Proc. Natl. Acad. Sci. USAVol. 81, pp. 3287-3291, June 1984Biochemistry

Structure of the Trg protein: Homologies with and differences fromother sensory transducers of Escherichia coli

(chemotaxis/receptors/transmembrane protein/protein carboxyl methylation/enzymatic deamidation)

JOHN BOLLINGER*, CHANKYU PARKt, SHIGEAKI HARAYAMA0§, AND GERALD L. HAZELBAUER*t

Programs in *Biochemistry/Biophysics and in tGenetics and Cell Biology, Washington State University, Pullman, WA 99164-4660; and 4iaboratory ofGenetics, Department of Biology, Faculty of Science, University of Tokyo, Hongo, Tokyo 113, Japan

Communicated by Julius Adler, February 1, 1984

ABSTRACT Transducer proteins are central to chemo-taxis in Escherichia coli. Three transducer genes comprise ahomologous gene family, while a fourth gene, trg, is more dis-tantly related. We have determined the nucleotide sequence ofbg. The deduced sequence of the Trg protein has features incommon with other transducers as well as regions of signifi-cant divergence. The protein sequence suggests the sametransmembrane structure postulated for other transducers: anextra cytoplasmic NH2-terminal domain connected by a mem-brane-spanning region to an intracellular COOH-terminal do-main. The COOH-terminal domain of Trg exhibits substantialsequence identity with the corresponding domains of the othertransducers, particularly near the sites of covalent modifica-tion. Trg appears to have the same five methyl-accepting sitesidentified in the Tsr protein. Two of those sites are glutaminesthat are deamidated to yield methyl-accepting glutamates,while the remainder are synthesized as glutamates. Conserva-tion in number but not in position of modified glutamines inTrg compared to the other transducers is consistent with thenotion that uncharged glutamines at a specific number of mod-ification sites serve to balance the signaling state of newly syn-thesized transducers. The N112-terminal domain of Trg exhib-its no significant homology with other transducers, implyingthat trg may be a fusion of the common COOH-terminal trans-ducer sequence with an unrelated NH2-terminal sequence. Thelocation of specific mutations within hg provides support forthe suggestion that ligand-binding sites are in the NH2-termi-nal domains.

Like many cells, motile Escherichia coli can detect changesin the chemical environment and respond appropriately.Central to the bacterial sensory system are the transducerproteins (1-4). These proteins are components in the path-way between ligand-recognition sites and flagellar motors,and are crucial in sensory adaptation. It appears that bindingof ligand or ligand-receptor complex to a transducer inducesa change in the transducer that, in an unknown manner, cre-ates an excitatory signal received by the flagellar motor. Co-valent modification of the excited transducer, which can oc-cur at several specific sites, counteracts the excitatorychange and thus results in adaptation to the stimulus. Theexcitatory state of a transducer is determined by two "signal-ing" parameters, occupancy of binding site and extent of co-valent modification. When the two are imbalanced, the cellis excited; when they are balanced, the cell is adapted. Threefunctional transducers, the products of the genes tsr, tar,and trg, have been identified in E. coli (1-4). A fourth gene,tap, codes for a product with features of a transducer, but notactic sensitivity has yet been linked to this protein (5).These 60-kDa proteins are methylated at specific glutamylresidues to create carboxyl methyl esters in a reaction cata-

lyzed by a specific methyl transferase. A specific demethyl-ase removes the methyl ester and also catalyzes a secondtype of modification, which has recently been shown to bedeamidation of specific glutamines to yield glutamates (6).The Tsr protein binds serine, and the Tar protein binds as-partate as well as ligand-occupied maltose-binding protein.Trg mediates response to sugars recognized by the galac-tose- and ribose-binding proteins, presumably by interactionwith the ligand-occupied proteins.

Recently, determination of the nucleotide sequences oftsr, tar, and tap from E. coli (5, 7), in conjunction with bio-chemical characterization of the sites of covalent modifica-tion (6, 8, 9), has provided a substantial amount of informa-tion about transducer proteins. The data suggest a simplemodel (5, 7) for organization of a native transducer protein inthe cytoplasmic membrane in which the membrane iscrossed by two hydrophobic regions, one near the NH2 ter-minus and the other 40% of the way along the sequence. TheNH2-terminal domain between the hydrophobic regionswould be on the periplasmic face of the membrane and theregion to the COOH-terminal side of the second hydropho-bic sequence would be on the cytoplasmic face. This organi-zation is consistent with the known location of the sites ofcovalent modification in the COOH-terminal region and withthe decreased level of sequence homology for the NH2-ter-minal domains, the regions expected to contain the differentligand-binding sites. The same model is consistent with in-formation about the Tar protein of Salmonella typhimurium(10, 11).The precise relationship of trg to the other transducer

