Salmonella typhimurium Mutants Generally Defective inChemotaxis
AMY L. TSUI COLLINS' AND B. A. D. STOCKER*
Department ofMedical Microbiology, Stanford University School of Medicine, Standford, California 94305
Received for publication 1 June 1976
The mutations of eight chemotaxis-deficient strains of Salmonella typhimu-rium, including flve new mutants in strain LT2, were mapped by P22 transduc-tion in relation to various fla mot deletions in S. abortus-equi. Seven recessiveche mutations mapped between motB and flaC: three, all nontumbling, in cheregion I, adjacent to motB, and four, including one ever-tumbling, in che regionII, adjacent to flaC. Mutant che-107, never-tumbling and dominant to wild type,mapped at flaAII, other mutations of which cause either absence of flagella orlack of locomotor function. We surmise that gene flaAII specifies a protein thatpolymerizes to form an essential component of the basal apparatus (so thatabsence of gene product prevents formation of flagella); that a component builtup from certain mutationally altered proteins cannot transmit (or generate)active rotation of the hook and flagellum, and so causes the Mot (paralysis)phenotype; and that a component built up from protein with the che-107 altera-tion permits only counterclockwise rotation, so that the tumble, normally pro-duced by transient clockwise rotation, cannot be effected.
Many mutants of Escherichia coli fail toshow the normal chemotactic response to anystimulus; by complementation and otherwisetheir mutations have been assigned to fourgenes (2, 18). Most mutants are affected at cheAor cheB, close together in the main cluster ofmotility genes. Mutants termed cheC arisefrom mutation at locus flaA, other alleles ofwhich cause absence of flagella, also within themain cluster (17, 22). Gene cheD is located else-where (18). Most chemotaxis-deficient mutantsfail to show the normal change in frequency of"tumbles" (i.e., abrupt changes in direction oftravel) when subjected to a chemotactic stimu-lus (i.e., change in concentration of an attrac-tant or repellent) and indeed seem unable totumble at all (15). The tumble is now known(15) to result from transient clockwise rotationof one or more of the flagella, and it is inferredthat nontumbling che mutants cannot rotatetheir flagella clockwise. Chemotaxis mutants ofanother phenotypic class show incessant tum-bling, a phenotype here termed "ever-tum-bling"; all of 26 ever-tumbling mutants of E.coli investigated by Parkinson (18) mapped atcheB. Chemotaxis-deficient mutants have alsobeen recognized in Salmonella species (5, 13,25, 26). Vary and Stocker (26) isolated an ever-tumbling mutant in Salmonella typhimurium
' Present address: Department of Therapeutic Radiol-ogy, University of Minnesota Medical School, Minneapolis,MN 55455.
LT2 and ascribed its abnormal motility to adefect in chemotactic behavior; Macnab andKoshland (16) showed that a sudden large in-crease in ambient concentration of a chemotac-tic attractant temporarily restored normal mo-tility. The purpose of the present investigationwas to isolate S. typhimurium mutants withgeneral defects in chemotaxis and to map thesites of mutation in them and other che mu-tants available in this species (5, 25, 26).
(This work is taken from the Ph.D. thesis ofA. L. Tsui Collins, Stanford University, 1975.)
MATERIALS AND METHODS
Bacterial strains and phages. The S. typhimu-rium strains used are listed in Table 1. Note that thechemotaxis-deficient strain SL2501, assigned allelenumber che-101, was obtained as a spontaneous mo-tile (fla+) mutant from SL12, identical to SW544,which is S. typhimurium Glasgow 0, a fla- strainisolated from a natural source and found to have alatent slow-swarming property (25). Strain SL4041,a che mutant of ever-tumbling phenotype, was iso-lated by Vary and Stocker (26) from an LT2 line.ST20 che-222, a nontumbling mutant in strain LT2(5), was received from Dana Aswad (Department ofBiochemistry, University of California at Berkeley).The fla, etc., deletion mutants used, some of thosedescribed by Yamaguchi and his colleagues (28), areshown, by mutation number, strain number, andline showing reported extent of deletion, in Fig. 1and 2. All of them stem from a strain of S. abortus-equi [antigenic formula 4, 12:(a); e, n, x] fixed in the
diethyl sulfate.b Mutant able to utilize D-malate (J. Stern and B. A. D. Stocker, unpublished data).c Additional characters of SL4213, not here relevant, are listed in ref. 11.d See description of parent strains, above, for characters other than chemotaxis.
normally unexpressed phase 1 by gene vh2- (12),some of them via sublines given various phase 1flagellar antigens by transduction (28). Thesestrains, though sensitive to the smooth-specificphage P22, were also sensitive to some rough-spe-cific phages and incompletely sensitive to FO phage;this suggests that they have incomplete defects inbiosynthesis of the lipopolysaccharide (LPS) core,resulting from leaky rfa mutations (27). Part-roughcharacter with diminished ability to adsorb phageP22 may account for the somewhat low yields ofmotile transductants sometimes obtained from thesestrains. Strain SL4213, an LT2 line used as parent ofchemotaxis mutants, has a gal mutation affectinggalE function, thus preventing the production ofsmooth (galactose-containing) LPS on ordinary me-dia (11, 27). In experiments involving adsorption ofphage P22, the effect of the mutation on LPS charac-ter was reversed by providing galactose (0.5%, wt/vol) in the medium (together with an equal concen-tration of glucose to prevent possible galactose toxic-ity).
