Growth of rth⁺ and rth-6 cells. Strains MG1655rr/pJK286-rth⁺ cdsA⁺ and MG1655rr/pJK286-rth-6 cdsA⁺ were grown in LB broth containing 0.5% NaCl at 30°C for 12 h. Then, 0.05 ml of these cultures was inoculated into LB broth containing 0.5% NaCl (A) or without NaCl (B). The cells were grown at 30 or 42°C with shaking. Optical densities at 660 nm (OD₆₆₀) were measured at 1-h intervals. Open squares, rth⁺ at 30°C; solid squares, rth⁺ at 42°C; open circles, rth-6 at 30°C; solid circles, rth-6 at 42°C.

Biosynthesis of isoprenoids.

E. coli rth gene. (A) Homology between the S. cerevisiae Rer2 protein and the E. coli Rth protein. The polypeptides of Rer2 and Rth are shown as boxes. The shaded and striped boxes represent homologous domains, and the percent values indicate identity and similarity (in parentheses) between the two domains. The numbers along the polypeptides indicate the amino acid (aa) residue numbers. (B) Chromosomal inserts in plasmids used for complementation test. The arrows indicate open reading frames in the rth region. The arrowheads represent the PCR primers used for cloning. The chromosomal regions cloned into the vector pJK2039 are shown as three open boxes: top, the PCR fragment amplified with primers 3 and 4; middle, the same PCR fragment with a Cm^r cassette inserted into the HpaI site within the rth gene; bottom, the PCR fragment lacking the 3′ region of the cdsA gene. Plus and minus signs denote complementation, positive and negative, respectively, for both rth-2 and rth-6 mutations.

Cell shapes of the rth-6 mutant. The upper panels show the rth⁺ strain, MG1655rr/pJK286-rth⁺ cdsA⁺; the lower panels show the rth-6 mutant, MG1655rr/pJK286-rth-6 cdsA⁺. The cells were incubated at the indicated temperatures for 2 h and observed without fixation under a microscope equipped with phase-contrast optics. The magnifications are the same for all of the panels (a 100× objective was used). The arrowheads in the panel with the rth mutant incubated at 42°C indicate ghosts.

Prenyltransferase activity. (A) Prenol products synthesized in an in vitro assay. Cell homogenates were prepared from the strains MG1655rr/pJK286 (mini-F Ap^r)-rth⁺ cdsA⁺ (lanes 1 and 2), MG1655rr/pJK286-rth-2 cdsA⁺ (lanes 3 and 4), MG1655rr/pJK286-rth-4 cdsA(Ts) (lanes 5 and 6), and MG1655rr/pJK286-rth-6 cdsA⁺ (lanes 7 and 8). The homogenates (0.1 mg of protein) were incubated with 10 μM [1-¹⁴C]IPP and 5 μM FPP in a final volume of 0.2 ml at 30°C (lanes 1, 3, 5, and 7) or at 40°C (lanes 2, 4, 6, and 8) for 30 min. The resulting prenols were extracted and analyzed by TLC on a silica gel 60F₂₅₄ plate with a benzene-ethyl acetate (4:1 [vol/vol]) solvent system and by autoradiography. For more details, see Materials and Methods. The arrowheads indicate the positions of authentic prenols: Fi, ficaprenols; So, solanesol (all-E-nonaprenol); Ori, origin; SF, solvent front. (B) Fractionation of cell homogenates. The homogenates were prepared from the same strains shown in panel A, rth⁺ (open circles), rth-2 (solid triangles), rth-4 (solid squares), and rth-6 (solid circles), and fractionated by DEAE-Toyopearl column chromatography. The prenyltransferase assay was carried out at 30°C with 50 μl of each fraction as the enzyme source in a final volume of 0.1 ml. The dotted line indicates the NaCl concentration of the elution buffer. (C) Prenol products of fraction 3. The cell homogenates prepared from the strains shown in panels A and B, rth⁺ (lanes 1 and 2), rth-2 (lanes 3 and 4), rth-4 (lanes 5 and 6), and rth-6 (lanes 7 and 8), were fractionated as described for panel B, and fraction 3 of each sample was incubated with [1-¹⁴C]IPP and FPP in the presence of 0.04 mg of Triton X-100 in the 0.2-ml mixture. The reaction was carried out at either 30 (lanes 1, 3, 5, and 7) or 40°C (lanes 2, 4, 6, and 8) for 30 min (lanes 1 and 2) and 3 h (lanes 3 to 8). The resulting prenols were analyzed by TLC on a reversed-phase LKC-18 plate with an acetone-water (19:1 [vol/vol]) solvent system and autoradiography. The arrowheads indicate the positions of authentic prenols: So, solanesol (all-E-nonaprenol); C₅₀, decaprenol (ficaprenol-50); C₅₅, undecaprenol (ficaprenol-55); C₆₀, dodecaprenol (ficaprenol-60); Ori, origin; SF, solvent front.