genes has not been clear. The Trg protein performs functionsin excitation and adaptation that are analogous to those per-formed by other transducers (12-14), yet the trg gene is notas closely related to other transducer genes as they are toeach other. Strong hybridization is observed between anypair in the tsr-tar-tap family (15), but hybridization of trg totar is only marginal under conditions of very low stringencyand is not detected at all to tsr (16). Precipitation of Tsr andTar proteins with anti-Trg antiserum demonstrates that thereis at least limited homology between Trg and each of theother transducers of known function (16). In discussing theirideas about the organization of transducer proteins in themembrane, Krikos et al. (5) predicted that the Trg proteinwould possess considerable amino acid sequence homologywith other transducers, and they proposed that the structureof Trg would constitute an important test of their model. Wepresent here the nucleotide sequence of trg. The deducedamino acid sequence of the Trg protein provides strong sup-port for the scheme proposed by Krikos et al. (5). In addi-tion, differences between Trg and the other transducers aswell as preliminary mapping of trg mutations that confer spe-

Abbreviations: kb, kilobase(s); kbp, kilobase pairs.§Present address: Department of Medical Biochemistry, Universityof Geneva, Geneva, Switzerland.

3287

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3288 Biochemistry: Bollinger et al.

cific phenotypes allow us to refine and extend the model oftransducer organization.

MATERIALS AND METHODSCloning and Sequencing. A 2.2-kilobase (kb), Bgl II/Hin-

cII fragment derived from pTH105, a pBR322 hybrid plasmidcontaining the Sal I/HindIII chromosomal segment ofpTH51 (17), was digested with Sau3A or Taq I, and the frag-ments were ligated to appropriately cleaved M13mp8. In ad-dition, a 1-kb Taq I fragment from pTH105 that containedthe Bgl II site was either ligated into M13mp9 or cleavedwith Bgl II or Sau3A and then ligated into the same vector.All restriction enzymes were from New England Biolabs.Recombinant phages were propagated in E. coli JM103

(18) and sequenced by the dideoxy method (19) using a kitfrom New England Biolabs. A 15-base primer or a 16-basereverse-sequencing primer from the same supplier was used.More than 75% of the trg sequence was determined on bothstrands. Each fragment was sequenced at least twice in sepa-rate experiments, and in regions with only one strand avail-able, at least four experiments were done.

In Vitro Recombination. Purified restriction fragments ofthe pBR322 hybrid plasmid pTH105 and of derivatives carry-ing mutations were obtained by electrophoresis in a 0.7%agarose gel and subsequent electroelution (20). Pvu II diges-tion produced a 4.9-kilobase-pair (kbp) fragment that con-tained the origin of replication and bla gene of pBR322 aswell as chromosomal DNA including the NH2-terminal 55%of trg, a 1.47-kbp fragment that contained the rest of trg anda 2.2-kbp fragment containing chromosomal and plasmidDNA. The 4.9-kbp fragment from one plasmid was mixedwith the 1.47-kbp fragment of a different plasmid, the frag-ments were ligated with T4 ligase and transformed into arecA trg-l::Tn5 host. Ampicillin-resistant transformantswere tested for a complete trg gene, whether wild type ormutant, by examining the ability to form a tactic ring on aribose swarm plate (14). This was an important consider-ation, because the probability of correct ligation of the twoblunt-ended fragments would be low. In fact, only 2%-3% ofampicillin-resistant transformants were ribose taxis-positive.In mapping trg-103, six of six ribose-taxis-positive transfor-mants containing a hybrid trg gene consisting of a wild-typeNH2 terminus and a mutant COOH terminus were galactose-taxis positive, while two of two ribose-taxis-positive trans-formants with a mutant NH2 terminus and a wild-typeCOOH terminus were galactose-taxis negative. In mappingtrg-104, two of two ribose-taxis-positive transformants con-taining a hybrid trg gene with the first configuration weregalactose-taxis-positive.

RESULTSNucleotide Sequence of trg. The strategy used to sequence

trg is outlined in Fig. 1 A and B. The gene was known toreside on a HindIII/Sal I fragment (17). The position of trgwas defined within narrow limits by the location of two trginsertion mutations and the observation that deletion ofDNA to the left of the Bgl II site did not affect trg expres-sion. Restriction fragments of the Bgl II/HinclI piece werecloned into M13 vectors (18), and the sequences were deter-mined (19).The nucleotide sequence of trg and the corresponding ami-

no acid sequence of the Trg protein are shown in Fig. 2. Thebeginning of the gene was identified as the only ATG codonbetween the Bgl II site and the position of the trg-l::TnSinsertion that was in phase with the extended open-readingframe. Preceding this ATG, there is a purine-rich region con-taining two potential 16S rRNA-binding sites (-12 to -10and -18 to -14). The reading frame is terminated by a TGAcodon, which is followed three codons later by a second ter-

