An archaeal photosignal-transducing module mediates phototaxis in Escherichia coli

Abstract

Halophilic archaea, such as Halobacterium salinarum and Natronobacterium pharaonis, alter their swimming behavior by phototaxis responses to changes in light intensity and color using visual pigment-like sensory rhodopsins (SRs). In N. pharaonis, SRII (NpSRII) mediates photorepellent responses through its transducer protein, NpHtrII. Here we report the expression of fusions of NpSRII and NpHtrII and fusion hybrids with eubacterial cytoplasmic domains and analyze their function in vivo in haloarchaea and in eubacteria. A fusion in which the C terminus of NpSRII is connected by a short flexible linker to NpHtrII is active in phototaxis signaling for H. salinarum, showing that the fusion does not inhibit functional receptor-transducer interactions. We replaced the cytoplasmic portions of this fusion protein with the cytoplasmic domains of Tar and Tsr, chemotaxis transducers from enteric eubacteria. Purification of the fusion protein from H. salinarum and Tar fusion chimera from Escherichia coli membranes shows that the proteins are not cleaved and exhibit absorption spectra characteristic of wild-type membranes. Their photochemical reaction cycles in H. salinarum and E. coli membranes, respectively, are similar to those of native NpSRII in N. pharaonis. These fusion chimeras mediate retinal-dependent phototaxis responses by Escherichia coli, establishing that the nine-helix membrane portion of the receptor-transducer complex is a modular functional unit able to signal in heterologous membranes. This result confirms a current model for SR-Htr signal transduction in which the Htr transducers are proposed to interact physically and functionally with their cognate sensory rhodopsins via helix-helix contacts between their transmembrane segments.

FIG. 1

Construction of the fusion proteins. Numbers indicate numbers of residues in the indicated segment. P, G, and M fusions (named for the final residue in the NpHtrII portion at the junctions) are defined by the different junctions between the haloarchaeal and eubacterial protein domains.

FIG. 2

Flash-induced absorption difference transients. Absorption transients of the halobacterial (A) and E. coli (B) membranes were measured at 400, 500, and 550 nm at 5-ms time resolution. Membranes containing the halobacterial NpSRII-NpHtrII fusion protein were resuspended at a concentration of 0.12 mg of protein/ml in a solution containing 4 M NaCl and 25 mM Tris, pH 6.8, and membranes containing the E. coli NpSRII-NpHtrII-StTar fusion were resuspended at a concentration of 0.11 mg of protein/ml in a solution containing 1% octyl-glucoside, 100 mM NaCl, and 100 mM MES, pH 6.8. The half-life values for O-rise and decay are, respectively, 29 and 362 ms for the NpSRII-NpHtrII fusion and 50 and 365 ms for the NpSRII-NpHtrII-StTar fusion.

FIG. 3

Expression of the His-tagged fusion proteins in H. salinarum (A) and E. coli (B) analyzed by SDS–8% PAGE. SM, solubilized membrane; FL, flowthrough after sample was bound to the resin; EL, eluate with imidazole. A comparable amount of protein (10 μg) eluted by imidazole was loaded in each case and was taken from the same samples that were used for the absorption spectra measurement of Fig. 5. An equal number and 2.5× the number of cell equivalents in the samples were loaded in the eluate lanes of panels A and B, respectively.

FIG. 4

Immunoblot analysis of the His-tagged fusion proteins in E. coli. An identical amount of membrane protein in each lane (4 μg) was separated by SDS–12% PAGE. The immunoblot used anti-His-tag antibody. Relative percent flash yields were calculated from the maximum laser flash-induced depletion values at 500 nm. P, G, and M fusions are defined by the fusion junction position as shown in Fig. 1.

FIG. 5

Absorption spectra of purified His-tagged fusion proteins. (A) NpSRII protein in E. coli membrane. The absorption maximum is at 503 nm. (B) NpSRII-NpHtrII fusion protein. The absorption maximum is at 501 nm. (C) NpSRII-NpHtrII-StTar fusion chimera. The absorption maximum at is 503 nm. Matching molar concentrations of the different proteins were used in the spectral measurements.

FIG. 6

Phototaxis responses for H. salinarum. Two seconds after initiation of data collection, a 500-nm photostimulus was delivered to the cells for 100 ms. Five points per second were collected and used to calculate the reversal frequency. Response data are the averages for 16 repetitive stimuli delivered every 1 min.

FIG. 7

Phototaxis responses by E. coli containing fusion chimeras. One-half second after initiation of data collection, cells with 1 μM retinal (except row A) were stimulated with 2 s of 500-nm continuous light for recording the attractant or repellent responses of Tar-M fusion (A and B), Tar-P and Tsr-P fusion (C and D), Tar-G and Tsr-G fusion (E and F), and Tar-M and Tsr-M fusion (G and H). Tracking was set at 15 frames per second, and the rcd (degrees/second) was plotted. Response data are the averages for 32 repetitive stimuli delivered every 30 s.