TrkH and its homolog, TrkG, determine the specificity and kinetics of cation transport by the Trk system of Escherichia coli

Abstract

The corrected sequence of the trkH gene of Escherichia coli predicts that the TrkH protein is a hydrophobic membrane protein of 483 amino acid residues, of which 41% are identical to those of the homologous and functionally analogous TrkG protein. Four proteins (TrkA, TrkE, TrkG, and TrkH), which are the products of widely separated genes, have been implicated as key components of Trk, the major constitutive K ϩ transport system of Escherichia coli (for a review, see reference 2). Of these four proteins, only TrkA, a 50.4-kDa peripheral membrane protein that binds NAD and is devoid of predicted membrane-spanning regions (19), is absolutely required for Trk activity. Either the TrkG or the TrkH product is necessary for activity; only when both are mutated is Trk activity abolished. The TrkE protein, which is encoded by the E. coli homolog of the sapD gene of Salmonella typhimurium (12, 13), is required when only TrkH is present. When TrkG is present, loss of TrkE alters the kinetics of transport, with a significant reduction in transport rate (3, 7).The fact that either TrkG or TrkH is required for Trk activity suggests that these two proteins perform similar functions in Trk and should therefore resemble each other. This expectation is confirmed by the DNA sequence of the trkH gene and the predicted structure of its product. Here, we report that these two proteins are homologous and that over 40% of their amino acid residues are identical.We were alerted to the initial report of the sequence of the trkH gene by Kenneth Rudd (17), who noticed that an open reading frame of 421 amino acids (11) was similar to TrkG. A subsequent report of DNA sequence data for this region indicated a slightly longer open reading frame of 431 amino acids (5). We found that the location of this open reading frame agreed perfectly with our transductional mapping of trkH (7) and that the sequence agreed with those of the restriction sites we had mapped in plasmid pWE211, which carries trkH (6, 7). However, it was obvious that the gene must be larger than that predicted from the published sequences, since the homology of TrkH with TrkG would end in the middle of a predicted membrane-spanning region. Further, plasmids that we constructed that ended at amino acid residue 458 did not complement trkH mutations, whereas plasmids carrying an additional 180 bp of distal sequence did complement trkH mutations. It seemed most likely that the published sequences had missing or extra bases toward the end of the gene, resulting in an erroneous frameshift.We have resequenced the distal part of the gene as well as other selected regions using the T7 sequencing system (Sequenase; U.S. Biochemicals) with double-stranded DNA. We used dITP in place of dGTP when band compression was a problem. Artifactual bands due to nonspecific termination were eliminated by treatment with terminal transferase (8).Our sequence data reveal the protein to have 483 residues with homology to trkG extending over the full lengths of the two proteins (Fig.

A Novel Sensor Kinase Is Required for Bordetella bronchiseptica To Colonize the Lower Respiratory Tract

“…BB0262 is predicted to encode a polypeptide with homology to 16S rRNA methyltransferases; BB0263 is predicted to encode an exported polypeptide of unknown function; and the BB0266 and BB0267 ORFs are similar to trkA and trkH, respectively, from several Gram-negative bacteria (3). In E. coli, trkA and trkH are involved with low-affinity transport of potassium into the cell (6,57). The expression of this transcript presumably is controlled by a promoter immediately 5Ј to BB0262, on the basis of the fact that the ORF that resides 5Ј to BB0262 is located 120 bp away and is divergently transcribed (47).…”

Metabolic Interdependence of Obligate Intracellular Bacteria and Their Insect Hosts

“…TrkA is a peripheral membrane protein bound to the cytoplasmic side of the membrane and is essential for transport activity. TrkH and TrkG are the K ϩ -translocating subunits, and TrkE seems to be involved in energy transfer (106). Interestingly, in Wigglesworthia only TrkA and TrkH are conserved, posing the question whether this system is functional.…”

Change to alanine of one out of four selectivity filter glycines in KtrB causes a two orders of magnitude decrease in the affinities for both K+ and Na+ of the Na+ dependent K+ uptake system KtrAB from Vibrio alginolyticus

“…Jan and Jan [2] have proposed that the K translocating subunit TrkG from one of the two Escherichia coli Trk K uptake systems also contains two P-loop regions. Subsequently it was recognized that TrkG belongs to a broad family of K symporter proteins [3^5], consisting of HKT1 from plants [6], Trk1 and Trk2 from fungi [7,8], KtrB (NtpJ) from bacteria [3,9], and TrkH (TrkG) from prokaryotes [10,11]. Independently, the Durell-Guy group and we recognized that these proteins consist of a basic structure of four consecutive M1-P-M2 motifs ( [5,12], see Fig.…”