Media. The nutrient broth and nutrient agar usedwere Oxoid nutrient extract broth no. 2 (CM67) andblood agar base (CM55). The medium used for selec-tion of motile or chemotaxis-proficient transduc-tants was nutrient broth with gelatin (Difco) (8%,wt/vol) and agar (Difco) (0.2 to 0.45%, wt/vol). This
flaE flaK motA|motB| chel |che/ll flaC- I SJ167
fla-291, SJ1672
fla-302, SJ1683
flaM
fla-1215, SJW215
fla-234, SJ
fla-1 140, S,
I
615
IW140
FIG. 1. Map ofthe right-hand part (i.e., end near-est trp) of the Hi-linked cluster of genes concernedwith motility. The previously reported (28) extent ofeach deletion is shown by a continuous line; dashedlines indicate extension of deletions of SJ1615 andSJ1683 into the che region; and dotted line indicatespart of che region not deleted but functionally im-paired in SJ1683, as inferred from data of Table 2.Entry above line is mutation number, followed bystrain number. All strains are S. abortus-equi.
medium is solid at 20°C but semisolid at 35 to 37°C.A rather soft medium, of agar content 0.25%, wasused when the recipient was a nonmotile S. abortus-equi strain; a somewhat stiffer medium, of agar
FIG. 2. Map of the left-hand part (i.e., end nearest his) of the Hi-linked cluster of genes concerned withmotility. The reported extent of each deletion (8, 28) is shown by a continuous line. Entry above line ismutation number, followed by strain number. All strains are S. abortus-equi.
content 0.3 or 0.45%, was used when the recipientwas motile but chemotaxis deficient.
Genetic methods. To induce mutations, broth cul-tures were exposed to ethyl methane sulfonate or tothe frame-shift mutagen, compound ICR-191. Chem-otaxis-deficient mutants were then sought by twomethods: (i) negative swarm selection, i.e., serialpassage in semisolid medium, the inoculum for eachpassage being taken from the site of inoculation ofthe previous passage, with the aim of obtainingenrichment of nonspreading mutants (4); and (ii)pour plates in semisolid medium, with inocula togive about 100 colonies, which were incubated for 4 hat 37°C to allow spreading growth and then over-
night at 20°C to allow further growth withoutspreading. Compact colonies were then picked (18,26). Mutants obtained by either procedure after sin-gle-colony reisolation were tested for rate of spreadin semisolid medium and by microscopy of brothcultures; clones that appeared motile by microscopybut spread only very slowly were retained as proba-ble che mutants. For transduction, phage P22.L4 (=int-4), grown on the strain to be used as donor, atabout 1010 plaque-forming units per ml, was mixedwith an equal volume of overnight, unshaken, 37°Cbroth culture of recipient, and the mixture was heldat 37°C for 20 min. Three standard loopful amountswere then steaked onto each of two 5-cm plates ofsemisolid medium. After 90 min, to allow adsorp-tion, the plates were transferred to the incubator,usually a programmed subambient incubator set torun at 35°C for 8 h and then at 20°C. The plates wereexamined with the naked eye, using a microscope
lamp placed below the glass slab supporting the dishto give dark-field illumination. The number andapproximate length of trails, and number and ap-proximate radius of spread of swarm, were recordedafter about 16 h at 20°C and again after a further dayat 20°C. On occasion, swarms spreading only slowlyor those spreading at normal speed were picked, andpurified transductant clones were tested for chemo-taxis character.
RESULTS
Isolation of chemotaxis-deficient mutants.Five clones spreading only very slowly in semi-solid medium but showing motility on micros-copy, therefore probably chemotaxis-deficientmutants, were isolated from mutagen-treatedcultures of two S. typhimurium LT2 lines (Ta-ble 1). Three of the mutants were isolated bynegative swarm selection from the restriction-negative galE, etc., line SL4213, two of them(SL2514 = che-105 and SL2515 = che-106) fromthe 10th passage of a culture treated with ethylmethane sulfonate, so that these two mutantsmay be identical. Another two (independent)mutants were found among 57 nonspreadingcolonies picked from pour plates in semisolidmedium made from a culture of strain SL1634exposed to the frame-shift mutagen, compoundICR-191. Picking a slow-spreading spontaneousswarm produced by the Fla- strain SL12 (=
Glasgow 0) yielded a fla+ mutant, SL2501,which appeared to be chemotaxis deficient, asexpected; symbol che-101 was assigned to theaffected gene. Strain ST20 che-222, isolated inan LT2 metE line (5), was likewise chemotaxisdeficient by the above criterion, as expected.Cells of strain SL4041 che-111 (previouslytermed che-411), the ever-tumbling mutant ofVary and Stocker (26), showed the nontransla-tional motility attributed to continuous tum-bles. Broth cultures of mutants che-105, che-106, and che-107, and of their che+ parent,were examined by microscopy of wet mountsand after staining of flagella by the method ofRyu (21). The proportion of motile and of flagel-late bacteria and the mean number of flagellaper flagellate bacterium were somewhat lowerfor the mutants than for the parent. The fla-gella appeared morphologically normal, exceptthat in some preparations of strain SL2516 che-107 many flagella unattached to cells wereseen, which suggested that flagella are shedabnormally easily in this strain.To determine tumble frequency, samples of
broth culture were transferred by loop to brothor defined medium and at once examined bymicroscopy. In experiments on the che+ parent,the median duration of travel of a motile cell,from moment of first observation to first suddenchange of direction (or cessation of motility orloss from observation), was 2.5 s for samplesdiluted into broth and less than 1 s for cellsdiluted into defined medium. Cells of the chemutants (except the ever-tumbling mutant che-111) did not show tumbles when similarlytested and are inferred to be chemotaxis defi-cient, of the nontumbling or smooth-swimmingvariety.Transductional crosses ofche mutants ofS.
