M the inversion of a single alanine-serine pair, was identified gions and specific residues of ..... Boyd, A., Krikos, A. & Simon, M. I. (1981) Cell 26, 333-343. 16.
Proc. Natl. Acad. Sci. USA Vol. 81, pp. 3287-3291, June 1984
Biochemistry
Structure of the Trg protein: Homologies with and differences from other 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 of Genetics, Department of Biology, Faculty of Science, University of Tokyo, Hongo, Tokyo 113, Japan
Communicated by Julius Adler, February 1, 1984
lyzed by a specific methyl transferase. A specific demethylase removes the methyl ester and also catalyzes a second type of modification, which has recently been shown to be deamidation of specific glutamines to yield glutamates (6). The Tsr protein binds serine, and the Tar protein binds aspartate as well as ligand-occupied maltose-binding protein. Trg mediates response to sugars recognized by the galactose- and ribose-binding proteins, presumably by interaction with the ligand-occupied proteins. Recently, determination of the nucleotide sequences of tsr, tar, and tap from E. coli (5, 7), in conjunction with biochemical characterization of the sites of covalent modification (6, 8, 9), has provided a substantial amount of information about transducer proteins. The data suggest a simple model (5, 7) for organization of a native transducer protein in the cytoplasmic membrane in which the membrane is crossed by two hydrophobic regions, one near the NH2 terminus and the other 40% of the way along the sequence. The NH2-terminal domain between the hydrophobic regions would be on the periplasmic face of the membrane and the region to the COOH-terminal side of the second hydrophobic sequence would be on the cytoplasmic face. This organization is consistent with the known location of the sites of covalent modification in the COOH-terminal region and with the decreased level of sequence homology for the NH2-terminal domains, the regions expected to contain the different ligand-binding sites. The same model is consistent with information 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 functions in excitation and adaptation that are analogous to those performed by other transducers (12-14), yet the trg gene is not as closely related to other transducer genes as they are to each other. Strong hybridization is observed between any pair in the tsr-tar-tap family (15), but hybridization of trg to tar is only marginal under conditions of very low stringency and is not detected at all to tsr (16). Precipitation of Tsr and Tar proteins with anti-Trg antiserum demonstrates that there is at least limited homology between Trg and each of the other transducers of known function (16). In discussing their ideas about the organization of transducer proteins in the membrane, Krikos et al. (5) predicted that the Trg protein would possess considerable amino acid sequence homology with other transducers, and they proposed that the structure of Trg would constitute an important test of their model. We present here the nucleotide sequence of trg. The deduced amino acid sequence of the Trg protein provides strong support for the scheme proposed by Krikos et al. (5). In addition, differences between Trg and the other transducers as well as preliminary mapping of trg mutations that confer spe-
Transducer proteins are central to chemoABSTRACT taxis in Escherichia coli. Three transducer genes comprise a homologous gene family, while a fourth gene, trg, is more distantly related. We have determined the nucleotide sequence of bg. The deduced sequence of the Trg protein has features in common with other transducers as well as regions of significant divergence. The protein sequence suggests the same transmembrane structure postulated for other transducers: an extra cytoplasmic NH2-terminal domain connected by a membrane-spanning region to an intracellular COOH-terminal domain. The COOH-terminal domain of Trg exhibits substantial sequence identity with the corresponding domains of the other transducers, particularly near the sites of covalent modification. Trg appears to have the same five methyl-accepting sites identified in the Tsr protein. Two of those sites are glutamines that are deamidated to yield methyl-accepting glutamates, while the remainder are synthesized as glutamates. Conservation in number but not in position of modified glutamines in Trg compared to the other transducers is consistent with the notion that uncharged glutamines at a specific number of modification sites serve to balance the signaling state of newly synthesized transducers. The N112-terminal domain of Trg exhibits no significant homology with other transducers, implying that trg may be a fusion of the common COOH-terminal transducer sequence with an unrelated NH2-terminal sequence. The location of specific mutations within hg provides support for the suggestion that ligand-binding sites are in the NH2-terminal domains.
