Ligand-specific activation of Escherichia coli chemoreceptor transmethylation

Abstract

Adaptation in the chemosensory pathways of bacteria like Escherichia coli is mediated by the enzyme-catalyzed methylation (and demethylation) of glutamate residues in the signaling domains of methyl-accepting chemotaxis proteins (MCPs). MCPs can be methylated in trans, where the methyltransferase (CheR) molecule catalyzing methyl group transfer is tethered to the C terminus of a neighboring receptor. Here, it was shown that E. coli cells exhibited adaptation to attractant stimuli mediated through either engineered or naturally occurring MCPs that were unable to tether CheR as long as another MCP capable of tethering CheR was also present, e.g., either the full-length aspartate or serine receptor (Tar or Tsr). Methylation of isolated membrane samples in which engineered tethering and substrate receptors were coexpressed demonstrated that the truncated substrate receptors (trTsr) were efficiently methylated in the presence of tethering receptors (Tar with methylation sites blocked) relative to samples in which none of the MCPs had tethering sites. The effects of ligand binding on methylation were investigated, and an increase in rate was produced only with serine (the ligand specific for the substrate receptor trTsr); no significant change in rate was produced by aspartate (the ligand specific for the tethering receptor Tar). Although the overall efficiency of methylation was lower, receptor-specific effects were also observed in trTar- and trTsr-containing samples, where neither Tar nor Tsr possessed the CheR binding site at the C terminus. Altogether, the results are consistent with a ligand-induced conformational change that is limited to the methylated receptor dimer and does not spread to adjacent receptor dimers.

FIG. 1.

(A) The aligned C termini of native (Tar, Tsr, and Trg) and engineered (trTar and trTsr) MCPs, with the CheR-docking site (NWETF) shown in boldface type. (B) Cartoon that depicts engineered interdimer methylation between homodimers of trTsr and Tar (left and right, respectively). CheR is bound to the Tar dimer at the docking site (depicted as the C-terminal filled rectangle), which is fully amidated (Tar_QQQQ) at the sites of methylation (depicted as filled circles). The trTsr dimer has one site available for methylation (site 3, depicted as an open circle) and is unable to bind CheR.

FIG. 2.

Swarm rates of cells expressing either full-length or truncated serine receptor (Tsr or trTsr) in combination with Trg (A) or Tar (B) on semisolid agar plates. Panel A (left to right): swarm rates of plasmid-containing HCB316 expressing Trg (HCB316/pBR322), Trg and trTsr (HCB316/pJL21), Trg and Tsr (HCB316/pHSe5.tsr_QEQE), and also RP437/pBR322 (wild type for chemotaxis). Open bars, without attractant; striped bars, 100 μM ribose; filled bars, 100 μM serine. Panel B (left to right): swarm rates of plasmid-containing HCB433 expressing Tar and Tap (HCB433/pBR322); Tar, Tap, and trTsr (HCB433/pJL21); Tar, Tap, and Tsr (HCB433/pHSe5.tsr_QEQE); and RP437/pBR322. Open bars, without attractant; striped bars, 100 μM aspartate; filled bars, 100 μM serine. Uncertainties are standard deviations.

FIG. 3.

Tumble frequencies as a function of time after the introduction of 50 μM aspartate (A) or 50 μM serine (B). HCB433/pBR322, □ (Tap and Tar); HCB433/pHSe5.tsr_QEQE, ○ (Tap, Tar, and Tsr); HCB433/pJL21, ▵ (Tap, Tar, and trTsr); HCB433/pBR322 (no attractant control), ▪ (A). The curves drawn through the data are either least-square lines or sigmoid functions to help guide the eyes.

FIG. 4.

SDS-PAGE (12.5% gels) of inner membrane samples expressing Tsr and/or Tar. Lanes: 1, molecular weight markers (in thousands); 2, Tar_QQQQ; 3, coexpressed Tar_QQQQ and trTsr_QQEQ; 4, trTsr_QQEQ; 5, coexpressed trTar_QQQQ and trTsr_QQEQ.

FIG. 5.

Methylation of inner membranes coexpressing Tar and Tsr. (A) Coexpressed Tar_QQQQ and trTsr_QQEQ (filled symbols) and a control sample (coexpressed Tar_QQQQ and trTsr_QQQQ [⋆]). (B) Coexpressed trTar_QQQQ and trTsr_QQEQ (open symbols). ▪ and □, no ligand; • and ○, 1 mM serine; ▴ and ▵, 1 mM aspartate; ▾ and ▿, both serine and aspartate. Reaction conditions included a solution containing 7 μM methylatable receptor (trTsr_QQEQ), 1 μM CheR, and 14 μM [³H]SAM.

FIG. 6.

Methylation rates as a function of the trTsr_QQEQ concentration in trTsr_QQEQ/Tar_QQQQ samples in the absence of ligand (▪) and in the presence of 1 mM serine (•) and in trTsr_QQEQ/trTar_QQQQ samples in the absence of ligand (□) and in the presence of 1 mM serine (○) are shown. Samples at the different trTsr_QQEQ concentrations also included 1 μM CheR and 14 μM [³H]SAM.

FIG. 7.

Illustrations of ligand-induced changes in methylation based on (A) changes in proximity and (B) changes in receptor dimer conformation. Each pair of circles represents a receptor dimer cytoplasmic domain in cross section (Tar_QQQQ, light gray; trTsr_QQEQ, dark gray), where the sites of methylation are depicted as circles on the surface of the domain (available, open circles; blocked, filled circles). Serine (Ser, ⋄) and aspartate (Asp, ) are shown bound to the dimer interface.