typhimurium to deletion mutants of S. abor-tus-equi. We wished to map our che mutations(all in S. typhimurium) in relation to the fla,mot, etc., deletions (28) available in S. abortus-equi. In earlier experiments (8), no or very fewmotile transductants, complete or abortive,were obtained in crosses of S. abortus-equi do-nor to S. typhimurium recipient, presumablybecause of the activity of the deoxyribonucleicacid restriction systems of the latter (7). Bycontrast, crosses with LT2 as donor and nonmo-tile S. abortus-equi as recipient gave manyabortive transductants (trails) but few com-plete transductants (swarms), presumably be-cause of infrequent crossing-over as a result ofincomplete genetic homology. We thereforeused the S. abortus-equi deletion mutants asrecipients and the che mutants of S. typhimu-rium as donors, and looked for both complete
and abortive transduction of motility. We ex-pected to recognize the phenotype, chemotaxisproficient (Che+) or chemotaxis deficient(Che-), of swarms by their rate of spread andhoped also to be able to determine the Chephenotype of abortive motile transductants.The path traced out by the abortive transduc-tant, if Che-, would be a "random walk," in-stead of the progression away from the site ofinoculation that is characteristic of Che+ abor-tive motile transductants, showing tactic re-sponse to gradients established by the highbacterial concentration at the site of inocula-tion. Abortivefla+ (or fla+ mot+) transductants,if Che-, would therefore be expected to pene-trate only a short distance into the semisolidmedium and to produce a more or less globularcluster of colonies, rather than the linear groupstretching away from site of inoculation, whichconstitutes the typical trail (24, 25).Phage P22 int-4 lysates of the eight che mu-
tants of S. typhimurium and control lysates, ofche+ relative (parent or revertant) of several ofthem, were applied to various deletion mutantsofS. abortus-equi. Table 2 records the results ofcrosses of the che donors to five S. abortus-equimutants with deletions which between themcover the whole of the flaEK-motAB-flaCMsegment (Fig. 1). The results defined threegroups. Group 3 compromised one mutant,SL2516 che-107; phage grown on it evoked bothswarms spreading at normal speed and trailsseveral millimeters long from all five recipi-ents. If the che-107 mutation of the donor werewithin the segment corresponding to the dele-tion of a recipient, all motile (fla+ or fla+ mot+)complete transductants would necessarily in-corporate the mutant che gene ofthe donor and,similarly, motile abortive transductants wouldpossess only the mutant form of the che genemutated in the donor and so would be expectedto give rise only to clusters (nonlinear trails).The production of rapid-spreading swarms andlong trails in these five crosses therefore showsthat che-107 is outside the segment, from flaEthrough flaM, affected by the deletions of thesefive recipients. Two of the five recipients haddeletions, fla-291 and fla-1215, extending frommotB, on the "left" (end nearest his), throughflaC, on the "right" (end nearest trp) (Fig. 1).Crosses ofthe seven che- donors of groups 1 and2 to these two recipients gave no rapid-spread-ing swarms; slow-spreading swarms were de-tected in the crosses involving the four chedonors of group 2. Several such slow-spreadingswarms, obtained in these or similar crosses,were picked and purified. Their slow-spreadingcharacter proved to be stable, and the motility
TABLE 2. Production of swarms (complete fla+ transductants) and trails (ubortive fla+ transductants) incrosses of che- mutants of S. typhimurium to fla deletion mutants of S. abortus-equi
Donors(S. typhimurium) Recipients (S. abortus-equi deletion mutants)
che-1 08 1 0 (c) 0 c ss cc (N) NN 0 NNche-222 1 0 (C) 0 (c) 0 (c) (N) NN (N) NNche-109 1 0 c 0 (c) N,s c (N) NN 0 NNche-1 01 2 (s) cc (s) c NN NN,c s cc (N) Nche-1 05 2 (s) cc (s) cc (N) NN (s) cc (N) NNche-106 2 (s) cc (s) cc N NN (s) cc (N) NNche-111 2 (s) c (s) cc NN NN ss cc (N) NNche-107 3 (N) NN (N) NN (N) NN (N) NN (N) NNche + (N) NN (N) NN (N) NN (N) NN (N) NN
a Number and character of swarms, per two plates. Symbols: 0, No swarms detected; s, slow-spreadingswarms, inferred che-; N, normal (fast-spreading) swarms, inferred che+; (s) or (N), 1 to 10; s or N, 11 to 20;ss or NN, more than 20.
b Number and character of trails, per two plates. Symbols: c, Clusters of subsurface colonies, i.e.,nonlinear trails ("short trails"); N, normal (linear) trails of normal length; (c) or (N), 1 to 10; c or N, 11 to 20;cc or NN, more than 20.