AHHi B P Hi PHI S

II11 1b

n5- Ikb

,' Tn5 Th/O -

B , P- Hi

B L' III 1l I al i _KXXXXXX)C<XXX)(Xt9 -XXXX )CXX)CO iQ.I,

Sam3A a 1 b-== =1

' kb

Toq I , . 4t " #l

c Trg Protein -6-

Pvu II

tA toL100- 200 300

residue number

400 500

FIG. 1. The trg region and the Trg protein. (A) Restriction mapof the chromosomal insert cloned into pBR322 to create pTH105.The positions of trg-J::Tn5 and trg-2::TnlO are indicated. Sites forHindIII (H), HincIl (Hi), Bgl II (B), Pvu II (P), and Sal I (S) areshown. (B) Sequencing strategy. The Taq I and Sau3A fragmentsthat were sequenced are shown. Arrowheads extend as far as unam-biguous sequence could be determined. (C) Diagram of deducedamino acid sequence of Trg. Areas indicated are hydrophobic re-gions (filled boxes) bracketed by hydrophilic (vertical line) or basic(vertical line with "+") residues; region of most extensive residueidentity (open box), and the K1 (open box including position 300)and R1 (open box including position 500) peptides with methyl-ac-cepting sites (vertical lines with asterisks).

mination codon. Translation of the putative trg sequenceyields a protein of 535 amino acids with a calculated size of57,965 Da, which is consistent with the apparent molecularsize of the Trg protein estimated using NaDodSO4/poly-acrylamide gel electrophoresis (13, 21). The translated pro-tein contains an excess of six negatively charged amino acidsas well as twelve histidines. Thus, the predicted pI would beclose to the pK of histidine, a value consistent with the ob-served pI of -6.5 for the Trg protein (21).Amino Acid Sequence of Trg. A number of distinct regions

can be identified along the deduced sequence of the Trg pro-tein (Fig. 1C). These regions correspond to similar regions ofthe other transducer proteins (5, 7). There are only twostretches of exclusively hydrophobic amino acids at least 10residues long. A hydrophobic region of 28 residues occursnear the NH2 terminus from amino acid 17 to amino acid 44and one of 25 residues between amino acids 197 and 221. Assuggested in consideration of the analogous regions of theother transducer proteins (5, 7, 10), the hydrophobic se-quences are likely to serve as membrane-spanning regions,probably assuming a-helical structures. Since the NH2 ter-mini of Tsr, Tar, and Tap strongly resemble the precursorsequences of exported proteins, it was possible that trans-ducer proteins were proteolytically processed (5, 7). Howev-er, the stoichiometry of [35S]methionines in the Tar proteinof S. typhimurium suggested that the NH2-terminal hydro-phobic region of that protein is not cleaved (10). The Trgsequence includes a single cysteine, which is within theNH2-terminal hydrophobic region (Fig. 2). [35S]Cysteine isstably incorporated into Trg protein (D. Nowlin, personalcommunication), indicating that the NH2-terminal hydro-phobic region is an integral part of the mature transducer.For the Tsr, Tar, and Tap proteins, the second hydrophobicregion separates NH2-terminal regions of moderate se-quence identity between any pair (10% to 60% over 29 resi-due sequences) and the COOH-terminal regions of high(60%-100%) sequence identity (7). The pattern of homologybetween the Trg sequence and any of the other transducerproteins exhibits the same separation of regions. There is nostatistically significant homology for the NH2-terminal do-mains but substantial (40%-90%) sequence identity betweenthe COOH-terminal domain of Trg and the corresponding re-gion of each of the other transducers. The pattern is illustrat-ed in Fig. 3, which presents the Trg sequence using the one-

Proc. NatL Acad Sci. USA 81 (1984)

Proc. Natl. Acad. Sci. USA 81 (1984) 3289

GATCATAAGTAATTACCGTCAAGTGCCGATGACTTTCTATCAGGAGTAAACCTGGACGAGAGACAACGGTA

ATG AAT ACA ACT CCC TCA CAG CGA TTA GGT TTT TTG CAT CAC ATC AGG TTG GTT CCG TTA TTT GCC TGC ATT CTA GGC GGT ATC TTA GTT

Met Asn Thr Thr Pro Ser Gln Arg Leu Gly Phe Leu His His Ile Arg Leu Val Pro Leu Phe Ala Cys Ile Leu Gly Gly Ile Leu Val

31CTA TTC GCA TTA AGT TCA GCC CTG GCT GGC TAT TTC CTC TGG CAG GCC GAT CGC GAT CAG CGT GAT GTT ACT CGC GAG ATT GAG ATC CGG

