ligase was from New England Biolabs. Electrophoresis supplies were from Bio-Rad (Hercules, CA). Bacterial Protein Extraction Reagent II (B-PER II) and Micro BCA reagents were from Pierce (Rockford, IL). Other chemicals were from Fisher Scientific (Pittsburgh, PA).

Bacterial Strains, Media, and Growth Conditions

     The bacterial strains used in this study are listed in Table 3-1. The rich medium used was Luria-Bertani (LB) medium (Difco Laboratories, Detroit, MI) (Miller 1972). The minimal media used was the No-carbon-E (NCE) medium (Vogel and Bonner 1956, Berkowitz et al. 1968, Marco and Orus 1993). Amino acids were provided at the following concentrations: valine, isoleucine, leucine, and threonine, 0.3 mM; and histidine, 0.1 mM. Antibiotics were provided in liquid or solid rich medium at the following concentrations unless otherwise stated: carbenicillin, 100 mg/ml; ampicillin, 100 mg/ml; kanamycin, 50 mg/ml; spectinomycin, 50 mg/ml; and tetracycline, 20 mg/ml. Ampicillin was used at 15 mg/ml in minimal media. Tetracycline was used at 2 mg/ml to induce expression out of TPOP insertions. IPTG was added to a final concentration of 1 mM for induction of genes cloned into pET vectors or placl'PO-BglII. MacConkey/PD/vitamin B12 indicator plates and aldehyde indicator plates were prepared as described previously (Conway et al. 1987, Bobik et al. 1999). General Protein and Molecular Methods

     Plasmid purification, bacterial transformation, electrophoresis, and other standard molecular and protein methods were performed as previously described (Sambrook et al. 1989, Johnson et al. 2001).