observed by microscopy corresponded to that ofthe che donor parent concerned. That is, S.abortus-equi transductants that had acquiredmotility and slow-spreading character from theever-tumbling mutant SL4041 che-lil showedever-tumbling motility, and those from crossesin which the donor was nontumbling showedsmooth-swimming cells. No trails of normallength were seen in any of these crosses, all ofwhich, however, yielded "short trails" (so calledfor convenience, although in fact what wascommonly observed were clusters of microcolo-nies a short distance, usually less than 1 mm,below the surface growth of the recipient, with-out obvious linear arrangement). All of thesecrosses were repeated at least once, with con-sistent results: control crosses, with che+ donor,included in each experiment, always gaverapid-spreading swarms and also trails of nor-mal length (2 to 3 mm after 8 h at 37°C and 16 hat 20°C). The absence of rapid-spreadingswarms and of linear trails (but presence ofclusters or short trails) in the crosses of theseven che mutants of groups 1 and 2 to deletionmutants fla-291 and fla-1215 corresponded toexpectation for the situation when the donorche is in a gene deleted in the recipient. Wetherefore infer that the che mutations con-cerned all lie within a region deleted in bothrecipients, i.e., between motB and flaC, or per-haps between flaC and flaM. (Motile recombi-nants of geneotype fla+ che- or fla+ mot+ che-would be expected in all such crosses, but thecorresponding slow-spreading swarms were de-
tected in only some of them [Table 2]. How-ever, slow-spreading swarms were sometimeshard to detect, in part because obscured by themuch more numerous clusters resulting fromabortive transduction, and we were mainlyconcerned to record the presence or absence ofrapid-spreading swarms and of trails of normallength. We think it probable that the apparentabsence of slow swarms in some of these crossesreflects our failure to detect motile but che-clones rather than their actual absence.)The seven che mutants inferred to map be-
tween motB and flaM were divided into twoclasses by the results of crosses to two mutantswith deletions ending between motB and flaC.The crosses of the four mutants of group 2 torecipients SJ1615 and SJW140 showed theirsites of mutation to be within the segment lostin deletion fla-234 of SJ1615, which includes allknown sites in flaC and flaM, but not withindeletion fla-1140 of SJW140, which covers partof flaC and all of flaM. Taken with the earlierinference, this means that the mutations of thefour group 2 mutants are in a part of the motB-to-flaC interval that is within deletion fla-234.Crosses of the four group 2 mutants to SJ1683,whose deletion, fla-302, extends from the leftthrough motB but not into flaC, gave rapid-spreading swarms and trails of normal length.Thus deletion fla-302 does not overlap, or affectthe function of, the gene(s) affected in the group2 mutants. (In the cross ofche-101 with fla-302,some of the trails produced were scored asshort; since the [presumed] che+ ancestor of
SL2501 che-101 is not available, we used arapid-spreading mutant of SL2501 as donor in acontrol cross and obtained the same result, i.e.,both long and short trails. We do not knowwhether the rapid-spreading variant used aroseby true reversion of che-1 01, by second-site mu-tation, or by external suppressor mutation, andthe mechanism of production of some shorttrails in these two crosses remains obscure;however, we do not think that the presence ofsome short trails affects the validity of the in-ference from the rapid-spreading swarms andlong trails also obtained in the cross of che-101to fla-302.) Phage grown on the three mutantsof group 1, che-108, che-109, and che-222,evoked rapid-spreading swarms and trails ofnormal length in crosses to SJ1615; therefore,their sites of mutation are not covered by itsdeletion, fla-234, and this deletion does notaffect the function of the gene(s) mutated inthese three strains. Crosses of the group 1 mu-tants to SJ1683 gave no trails of normal lengthbut many short trails. Thus deletion fla-302 ofSJ1683 extends through motB to the right so asto destroy the function of the cistron(s) affectedby mutations che-108, che-109, and che-222.The cross ofche-108 to SJ1683 fla-302 gave onlyslow-spreading swarms and we did not detectany swarms in the cross of che-222 to fla-302,but both slow-spreading and normal swarmswere obtained when the donor was che-1 09.Thus deletion fla-302, though it affects thefunction of the gene mutated in che-109, doesnot overlap the site of this mutation, but proba-bly does overlap the che-108 and che-222 sites.Taken together, these results indicate the
gene order:
ment (Fig. 2, Table 3), compared with theirpresence in control crosses with che+ donor,suggested that che-107 is located in this seg-ment. Rapid swarms in the cross to SJ1608showed that che-107 is not in flaAI or in theright-hand end, segments iv-x, of flaAII, andtheir presence in crosses with SJW212,SJW234, and SJW328 similarly excluded all theknown sites (segments i-iv) offlaAIII. It there-fore seems that che-107 is within the left-handend, segments i-iii, of flaAII (or possibly in apreviously undetected gene betweeen flaAIIand flaAIII or in a segment of flaAIII to theright of segment iv). Crosses with several otherdeletion mutants in the left-hand cluster gaveresults compatible with this conclusion.
Since mutant che-107 had been obtained inan S. typhimurium LT2 line made restrictionnegative for the LT and S systems (7, 11), itcould be used as transductional recipient withS. abortus-equi donors. Since SL2516 che-107 iscapable of slow spread through soft semisolidmedium, a stiffer medium, of agar content0.3%, allowing virtually no spread of chemo-taxis-deficient strains, was used. Inocula ofche-107 treated with P22 lysates ofche+ donors,S. typhimurium or S. abortus-equi, gave nu-merous fast-spreading swarms (che+ completetransductants), but no or very few traits (Table3); the other seven che mutants when treatedwith phage grown on wild-type S. typhimu-rium gave not only swarms but also trails(abortive che+ transductants), as described be-low in our account of complementation tests.Such failure to produce trails, previously re-
motB-[(che-108,che-222),che-109]-(che-101 ,che-105,che-106,che-111 )-fZaC4- che regionI--4 - che region II-
. . . . . . .
deletion fla-302 of SJ1683
where the dotted line indicates loss of functionof a nondeleted che gene or operon caused bydeletion fla-302 of SJ1683, and the segmentscontaining the mutations of group 1 and group2 mutants are indicated, respectively, as cheregion I and che region II.
Since it appeared that che-107 was not lo-cated in the right-hand gene cluster, lysates ofche-107 were applied to S. abortus-equi recipi-ents with deletions in the left-hand gene clus-ter. Mutation che-107 proved to be dominant,and discussion of production of trials in thesecrosses is therefore deferred. The absence ofrapid-spreading swarms in crosses ofche-107 tofour recipients with deletions each includingthe whole of the flaR-ftaAII-tlaAII-flaAI seg-
deletion fla-234 of SJ1615
corded for some classes of fla and che mutants(14, 18, 28), indicates dominance of the mutantgene (as endogenote) to its wild-type allele (asexogenote). The presence or absence of swarms(complete che+ trandsuctants) in these crosses(Table 3) corresponded with the inference abovethat che-107 is located in the left-hand end oftlaAII. Thus no swarms were seen when thedonor was 5J1687, deleted for segments ii-v, orSJ1667, deleted for segments i-ix, of flaAII,whereas numerous swarms were obtained incrosses in which the donor was (i) SJ1728, witha deletion of all of the left-hand gene clusterexcept the flaA genes; (ii) either of two strains
TABLE 3. Production of swarms and trails in transductional crosses, in each direction, of S. typhimuriumLT2 mutant che-107 with fla deletion mutants in S. abortus-equi
SJ1727 fla-373 flaD-AI ss cc N NN 0 0SJW319 fZa-1319 flaR-L (s) cc (N) NNe 1 0SJW238 fla-1238 flaR-AI s cc (N) NN 0 0SJW270 fZa-1270 flaB-AI ss cc N NN 0 0SJ1602 fla-221 flaD-B N NN - - - -SJW383 ha-1383 flaAII(iii-x)-AI s c (N) NN 16 0SJ1608 fla-227 flaAII(iv-x)-AI N,s cc (N) NN 7 0SJ1728 fla-375 flaD-R - - (N) NNf 16 0SJ1687 fla-306 flaAII(ii-v) (s) (c) (N) N 0 0SJW330 fla-1330 flaAII(v-vii) N (N,c) NN NN 9 0SJW336 fla-1336 flaAII(vii-viii) NN (N,c) NN NN 8 0SJ1667 fla-286 flaAII(i-ix) s cc - - 0 05JW2349 fla-1234 flaAIII(i) NN,s NN,c NN NN,c 15 2SJW3289 fla-1328 flaAIII(iii-iv) (N,s) cc (N) N,c 2 0SJW212" fla-1212 flaAIII(ii-iii) (N) c (N) (N) 8 0SJ1615 fla-234 flaC-M (N) NN (N) NN 87 3SJ1683 fla-302 flaE-motB (N) NN (N) NN 86 11LT2h flaA41 flaAIII-AI - - - - 0 0SL1634h fla+ 23 3
a S. typhimurium strains used as transductional donor in cross to S. abortus-equi deletion mutant shownin column 1 as recipient.