Leu Phe Ala Leu Ser Ser Ala Leu Ala Gly Tyr Phe Leu Trp Gln Ala Asp Arg Asp Gln Arg Asp Val Thr Ala Glu Ile Glu Ile Arg61

ACC GGG TTA GCG MC AGT TCA GAT TTT TTG CGT TCA GCC CGG ATC MT ATG ATT CAG GCC GGG GCT GCG AGT CGT ATT GCG GM ATG GMThr Gly Leu Ala Asn Ser Ser Asp Phe Leu Arg Ser Ala Arg Ile Asn Met Ile Gln Ala Gly Ala Ala Ser Arg Ile Ala Glu Met Glu91

GCA ATG MG CGA AAT ATT GCG CM GCC GM TCG GAG ATT AAA CAG TCG CAG CM GGT TAT CGT GCT TAT CAG MT CGA CCG GTG AM ACAAla Met Lys Arg Asn Ile Ala Gln Ala Glu Ser Glu Ile Lys Gln Ser Gln Gln Gly Tyr Arg Ala Tyr Gin Asn Arg Pro Val Lys Thr

121CCT GCT GAT GM GCC CTC GAC ACT GM TTA AAT CM CGC TTT CAG GCT TAT ATC ACG GGT ATG CM CCT ATG TTG MA TAT GCC MA MT

Pro Ala Asp Glu Ala Leu Asp Thr Glu Leu Asn Gln Arg Phe Gln Ala Tyr Ile Thr Gly Met Gln Pro Met Leu Lys Tyr Ala Lys Asn

151GGC ATG TTT GM GCG ATT ATC MT CAT GM AGT GAG CAG ATC CGA CCG CTG GAT MT GCT TAT ACC GAT ATT TTG MC MA GCC GTT MGGly Met Phe Glu Ala Ile Ile Asn His Glu Ser Glu Gln Ile Arg Pro Leu Asp Asn Ala Tyr Thr Asp Ile Leu Asn Lys Ala Val Lys181ATA CGT AGC ACC AGA GCC MC CM CTG GCG MC TTG GCC CAT CAG CGC ACC GCC TGG GTG ATG TTC ATG ATC GGC GCG TTT GTG CTT GCC

Ile Arg Ser Thr Arg Ala Asn Gln Leu Ala Asn Leu Ala His Gln Arg Thr Ala Trp Vai Met Phe Met Ile Gly Ala Phe Val Leu Ala

211CTG GTC ATG ACG CTG ATA ACA TTT ATG GTG CTA CGT CGG ATC GTC ATT CGT CCA CTG CM CAT GCC GCA CM CGG ATT GM MA ATC GCCLeu Val Met Thr Leu Ile Thr Phe Met Val Leu Arg Arg Ile Val Ile Arg Pro Leu Gln His Ala Ala Gln Arg Ile Glu Lys Ile Ala

241AGT GGC GAT CTG ACG ATG MT GAT GM CCG GCG GGT CGT MT GM ATC GGT CGC TTA AGT CGT CAT TTA CAG CM ATG CAG CAT TCA CTGSer Gly Asp Leu Thr Met Asn Asp Glu Pro Ala Gly Arg Asn Glu Ile Gly Arg Leu Ser Arg His Leu Gln Gln Met Gln His Ser Leu271GGG ATG ACA GTA GGG ACT GTT CGA CAG GGC GCG GMA GAG ATT TAT CGT GGC ACC AGC GM ATT TCA GCT GGC MT GCG GAT CTG TCA TCTGly Met Thr Val Gly Thr Val Arg Gln Gly Ala Glu Glu Ile Tyr Arg Gly Thr Ser Glu Ile Ser Ala Gly Asn Ala Asp Leu Ser Ser

301CGC ACC GM GM CM GCG GCG GCT ATC GM CM ACT GCC GCC AGC ATG GAG CM CTC ACT GCG ACG GTG MA CAG MT GCG GAT MC GCGArg Thr Glu Gin Gln Ala Ala Ala Ile Glu Gln Thr Ala Ala Ser Met Glu Gln Leu Thr Ala Thr Val Lys Gln Asn Ala Asp Asn Ala

331CAT CAT GCC AGC MA CTG GCG CM GAG GCT TCT ATT AM GCC AGC GAT GGC GGG CAG ACG GTT TCC GGT GTA GTA AM ACG ATG GGC GCT

His His Ala Ser Lys Leu Ala Gln Glu Ala Ser Ile Lys Ala Ser Asp Gly Gly Gln Thr Val Ser Gly Val Val Lys Thr Met Gly Ala

