs strains of Sal-

a) E.coli x E.coli

B- Z+

E> > =., D Mutant complexis dominant to

D~~ 'Rur" =EZ wild type complex.

,4, ,EZ,IDB* Z~

b) E. coli x S. typhimurium

BE ZE

Preferential subunitassociation leads toinactive complexes.

Xs TSFIG. 4. Subunit interaction models for explaining weak complementation of noncorresponding genes. It is

proposed that theproducts ofthe cheBE (cheXs) gene and the cheZE (cheTs) gene interact to form a functionallyactive protein. (a) Weak complementation in E. coli x E. coli heterozygotes is believed to result fromdominance of a mutant complex. This only occurs with specific allele pairs. (b) In E. coli x Salnonellaheterozygotes, weak complementation is more prevalent and could be due to species-specific association ofsubunits. Since it was possible to obtain functional interaction of E. coli and S. typhimurium subunits byusing a polar cheYE mutation to eliminate cheZE expression completely, poor complementation is evidentlydependent on the presence ofmutant (presumably missense) gene products.

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monella. These instances of poor complemen-tation could be due to formation of dominantcomplexes comparable to those thought to occurin E. coli x E. coli tests (Fig. 4a). However, asomewhat different possibility is suggested bythe fact that poor complementation in interspe-cies tests was not allele specific, but rather oc-curred with all combinations of mutant alleles.Full complementation was only observed whenthe members of an interacting gene pair origi-nating from the same species (e.g., cheBE-cheZE)were both wild type, implying that E. coli sub-units may not interact properly with Salmonellasubunits to form a functional product complex(Fig. 4b). If this analysis is proven to be correct,it must mean that individual chemotaxis geneproducts of E. coli are not necessarily inter-changeable with those of S. typhimurium. Atsome level, however, product complexes must befunctionally equivalent because Salmonella chedefects can be fully corrected by furnishing anappropriate ensemble of che fimctions from E.coli. The level at which interchangeability canoccur is not obvious because the F-prime ele-ments used to perform these interspecies com-plementation tests probably carried genes spec-ifying a major portion of the flagellar and chem-otaxis machinery of E. coli. Further studies willbe needed to assess the true extent of functionalhomology in these two species. It may be thatsome single products and perhaps even smallproduct complexes are not readily interchange-able, but in any event, the initial studies reportedhere demonstrate considerable similarity inoverall organization and operation of the chem-otaxis systems in these two organisms.

ACKNOWLEDGMENTSWe thank Barry Taylor for sending us the new cheTstrains

and for communicating some of his results with them. Thiswork was supported by Public Health Service grants AM-09765 (to D.E.K.) and GM19559 (to J.S.P.) from the NationalInstitutes of Health. A.L.D. was supported by National Sci-ence Foundation Graduate Fellowship 1-782000-21646-3.

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typhimurium chemotaxis mutants. J. Mol. Biol. 97:225-235.

4. Bachmann, B. J., K. B. Low, and A. L Taylor. 1976.Recalibrated linkage map ofEscherichia coli. Bacteriol.Rev. 40:116-167.

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8. Kort, E. N., M. F. Goy, S. H. Larsen, and J. Adler.1975. Methylation of a protein involved in bacterialchemotaxis. Proc. Natl. Acad. Sci. U.S.A. 72:3939.3943.

9. Koshland, D. E., Jr. 1977. A response regulator model ina simple sensory system. Science 196:1065-1063.

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11. Miller, J. H. 1972. Experiments in molecular genetics.Cold Spring Harbor Laboratory, New York.

12. Parkinson, J. S. 1974. Data procesing by the chemotaxismachinery of Escherichia coli. Nature (London) 252:317-319.

13. Parkinson, J. S. 1976. The cheA, cheB, and cheC genesof Escherichia coli and their role in chemotaxis. J.Bacteriol. 126:758-770.

14. Parkinson, J. S. 1977. Behavioral genetics in bacteria.Annu. Rev. Genet. 11:397-414.

15. Parkinson, J. S. 1978. Complementation analysis anddeletion mapping of Escherichia coli mutants defectivein chemotaxis. J. Bacteriol. 135:45-53.

15a.Parkinson, J. S., and S. R. Parker. 1979. Interaction ofthe cheC and che2 products is requited for chemotacticbehavior in Escherichia coli. Proc. Natl. Acad. Sci.U.S.A. 76:2390-2394.

16. Rubik, B., and D. E. Koshland, Jr. 1978. Potentiation,desensitization, and inversion of responses in bacterialsensing of chemical stimuli. Proc. Natl. Acad. Sci.U.S.A. 75:2820-2824.

17. Silverman, M., and M. Simon. 1973. Genetic analysis ofbacteriophage Mu-induced flagellar mutants in Esche-richia coli. J. Bacteriol. 116:114-122.

18. Silverman, M., and M. Simon. 1977. Identification ofpolypeptides necessary for chemotaxiis in Escherichiacoli. J. Bacteriol. 130:1317-1325.

19. Springer, W. R., and D. E. Koshland, Jr. 1977. Iden-tification of a protein methyltransferase as the cheRgene product in the bacterial sensing system. Proc. Natl.Acad. Sci. U.S.A. 74:533-537.

20. Stock, J. R., and D. E. Koshland, Jr. 1978. A proteinmethylesterase involved in bacterial sensing. Proc. Natl.Acad. Sci. U.S.A. 75:3659-3663.

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