b S. typhimurium motile but Che- recipient in transductional crosses with S. abortus-equi deletionmutants shown in column 1 as donors. Che+ transductants were selected on fairly stiff medium, allowingdetection of complete che+ transductants as swarms and of abortive che+ transductants as trails. Numbersare numbers of swarms and trails per two plates.
c Number and character of swarms, per two plates, in crosses with che-107 or che+ donor and nonmotilerecipient. Symbols are as in Table 2. -, Not tested.
d Number and character of trails, per two plates, in crosses with che-107 or che+ donor and nonmotilerecipient. Symbols are as in Table 2. -, Not tested.
e The donor in this cross is che-101, not SL4012.f The donor in this cross is che-108, not SL4012." These three recipients, with deletions of segments of flaAIII, produced some groups of microcolonies
below agar surface even on control plates without phage.h These two strains are S. typhimurium LT2 derivatives, not S. abortus-equi.
with deletions in flaAIII; or (iii) SJ1608, deletedfor segments iv-ix offlaAII and for all offlaAI.The appearance of 16 swarms in the cross withdonor SJW383, deleted for regions iii-x offlaAII and all of flaAI, excluded segment iii offlaAII as a possible site of che-107, althoughthe reverse cross had given no rapid-spreadingswarms. One cross gave a result apparentlyinconsistent with the inferred location of che-107 within flaAII. Strain SJW319 fla-1319 hasa deletion extending from flaR through allthree of the flaA genes, Hi, and flaL. In crosseswith this strain as donor and che-107 as recipi-ent, a single swarm was obtained in each oftwoexperiments. Though no swarms were seen onthe control plates without phage, we think thateach swarm may have resulted from a sponta-neous reverse (or suppressor) mutation in the
recipient, che-107. We had available a singledeletion mutant in the flaA region derived fromS. typhimurium strain LT2, mutant flaA41,whose deletion is inferred to affect all three ofthe flaA genes since it neither complements norrecombines to give wild type with S. abortus-equi mutants with deletion or stable point mu-tations in any one of these genes (28; S. Yama-guchi, personal communication). In the cross offlaA41 as donor to che-107, no swarms wereobtained, compared with 23 obtained in thecontrol cross with an LT2 fla+ che+ donor. Insummary, the presence or absence of rapid-spreading swarms (che+ complete transduc-tants) in crosses, in each direction, ofche-107 todeletion mutants ofS. abortus-equi or S. typhi-murium indicates that che-107 is located in theleft-hand segments, i-ui, of gene flaAII, or pos-
sibly in a heretofore unidentified gene betweenflaAIII and flaAII, or in a right-hand extremityof gene flaAIII, not removed by deletion fla-1328.
Table 3 includes the scoring for production oftrails, either of normal length or short, incrosses ofche-107 (and che+ control) donor to S.abortus-equi deletion mutant recipients. Theproduction of only short trails in nearly allcrosses to recipients with deletions involvingflaAII or flaAIII, compared with trails of nor-mal length in crosses to other recipients (e.g.,SJ1602, with a deletion of flaDB), indicatedthat che-107 failed to complement flaAII andflaAIII deletion mutants with respect to chemo-taxis. We suspected that in at least somecrosses this failure to complement might reflectonly dominance of che-107 as exogenote to itswild-type allele in the chromosome. If in agiven cross all the donor chromosome frag-ments carrying the relevant fla+ genes (i.e.,those corresponding to the fla deletion of therecipient) included also che-107, dominance orpartial dominance of che-107 would result inabsence of linear trails or in trails of less thannormal length. To test this possibility, we usedas recipient strain SL4045, which is an S. typhi-murium LT2 derivative fixed in phase 1 andnonflagellate because of an amber mutation inHl, the structural gene for phase 1 flagellin(26). Gene Hl is closely lined to the flaA genes,but certainly not in the same complementationgroup as any of them. It is known that nearlyall (>98%) transduced chromosomal fragmentsthat include a flaA + gene also include Hl (19),and the converse may also be true. The che-107and che+ lysates evoked about equal numbersof trails (20 to 30 per plate of semisolid medium,agar content 0.3%) from SL4045. The groupsevoked by the che+ lysate were linear, 2 to 3mm long; by contrast, about 85% of thoseevoked by the che-107 lysate were short trails,i.e., groups without any definite linear ar-rangement. We interpret this result as showingdominance, with respect to chemotaxis, of exo-ogenote che-107 to its wild-type homologue inthe chromosome, which is probably flaAII+.Though the crosses of che-107 to other strainswith deletions affecting flaAII gave only shorttrails, some trails of apparently normal lengthwere observed in the crosses to SJW330, deletedfor segments v-vii, and SJW336, deleted forsegments vii and viii (Table 3). On repetition ofthese two crosses using a very soft semisolidmedium (with 0.15% agar), trails of linear formwere again observed, but they were muchshorter than the trails in the control cross, withche+ donor. Thus recipient SJW330 treated