361ATC TCC ACG AGT TCG MG MA ATT TCT GAG ATC ACC GCC GTC ATC MC AGT ATT GCT TTC CAG ACG AAT ATT CTG GCA CTG MT GCT GCC

Ile Ser Thr Ser Ser Lys Lys Ile Ser Glu Ile Thr Ala Val Ile Asn Ser Ile Ala Phe Gln Thr Asn Ile Leu Ala Leu Asn Ala Ala

391GTT GM GCC GCG CGC GGT GAG CM GGG CGT GGA TTT GCC GTT GTC GCC AGC GM GTA CGG ACA CTC GCA AGT CGC AGC GCT CAG GCG GCGVal Glu Ala Ala Arg Gly Glu Gln Gly Arg Gly Phe Ala Val Val Ala Ser Glu Val Arg Thr Leu Ala Ser Arg Ser Ala Gin Ala Ala

421MA GAG ATT GAA GGC TTG ATC AGT GM TCA GTC AGG TTA ATT GAC CTG GGG TCG GAT GAG GTG GCC MC GGC GGG AM ACC ATG AGC ACTLys Glu Ile Glu Gly Leu Ile Ser Glu Ser Val Arg Leu Ile Asp Leu Gly Ser Asp Glu Val Ala Asn Gly Gly Lys Thr Met Ser Thr451ATT GTT GAT GCC GTC GCG AGT GTC ACA CAT ATC ATG CAG GAA ATC GCC GCC GCC TCG GAT GAM CM AGT AGA GGC ATA ACG CAG GTT AGCIle Val Asp Ala Val Ala Ser Val Thr His Ile Met Gln Glu Ile Ala Ala Ala Ser Asp Glu Gln Ser Arg Gly Ile Thr Gln Val Ser481CAG GCG ATC TCT GM ATG GAT AAG GTG ACG CAA CAG MT GCT TCT CTG GTA GM GAG GCC TCA GCG GCG GCG GTG TCC CTT GM GAA CAGGln Ala Ile Ser Glu Met Asp Lys Val Thr Gln Gln Asn Ala Ser Leu Val Glu Glu Ala Ser Ala Ala Ala Val Ser Leu Glu Glu Gln511GCG GCA CGA TTA ACT GAG GCG GTG GAT GTA TTC CGT CTG CAC AAA CAT TCT GTG TCG GCA GM CCT GCG GAG CGG TGA ACCAGTTAGTTTCGCAla Ala Arg Leu Thr Glu Ala Val Asp Val Phe Arg Leu His Lys His Ser Val Ser Ala Glu Pro Ala Glu Arg ---

TACGGTGTGAAAATGTTAAGGAGATCGA

letter code for amino acids. The hydrophobic regions as well gins 148 residues from the eas the sites of methylation (see below) are indicated. sequence. The corresponding

Sites of Covalent Modification. Like Tsr and Tar (8, 9), the gous to this "highly conserv

Trg protein contains multiple methyl-accepting sites and the 45 positions, including a s

multiple sites for CheB-dependent deamidation (17, 22). A In the COOH-terminal dom;methionine- and lysine-containing tryptic peptide of Trg con- quences exhibit residue identtains two sites for the CheB-dependent modification and at the end of the hydrophobic r(

least two methyl-accepting sites (22). This peptide, called ment 302 amino acids long. I

K1, is closely related in chemical properties to the analogous of these positions. It seems

K1 peptide of Tsr (8, 9). The methyl-accepting sites (9) and tions (Fig. 3) are of critical inamino acid sequence (7) of the K1 peptide of Tsr have been tions performed by the COOdetermined and an almost identical peptide, differing only in Mapping trg Mutations. Mthe inversion of a single alanine-serine pair, was identified gions and specific residues offor the Tar protein of E. coli (5) and of S. typhimurium (10). for each of the functions peThe methyl-accepting sites are at positions of glutamates, or have isolated trg mutants witof glutamines that are apparently deamidated before serving mutations were generated byas methyl-accepting residues. In Fig. 4A, the sequences of with N-methyl-N'-nitro-N-nthe Tsr and Tar K1 peptide are compared to the correspond- by surveying for defective bing sequence of Trg (amino acids are represented by stan- (unpublished data). Two m

dard one-letter abbreviations). All three of these sequences drastically decreased respon

are precisely the same number of residues from the end of a significant effect on resporthe second hydrophobic sequence. In the Trg sequence, at ation in unstimulated cells o

the positions corresponding to the methyl-accepting sites of tion by ribose were indistinTsr, there are two glutamines and one glutamate, correlating sponding wild-type cells, whwell with the previous identification of two CheB-dependent sulted in little change in medeamidation sites and at least two methyl-accepting sites on must affect a region of the Ithe Trg peptide (22). A second, dimethylated peptide has tactic response to galactose talso been identified among Trg tryptic fragments (22), and simple idea is that the defecFig. 4A shows that this peptide is likely to correspond to the ligand-occupied galactose-bi