Like many cells, motile Escherichia coli can detect changes in the chemical environment and respond appropriately. Central to the bacterial sensory system are the transducer proteins (1-4). These proteins are components in the pathway between ligand-recognition sites and flagellar motors, and are crucial in sensory adaptation. It appears that binding of ligand or ligand-receptor complex to a transducer induces a change in the transducer that, in an unknown manner, creates an excitatory signal received by the flagellar motor. Covalent modification of the excited transducer, which can occur at several specific sites, counteracts the excitatory change and thus results in adaptation to the stimulus. The excitatory state of a transducer is determined by two "signaling" parameters, occupancy of binding site and extent of covalent modification. When the two are imbalanced, the cell is excited; when they are balanced, the cell is adapted. Three functional 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 no tactic sensitivity has yet been linked to this protein (5). These 60-kDa proteins are methylated at specific glutamyl residues to create carboxyl methyl esters in a reaction cataThe publication costs of this article were defrayed in part by page charge
Abbreviations: kb, kilobase(s); kbp, kilobase pairs. §Present address: Department of Medical Biochemistry, University of Geneva, Geneva, Switzerland.
payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 3287
3288
Biochemistry: Bollinger et al.
cific phenotypes allow us to refine and extend the model of transducer organization.
MATERIALS AND METHODS Sequencing. A 2.2-kilobase (kb), Bgl II/Hinand Cloning cII fragment derived from pTH105, a pBR322 hybrid plasmid containing the Sal I/HindIII chromosomal segment of pTH51 (17), was digested with Sau3A or Taq I, and the fragments were ligated to appropriately cleaved M13mp8. In addition, a 1-kb Taq I fragment from pTH105 that contained the Bgl II site was either ligated into M13mp9 or cleaved with 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 kit from New England Biolabs. A 15-base primer or a 16-base reverse-sequencing primer from the same supplier was used. More than 75% of the trg sequence was determined on both strands. Each fragment was sequenced at least twice in separate experiments, and in regions with only one strand available, at least four experiments were done. In Vitro Recombination. Purified restriction fragments of the pBR322 hybrid plasmid pTH105 and of derivatives carrying mutations were obtained by electrophoresis in a 0.7% agarose gel and subsequent electroelution (20). Pvu II digestion produced a 4.9-kilobase-pair (kbp) fragment that contained the origin of replication and bla gene of pBR322 as well as chromosomal DNA including the NH2-terminal 55% of trg, a 1.47-kbp fragment that contained the rest of trg and a 2.2-kbp fragment containing chromosomal and plasmid DNA. The 4.9-kbp fragment from one plasmid was mixed with the 1.47-kbp fragment of a different plasmid, the fragments were ligated with T4 ligase and transformed into a recA trg-l::Tn5 host. Ampicillin-resistant transformants were tested for a complete trg gene, whether wild type or mutant, by examining the ability to form a tactic ring on a ribose swarm plate (14). This was an important consideration, because the probability of correct ligation of the two blunt-ended fragments would be low. In fact, only 2%-3% of ampicillin-resistant transformants were ribose taxis-positive. In mapping trg-103, six of six ribose-taxis-positive transformants containing a hybrid trg gene consisting of a wild-type NH2 terminus and a mutant COOH terminus were galactosetaxis positive, while two of two ribose-taxis-positive transformants with a mutant NH2 terminus and a wild-type COOH terminus were galactose-taxis negative. In mapping trg-104, two of two ribose-taxis-positive transformants containing a hybrid trg gene with the first configuration were galactose-taxis-positive. RESULTS Nucleotide Sequence of trg. The strategy used to sequence trg is outlined in Fig. 1 A and B. The gene was known to reside on a HindIII/Sal I fragment (17). The position of trg was defined within narrow limits by the location of two trg insertion mutations and the observation that deletion of DNA to the left of the Bgl II site did not affect trg expression. Restriction fragments of the Bgl II/HinclI piece were cloned into M13 vectors (18), and the sequences were determined (19). The nucleotide sequence of trg and the corresponding amino acid sequence of the Trg protein are shown in Fig. 2. The beginning of the gene was identified as the only ATG codon between the Bgl II site and the position of the trg-l::TnS insertion that was in phase with the extended open-reading frame. Preceding this ATG, there is a purine-rich region containing two potential 16S rRNA-binding sites (-12 to -10 and -18 to -14). The reading frame is terminated by a TGA codon, which is followed three codons later by a second ter-
Proc. NatL Acad Sci. USA 81
,'
,
B
L'
B Sam 3A Toq I
PHI 1I
Hi
P
B HHi 11
A
S Ikbb
-
n5
(1984)
-
Tn 5
Th/O
-
P
Hi
i I al III 1l KXXXXXX)C