with phage grown on che-107 gave trails 1 to 2mm long, compared with 4 to 8 mm for trailsevoked by phage grown on the che+ donor. Itthus appears that these two flaAII deletion mu-tants are partly complemented, with respect tochemotaxis function, by che-107, which mayindicate that the chemotaxis defect ofche-107 isnot quite complete. In summary, our observa-tions on trails indicate that che-107, even asexogenote, is dominant to its wild-type allele,but that the defect in chemotaxis that it causesis not complete, and they are compatible withlocation of che-107 in either flaAII or flaAIII(or in an unnamed gene between them). (In thecourse of our crosses of che-107 to S. abortus-equi deletion mutants as recipients [Table 3],we found that control inocula [without phage]of all the three reported mutants with deletionsof segments of flaAIII, viz., SJW212, SJW234,and SJW328 [Fig. 2], produced a few groups ofmicrocolonies just below the surface of thesemisolid medium, as do many fla and motpoint mutants, including some fla amber mu-tants [20, 26]. S. Yamaguchi [personal commu-nication] has also noticed this surprising prop-erty of flaAIII deletion mutants. Because oftheir small numbers, the spontaneous clustersproduced by flaAIII deletion recipients did notinterfere with detection of clusters attributableto Che- abortive fla+ transductants.)Complementation and recombination of
pairs of che mutants. Four che mutants, twowith defects in che region I and two with defectsin che region II, were intercrossed by transduc-tion in all pairwise combinations. Plates ofsemisolid medium, of agar content 0.45%, inoc-ulated from the transduction mixtures were in-cubated for 8 h at 37°C and then for about 20 hat 240C. The crosses, including controls (nophage or with wild-type donor), were donethree times, with essentially consistent results(Table 4). On the rather stiff semisolid me-dium, the che- recipients did not spread to anappreciable extent during the incubation periodat 370C. Only a very few swarms, attributed tospontaneous reversion, were seen on the controlplates, without phage, or in the "selfed" combi-nations. All four mutants produced abundantswarms, i.e, che+ complete transductants,when treated with phage grown on wild-typedonor or on any of the other three mutantstested. Thus no one of these four mutants isidentical with any other. In the crosses withwild-type donor, all four recipients also pro-duced numerous trails, attributed to abortivetransduction ofche+ to the che- (though motile)recipients. These trails had a somewhat fuzzyappearance, each component colony (of motile
a Plates of stiff semisolid medium (agar content, 0.45%) were inoculated with 3 loopfuls of transductionmixture. Symbols before slash are number ofswarms and symbols after slash are number of trails, recordedafter 8 h at 37°C and then 20 h at 24°C. Symbols: +, 1 to 10; +, 11 to 20; + +, 20 or more, per two plates.
b Suffix (I) or (II) indicates inferred site of mutation, che region I or region II. No trails and an average of<1 swarm/plate were observed on control plates of each recipient without phage.
but chemotaxis-deficient bacteria) having a dif-fuse margin instead of the sharp outline of a
colony of nonmotile bacteria such as those intrails produced by fla (or mot) recipients. Thetrails showed that the four che mutations testedwere all recessive to che+. As described above,strain SL2516 che-107 gave no or very few trailswhen treated with phage grown on a wild-typedonor (Table 3), and che-107 is inferred to bedominant to its wild-type allele. The other mu-tants not included in Table 4 produced bothswarms and trails when treated with phagegrown on wild-type donor, and their che allelesare inferred to be recessive (in confirmation ofthe results of Aswad and Koshland [5] for che-222). All the che mutants tested were known tohave originated by independent mutation ex-
cept for SL2514 (che-105) and SL2515 (che-106),both isolated from the same mutagen-treatedculture after several cycles of negative swarm
selection (Table 1). No trails and no swarms
were obtained when these two mutants were
intercrossed in each direction, and they may
therefore have the same mutation, or differentmutations, close together in region I. The com-
plementation results for the four mutantstested in all combinations (Table 4) did notcorrespond to what might have been expected ifche regions I and II each corresponded to a
cistron. Thus the two mutants with defects inregion I complemented well in one direction ofcrossing and poorly in the other; the two mu-
tants with mutations in region II gave a similarresult. Furthermore, in some of the four combi-nations of region I mutant with region II mu-
tant, there was only partial complementation(few trails) in at least one direction of crossing.