R1 peptide that was previously characterized as dimethylat- site is at least partially separzed in Tsr (8, 9) and monomethylated in Tar (8, 9, 11). with ribose-binding protein.Homologous Regions. The longest uninterrupted stretch of these mutations are located

amino acid identity for the Tsr, Tar, and Tap sequences be- by in vitro recombination us

FIG. 2. Nucleotide se-

quences of trg. The sequence in-cludes nucleotides from the firstSau3A site to the final Taq I siteshown in Fig. 1. The amino acidsequence of the primary geneproduct, derived from the DNAsequence, is also shown. Therewere four places in the sequencethat were beginnings or ends offragments generated by Sau3Aand cloned into the M13mp8BamHI site, yet did not yieldthe expected G-A-T-C sequenceeither in the sequencing of thatparticular fragment or in the se-

quencing of fragments in whichthose positions were internal.We feel that the combination ofconsiderations of enzyme speci-ficity and of base pairing re-

quired for cloning argue that thecorrect sequences are all G-A-T-C, and they are so listed. For-tunately, the ambiguities wereall in third base positions that donot alter the amino acid calledfor by the codon. The question-able positions are 177, C/T; 612,C/T; 891, T/C; and 1449, C/T.

nd of the second hydrophobicg region of Trg is quite homolo-,ed" region but diverges at 8 of;ingle residue deletion (Fig. 4C).ain, the Tsr, Tar, and Tap se-

tity at 181 positions beginning ategion and extending over a seg-'he Trg sequence diverges at 27likely that the conserved posi-mportance in the common func-)H-terminal domain.Vith the goal of identifying re-f the Trg protein that are criticalrformed by the transducer, weh subtle or unusual defects. Themutagenesis of the cloned gene

iitrosoguanidine and identified'ehavior without prior selectionMutations, trg-103 and trg-104,se to galactose but did not havense to ribose. Levels of methyl-)r in adapted cells after stimula-iguishable from those in corre-iile stimulation by galactose re-

sthylation. Thus, the mutationsrrg protein that is necessary forbut not for response to ribose. ASt is in the site where Trg bindsnding protein, implying that this'able from the site for interactionWe have shown that both of

in the NH2-terminal half of trgsing the single Pvu II site in trg

Biochemistry: Bollinger et al.

3290 Biochemistry: Bollinger et al.

50MNTTPSORLGFLHHnIRLVP;*FAC IIG L'kFALESALAGYFLWQADRDQRDVTAEI EI R

3aa 100* * ~~~~~~gap.*

TG3ANSSDFLRSARI NMIQAGAASRIAEMEAMKRNI'AQAESEI KQS0GGYRAYQNRPVKT

ISO* * * 0* 0

PADEALDTELNORFQAYITGMOPMLKYAKNGMFEAI I NHESEOI RPLDNAYTDILNKAVK

200* * 0 0 . 0

I RSTRANQLANLAHQRTAWVMFMI GAFVLALVMTLITFMVLRRIV RKOQHAAQRI EKIFA250 300

SGDQTMNDEPA&NI GRLSRHLQG"HSffGGTI&EEIYRSS iS

350

RTpAI E QWA rELY VN gK OQE& P8SDOOQTMSGVVKW#3Agap 400

D]STiKHSEETAE NS|I AOFOTN LALNAA' GFAVVCQGRtKNVA|I450

_CEG13 SEARL D ANG G T HLTHENA4;iSHTd500

* 9 0 0

QISSS4V;TQQNRA SaAS A&VS3Etk" TE_&VHKHSVSAEPAER* *

FIG. 3. Comparison of the Trg sequence to the sequences of Tsr,Tar, and Tap. The Trg sequence was matched with the maximal ho-mology alignment of Tsr, Tar, and Tap (5) by aligning the Trg resi-due (R-16) preceding the first hydrophobic region and following thesecond hydrophobic region (R-222) with the corresponding positionsin the Tsr-Tar-Tap alignment. Maximum sequence identity wasachieved by introducing into the Trg sequence a 3-residue gap afterposition 96 [corresponding to gaps introduced in Tar and Tap (5)]and a 1-residue gap after position 395. Boxes are placed around posi-tions at which the identical amino acid is found in all four transduc-ers from E. coli. Underlined positions are those at which the Trgresidue matches the amino acid in two other transducer sequences.Hydrophobic regions are indicated by horizontal lines, and methyl-accepting sites are indicated by vertical lines with asterisks.

