white resin, and polymerized in gelatin capsules at 50'C for 5 days (Aldrich et al. 1992). Thin sections were placed on 100 mesh Formvar-coated nickel grids, blocked for 30 min. on 1% powdered milk in phosphate-buffered saline (PBS) at pH 7.2, and floated overnight on rabbit polyclonal antibody against purified diol dehydratase from Klebsiella oxytoca (provided by Tetsuo Toraya) diluted 1:1000 in PBS. After labeling with the primary antibody, the grids were washed two times with high-salt Tris-Tween buffer, followed by two washes with water and then floated on goat anti-rabbit antibody conjugated to 12-nm diameter colloidal gold particles (Jackson ImmunoResearch Laboratories, Inc., West Grove, Pa.). Samples were washed as done for primary labeling, fixed for 10 minutes by floating on Trump's reagent(McDowell and Trump 1976), and then post-stained with 0.5% uranyl acetate followed by staining with lead citrate. Construction of In-Frame pduCDE Deletion

      Restriction digestion was used to delete pduD and fuse the remaining 5'pduC and 3' pduE DNA sequence (Figure 2-1). Plasmid pVJ70 containing thepduABCDEFG genes was digested with NruJ releasing two fragments, of 1123 and 2448 bp, between pduC andpduE, leaving 90 bp of the 5' region containing the translational start site of pduC and 225 bp of the 3' region containing the translational stop site ofpduE intact. Next a 10 bp linker (GAAGATCTTC) was ligated into the digested vector in such a manner that the reading frame between the remaining pduC and pduE sequences was retained. The ligation reaction was used to transform E. co/i HB 101 via electroporation, and DNA sequencing was performed on subsequent clones to verify that only one linker had been introduced into the plasmid. One clone (plasmid pGH1) containing an insert of desired sequence, was digested with XbaJ and SphJ and then ligated into pCVD442 using