DISCUSSIONWe tested eight S. typhimurium mutants
generally defective in chemotaxis, seven non-
tumbling and one ever-tumbling. All could re-
vert and therefore presumably result frompoint mutation, and all except one were reces-sive. The results of transductional crosses of theS. typhimurium mutants as donors to S. abor-tus-equi recipients with deletions in the motB-flaC region (Fig. 1, Table 2) showed that theseven recessive mutants had mutations be-tween motB and flaC, a segment we shall callthe che region. The results of crosses of theseseven mutants to two S. abortus-equi recipientswith deletions ending between motB and flaC(Fig. 1, Table 2) defined two classes: class I,three strains with mutations in che region I (onthe left, motB, side), containing a che gene(s)either deleted or rendered nonfunctional bydeletion fla-302 of SJ1683 but unaffected bydeletion fla-234 of SJ1615; and class II, fourmutants (including the ever-tumbling che-111)with mutations in region II (on the right, flaC,side), containing a che gene(s) unaffected byfla-302 but deleted in fla-234. Several earlierreports of Salmonella mutants with a generaldefect in chemotaxis (recognized or unrecog-nized) likewise concern mutations in che regionI or II. Iino and Oguchi (13) briefly describe ache point mutant in S. abortus-equi and, bycrosses with deletion mutants as donors (in-stead of as recipients, as in our intergenericcrosses), show that it maps between motB andflaC, overlapped by deletion fla-234 but not byfla-302, i.e., in what we term che region II.Enomoto (9) described a slow-spreading mutantof S. typhimurium LT2 whose mutation ap-peared to map at motB. His observations andthe results of recent examination of the strain(B. Stocker, unpublished data) are compatiblewith mutation in che region I, determining thenontumbling Che- phenotype. The (latent)slow-spreading character of the nonflagellateS. typhimurium strain Glasgow 0, reported in1953 (25) as cotransducible with a fla genemutated in S. typhimurium strain SW548 andsurmised (J. Adler, personal communication to
B. Stocker) to indicate a chemotaxis defect, hasnow been shown (Table 2) to result from amutation in che region II. Transductionalcrosses of SW548 (using phage KB1 [6], sincethis strain is immune to P22) have shown itsfla mutation to be, by the criterion of comple-mentation, in flaC, adjacent to che region II(A. L. Tsui Collins, Ph.D. thesis, StanfordUniversity, Stanford, Calif., 1975).
Since the products of the che genes in regionsI and II are not known, recognition of separategenes must depend mainly on complementationtests. The two deletion mutants defining re-gions I and II complement each other, withrespect not only to motility but also to chemo-taxis, as shown by production of trails of normallength in transductional crosses (28; Stocker,unpublished data). This indicates that the chegene(s) in region I and those in region II arefunctionally independent, not in a single op-eron. In E. coli, nearly all che mutations havebeen assigned to either cheA or cheB, genesdefined by complementation groups (2, 17, 18).The E. coli map order hag-flaI-mot-cheA-cheB(3, 18) appears to correspond to the Salmonellaorder H1-flaEK-motAB-che(I)-che(II)-flaCM.Perhaps che regions I and II of Salmonellacorrespond, respectively, to cheA and cheB ofE. coli, but this surmise remains to be checkedby appropriate intergeneric complementationtests. Tests of complementation with respect tochemotaxis present some difficulty, because intransductional crosses ofche point mutants therecipient strain itself is motile and may spreadsufficiently in the semisolid medium to obscurethe production of trails. In the investigationthat defined che complementation classes A, B,and C in E. coli, Armstrong and Adler (2)avoided this difficulty by using nonmotile (flaor mot) derivatives of che mutants, chosen ashaving mutations cotransducible with che. Inmost of our experiments we used nonmotilerecipients which, however, were nonflagellatebecause of deletions covering both fla and chegenes. Aswad and Koshland (5) used motileche- recipients (S. typhimurium) made recA-to avoid the swamping of trails by che+ recom-binant swarms and deposited their inocula insemisolid medium instead of on the surface. Inour experiments with motile but che- recipients(Tables 4 and 3, che-107 as recipient), we used afairly stiff semisolid medium and incubatedfor only 8 h at 37°C, at which temperature themedium is semisolid, and then at 20°C to allowcontinued growth of cells in trails without fur-ther spreading. Trails several millimeters long,attributable to che+ abortive transductants,were easily observed in these experiments forall recipients except che-107, inferred to be
dominant. In our few crosses of pairs of che-mutants, we observed some differences in com-plementation according to direction of crossing(Table 4); we do not know how to interpret suchresults and did not extend this part ofour inves-tigation. Parkinson (17, 18) has tested comple-mentation of pairs of che mutants in E. coli bydetermining the chemotaxis phenotype of mer-odiploids carrying an F' factor with a mutatedche region. Nearly all of a large number ofmutants tested could be assigned to one of twomajor complementation groups, A and B, byfailure to complement appropriate tester mu-tants. However, within each major group sev-eral subgroups could be defined by the ability ofmutants of one subgroup to complement mu-tants of another subgroup, a result he attri-butes to intragenic complementation. Aswadand Koshland (5) isolated 72 che mutants of S.typhimurium by a new selection method andassigned them to six classes, mainly by comple-mentation tests. In a continuation of this inves-tigation (H. M. Warrick, B. L. Taylor, and D.E. Koshland, Jr., personal communication),further mutants were isolated and the numberof classes required to accommodate them (andtwo mutants from this laboratory) was in-creased to nine. Crosses of representative mu-tants to S. abortus-equi deletion mutants(Koshland, personal communication; Stocker,unpublished data) indicate that five classes,p, q, r, w, and x, result from mutation be-tween motB and flaC, classes w and p in cheregion I and classes q and r in region II. Class xcomprises only strain SL4041, the ever-tum-bling amber che-ll mutant of Vary andStocker (26). We observed no trails of normallength (and no fast swarms) in crosses of che-111 as donor to deletion mutant fla-234 (Table2) and assigned che-ill to region II. Warrickand his colleagues (personal communication)report production of trails in this cross andtherefore locate che-lil between the ends ofdeletions fla-302 and fla-234 (Fig. 1). The in-complete functional defect ofche-l ll and differ-ent criteria for complementation may accountfor this apparent difference. Koshland and hiscolleagues (5; personal communication) think itlikely that their five complementation classescorrespond to five separate genes in the cheregion. If so, the situation in Salmonella ismore complex than that in E. coli, since Par-kinson (18) interprets his observations as indi-cating the existence of only two genes in thecorresponding region, with much intrageniccomplementation.Mutant che-107, SL2516, was phenotypically