located just past the middle of the gene (Fig. 1C). Specifical-ly, we constructed pBR322-derived plasmids carrying hybridtrg genes in which the trg DNA on one side of the Pvu II sitewas from the wild-type gene and the DNA on the other sidewas from a mutant gene. Neither mutation affected ribosetaxis, so plasmids carrying a complete trg, whether mutantor wild type, were identified by their ability to confer a ri-bose-taxis-positive phenotype on a Trg-negative host trans-formed with the plasmid. Ribose-taxis-positive transfor-mants were then examined for tactic response to galactose.The hybrids in which the NH2-terminal half of trg was fromthe wild-type gene and the COOH-terminal half was fromeither mutant gene restored galactose taxis in a transformedhost containing a null mutation in the chromosomal copy oftrg. Hybrids of the opposite configuration did not restoregalactose taxis. Thus both- mutations were located to theNH2-terminal side of the Pvu II site. It has been suggestedthat because the NH2-terminal domains of the transducersdiverge in amino acid sequences, these regions are likely tocontain the ligand-recognition sites and thus be exposed onthe periplasmic face of the cytoplasmic membrane (5). Ourobservations substantially strengthen that idea.

DISCUSSIONThe observations reported here provide strong support forthe model of transmembrane disposition of transducer pro-teins proposed by Krikos-et al. (5). The amino acid sequenceof Trg exhibits the same general features found in the pri-mary sequences of other transducers; thus, the model basedon those features also applies to Trg. Mapping of galactose-taxis negative, ribose-taxis-positive mutations in the firsthalf of trg supports the idea that the divergent, NH2-terminaldomains contain the different ligand-binding sites. The ex-

A. Methyl - Accepting Regions

KI Peptide RI Peptide

TsrZ/Tar TE A LA AASMEQLTATVK V NLV ;S AAATrg TEAI EN AASMEQLTATVK VTOQNASLVEEASAVS EEQAF

B. Amino Terminal Regions

Top MF RIRI T FLIL ILCGI 0QI GMS FWAFRTsr M RIIVT L L VFGLL T G FFNALKTar _IR VT MVLGVFAL QLII SL F S SLHTrg MNTTPSQRLGFLHHI LFACI LGILVgAL S AGYFLWQ

Tsr C. "Highly Conserved" Regions

Tapr lDIIS1DGIAFQTNILALNAAVEAARWGEQGRGFAVVAS VLTrg ETSIAFOTNIILALNAAVEAARI EOGRGFAVVASVT

FIG. 4. Comparison of selected regions of the four transducers.Boxes indicate positions at which the sequences compared containidentical residues. (A) Methyl-accepting regions. At the positionswhere the Tsr and Tar sequences differ, the upper letter is the Tsrresidue and the lower letter is the Tar residue. The positions ofmethyl-accepting sites in Tsr (6) are indicated by asterisks. Themethyl-accepting positions at which Trg contains a glutamate inplace of a glutamine or vice-versa are indicated by arrows. (B) Ami-no terminal regions. Sequences were aligned using the basic residuethat precedes the extended hydrophobic region. (C) Highly con-served regions. The longest sequence of residue identity for Tsr,Tar, and Tap is compared to the corresponding sequence of Trg. Asingle residue gap is included in the Trg sequence.

tensive homology among the COOH-terminal domains of allfour transducers argues strongly for a common evolutionaryorigin. Comparison of COOH-terminal domains reveals thatTrg is not as related to Tsr, Tar, or Tap as those proteins areto each other, but that Trg is slightly more related to Tsr andTar, the transducers of known function, than it is to Tap.Slight differences in extent of homology are also observed incomparison of the nucleotide sequences and, as discussedelsewhere (16), appear to account for detection of trg:tar butnot trg:tsr hybridization.A combination of biochemical (6, 8, 9) and nucleotide se-

quence (7) data for the K1 peptide of Tsr has provided per-suasive evidence that the CheB-dependent modification isdeamidation of specific glutamine residues that then serve asmethyl-accepting glutamates. However, the amino acid (6)and nucleotide (7) sequence data indicate that two of thethree (or four) methyl-accepting sites, all of which can bemethylated on a single molecule, are deamidated glutamines(Fig. 4A), while analysis by high-performance liquid chroma-tography detected only one CheB-dependent modificationon the Tsr (or Tar) K1 peptide (7, 8). This conflict of datadoes not occur when the K1 peptide of Trg is analyzed. Twodeamidation sites are indicated, both by the nucleotide se-quence (this study) and by analysis of the peptide using high-performance liquid chromatography (22). The K1 peptide ofTrg differs from the corresponding Tsr and Tar peptides byonly four conservative substitutions in the amino acid se-quence, and so it seems likely that the pattern of covalentmodification is the same for all three proteins. Thus, we pre-sume that double CheB-dependent modification observedfor the K1 peptide of Trg also occurs in Tsr and Tar butminor chemical differences between the peptides or their ori-gins (minicells versus UV-irradiated phage-infected cells) re-sulted in peptides in which the double deamidation was un-detectable for Tsr and Tar.The functional significance of the CheB-dependent deami-

dation is not known. A simple question is: if the glutaminesmust be changed to glutamates to serve as methyl-acceptingsites, and three of the five identified sites are already gluta-

Proc. NatL Acad Sci. USA 81 (1984)

Proc. NatL. Acad. Sci. USA 81 (1984) 3291

mates in the newly synthesized protein, then why are not allof the sites glutamates to begin with? There must be a pres-sure that maintains the two glutamine codons. Comparisonof the methyl-accepting sites in the K1 peptide of Trg to the

corresponding sites in the other transducers provides an in-

teresting hint about the nature of that pressure. In Tsr, Tar,and Tap, the first and third methyl-accepting sites are also

deamidation sites, but the middle methyl-accepting site is

not (in Tap, the middle position is not preceded by a gluta-mate, so it is probably not modified at all). One interpreta-tion of this pattern is that there is a specific functional re-

quirement for the middle site of modification to be a gluta-mate, not a glutamine, and for the two flanking sites to be

synthesized as glutamines. The Trg sequence argues againstthat idea, because the first site is a glutamate, not a gluta-mine, and the middle site is a glutamine, not a glutamate.However, it is striking that the total number of glutamines at

functional modification sites is the same for all the transduc-

ers. This conservation can be interpreted in the context of

the idea, introduced to us by F. W. Dahlquist, that mainte-

nance of the adapted state in actively growing cells requiresthat newly synthesized transducers are in a signaling statebalanced between the extremes. A number of observations(23) indicate that the signaling state of the methyl-acceptingdomain of a transducer is determined by the number of nega-tive charges (glutamate residues) versus neutral groups (glu-tamyl methyl esters or glutamines) at the modification sites.In a rapidly growing unstimulated cell, entrance of a substan-tial proportion of newly synthesized unmethylated mole-cules into the transducer pool could shift the behavior of thecell toward exclusive counterclockwise rotation if all siteswere charged. Instead, the presence of glutamines at twosites means that newly synthesized transducers are in a sig-naling state much closer to the usual balance of an unstimu-lated or adapted cell.The NH2-terminal domains of Tsr, Tar, and Tap are signif-

icantly less related to each other than are the correspondingCOOH-terminal domains, but sufficient homology is evidentto indicate that like the COOH-terminal domains, the NH2-terminal domains of all three proteins are derived from a

common ancestor (5, 7). In contrast, when the sequence ofthe NH2-terminal domain of Trg is compared to the othertransducers, the number of amino acid identities is onlyslightly greater than expected by chance. Most of the identi-ties are in or near the NH2-terminal hydrophobic region andthus may reflect a common function in membrane localiza-tion rather than a common evolutionary origin. In fact, theTrg sequence at the extreme NH2 terminus is quite differentfrom the closely related sequences of the other three trans-ducers (Fig. 4B). For those transducers, the first 34 residuesexhibit a substantial number of amino acid identities, and thesequences are very similar to precursor sequences found on

exported proteins (5, 7, 10). Although Trg has a region ofhydrophobic amino acids near the NH2 terminus, it is notprecisely the same length as that found in the other transduc-ers and is preceded by a substantial length of polypeptidewith an amino acid sequence quite different from those ob-served for the other transducers or for precursor sequences(Fig. 4B). Thus, there is no significant indication that theNH2-terminal domain of Trg is derived from the same originas the other transducers. Instead, it is quite possible that trgoriginated as the fusion of a sequence coding for the COOH-terminal domain common to all transducers and a gene or

gene fragment coding for a ligand-recognition domain unre-

lated to other transducers. The origin of this fragment is un-

known, but a likely candidate would be a gene coding for a

protein that interacted with one of the sugar-binding proteinsin the process of transport. Unfortunately, sequences of therelevant components of the ribose or galactose transport sys-tems are not available. However, sequences are available forall the components of the transport system of S. typhimur-ium, which utilizes the histidine-binding protein (24). One ofthose components, the HisM protein, contains at the NH2terminus a 28-residue hydrophobic region preceded by a 17-residue hydrophilic region, a pattern that corresponds to theTrg NH2 terminus with a hydrophobic region of the same

length preceded by a 16-residue hydrophilic region. Thereare few identical residues in the two sequences, but the simi-larity in the general organization is tantalizing.

We thank E. Kofoid and J. S. Parkinson for advice about DNAsequencing. This work was supported by grants from the McKnightFoundation (to G.L.H.); the U.S. Public Health Service (GM 29963to G.L.H.); and the Ministry of Education, Sciences and Culture ofJapan (to S.H.).

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Biochemistry: Bollinger et aL