similar to the six other nontumbling chemo-taxis-deficient mutants. Genetic analysis
showed that it differed in two respects, domi-nance and map position. When tested as a re-cipient (on semisolid medium of consistencystiff enough to prevent spreading of Che- Fla+cells) and treated with phage grown on a wild-type donor, it produced no or very few trails(Table 3). Rapid-spreading swarms, i.e., Che+complete transductants, were obtained in thesame crosses, so absence of trails cannot haveresulted from che-107 having two che muta-tions at loci too far apart to be cotransduced.Such failure to produce trails, attributable todominance of endogenote mutant allele overwild-type exogenote, has been reported forsome flaA, flaC, and mot mutants of S. typhi-murium (10, 14), for some mot and che mutantsof E. coli (1, 2, 18), for flaAII and flaN pointmutants in S. abortus-equi (28), and for someche mutants in S. typhimurium (5). A reasona-ble hypothesis is that each gene concerned spec-ifies a protein that polymerizes to produce theeffective product (perhaps one of the compo-nents of the basal body of the flagellum) andthat incorporation of mutant protein (specifiedby a missense allele) along with normal proteininterferes with function. In the cited instancesthe absence of trails, i.e., dominance of mutantallele to wild type, was observed in transduc-tional crosses when the mutant allele was pres-ent in the recipient chromosome but was notdetected in the reverse cross. In our experi-ments using as recipient an Hi (amber) mutantfixed in phase 1, we observed dominance ofexogenote che-107 over its chromosomal homo-logue (inferred to be flaAII+) in that about 85%of abortive motile transductants produced onlynonlinear gorups of microcolonies instead oftrails 2 to 3 mm long, such as were producedwith a che+ donor. The minority of linear trailsevoked by phage grown on che-107 as donormay have been produced by abortive transduc-tants with a chromosome fragment containingan "active" allele of the unlinked flagellin-spec-ifyingH2 gene of the donor, able to replace thefunction of the defective flagellin-specifyinggene, Hi, of the effectively monophasic recipi-ent, or they may have been produced by a mi-nority of abortive transductants containingchromosome fragments carrying Hi + but notthe adjacent che-107. The dominance of endo-genote che-107 over exogenote wild type hasbeen observed also in merodiploids obtained bytransfer of plasmid F'1338, carrying the wild-type hag-fla-mot-che region of E. coli origin(22), to a his mutant of SL2516 che-107 (A. L.Tsui Collins, Ph.D. thesis). We consider belowthe possible nature of the product of the geneaffected by mutation che-107.
Crosses of che-107 to S. abortus-equi mu-tants with deletions in the motB-che-flaCM re-gion (Table 2, Fig. 1) showed that che-107 doesnot map in segment I or II of the che region.The presence or absence of rapid-spreadingswarms in crosses of che-107, as donor or asrecipient, with S. abortus-equi and S. typhimu-rium mutants with deletions of varying extentsin the flaD-flaAI segment (Table 3, Fig. 2)strongly indicates that che-107 is located in ornear the left-hand end of gene flaAII, in eithersegment i or segment ii of this gene (Fig. 1), orpossibly in a previously undetected gene be-tween flaAII and flaAIII or in a previouslyundetected segment of gene flaAIII to the rightof segment iv. The one inconsistent result, pro-duction of a fast-spreading swarm by che-107treated with phage grown onfla-1319, probablyresulted from spontaneous reversion in the re-cipient. Tests of complementation of che-107(Table 3) gave little information because of thedominance of this mutation. However, wethink them compatible with location ofche-107in gene flaAII or flaAIII or between them.Some linear trails noted in crosses to recipientswith deletions in flaAII (Table 3) may haveresulted from the incomplete defect of chemo-taxis function of che-107.Gene flaAII is exceptional in two respects:
first, point mutants as endogenote are domi-nant to wild type (28); second, some point mu-tants in flaAII, though nonmotile, are flagel-late (paralysis or Mot phenotype), whereas oth-ers (and all deletion mutants) are nonflagellate(28). All the known flaAII mutants of Mot phe-notype in S. abortus-equi or S. typhimuriummap in the left-hand end, segments i-iii, of thegene, whereas many point mutants in seg-ments iii-x are nonflagellate (28). If, as wethink, che-107 maps in the left-hand end ofgene flaAII, it appears that mutation in thispart of the gene may cause any of three mutantphenotypes, absence of flagella, presence ofmorphologically normal flagella that are non-functional, or chemotaxis defect, of the non-tumbling type. Normal translational motilityresults from counterclockwise rotation of eachflagellum, and tumbles result from reversal ofrotational direction of one or more flagella (15,23). It is not known how rotation is generated,but the component structures of the basal bodymay be involved. Possibly each such structureis a polymer of a protein specified by a fla (ormot or che) gene. If so, then absence of a certaingene product, resulting from deletion or non-sense mutation, would prevent assembly of thebasal body and so cause the Fla- phenotype(the null phenotype in the sense of Parkinson
[18]); polymerization ofsome abnormal forms ofthe same protein, resulting from certain non-sense mutations, might permit assembly ofbasal body and flagellum but interfere withgeneration or transmission of any rotationalmovement, producing the Mot- phenotype;and polymerization of some other mutated pro-tein might permit assembly and counterclock-wise but not clockwise rotation, so producingthe nontumbling Che- phenotype. Armstrongand Adler (2) reported an E. coli chemotaxis-deficient mutant that was of nontumbling typeand did not fall in complementation classes Aor B and was therefore termed cheC. Parkinson(18) described similar mutants and, despitetheir partial dominance, showed that theyfailed to complement the original cheC mutant.Silverman and Simon (22) found that cheC be-haves as an allele of flaA (of E. coli, corre-spondence to any fla gene ofSalmonella not yetdetermined). In S. typhimurium a class, u, ofchemotaxis-deficient mutant, dominant orpartly dominant, seems to result from mutationat or near flaQ (H. M. Warrick, B. L. Taylor,and D. E. Koshland, Jr., personal communica-tion). A similar explanation may apply to allthese situations.
ACKNOWLEDGMENTSThis investigation was supported by Public Health Serv-
ice research grant A107168 from the National Institute ofAllergy and Infectious Diseases.We thank our colleagues named above for providing
bacterial strains and unpublished information.
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